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![JEM Vol. 211, No. 8
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Histological and immunohistochemical analysis. Spinal columns were
removed after mice were perfused with 4% paraformaldehyde (PFA). For im-munofluorescence
assay, free floating sections of the lumbar spinal cord were
prepared as previously described (Huang et al., 2006). For immunofluores-cence
assay, sections were blocked with 10% normal serum for 2 h and stained
with primary antibodies at 4°C for 24–48 h. After washing with PBS-T (PBS
with 0.1% Triton X-100; Sigma-Aldrich) three times, the sections were incu-bated
with secondary antibodies at room temperature for 2 h and mounted
in ProLong Gold antifade reagent (Invitrogen). Antibodies used include rat
anti-CD11b (BD), mouse anti-GFP (Abcam), rabbit anti-RFP (Abcam), Alexa
Fluor 488 goat anti–mouse IgG (Invitrogen), Alexa Fluor 594 goat anti–rabbit
IgG (Invitrogen), and Alexa Fluor 647 goat anti–rat IgG (Invitrogen). Nuclei
were labeled by DAPI. Images were collected by confocal laser-scanning mi-croscope
1547
(SP5; Leica).
Quantitative 3D morphology. Quantitative 3D morphology of MDMs
and MiDMs was analyzed in confocal images from spinal cord of mice at
EAE onset. Free floating sections of the lumbar spinal cord were stained with
RFP for MDMs, GFP for MiDMs, and DAPI for nuclei. Stack images were
taken at 0.2-μm step size along the z-direction with a 63× objective (numer-ical
aperture [NA] = 1.4) and zoom factor 2. A square (1,024 × 1,024 pixels)
corresponding to 123 × 123 μm2 was used for the analysis. Cells were 3D re-constructed
by ImageJ software and all analyses were performed using ImageJ
with 3D Convex Hull plugin. The parameters analyzed include voxel (volu-metric
pixel), convex voxel, volume, convex volume, surface, and convex sur-face
area. Other calculated parameters were: Solidity3D = volume/convex
volume; Convexity3D = convex surface area/surface area; Formfactor3D =
36 3 π × volume2 surface area3. The number of primary processes was esti-mated
visually. We included 5 mice, 54 MDMs; 51 MiDMs in this assay with
2 sections/mouse, 4–6 cells/section and 8–12 cells/mouse. Those mice came
from three EAE inductions.
SBF-SEM. Spinal cords were removed after mice were perfusion-fixed
using 4% PFA with 1% glutaraldehyde. Lumbar spinal cord sections were
made on a vibratome (Leica). Sections were stained with 0.4% OsO4, uranyl
acetate and lead aspartate, then embedded in epon resin (Electronic Micros-copy
Sciences). SBF-SEM images were acquired using a Sigma VP SEM
(Carl Zeiss) with 3View (Gatan). Serial image stacks of images at 100-nm
steps were obtained by sectioning 48 × 48 × 20 μm3 tissue blocks (length ×
width × depth) at a resolution of 8192 × 8192 pixels. Image stacks were pro-cessed
for 3D reconstruction by TrakEM2 in FIJI software (National Institutes
of Health). Alternating sections from the same stacked images were chosen to
make stacks for 3D reconstructions which matched the 0.2-μm step size used
for acquiring confocal stacked images. In SBF-SEM images, we discriminated
MDMs and MiDMs using the volume/primary processes model (Fig. 1 E)
generated from analyzing confocal images. Quantifications of myeloid-cell
spatial relationships to axoglial units, including myelin incorporation, were
done in SBF-SEM images.
Quantification of nuclei and mitochondria. Characterizations of nu-clear
shapes were conducted in SBF-SEM images. Nuclei were categorized
as follows: round, round shape and smooth surface with ratio of length/
width ≤1.5; elongated, elongated or oval shape with length/width 1.5, and
may have small indentations; Bilobulated: two connected lobes with single
intervening large indentation; Irregular: complicated shape with corrugated
surface, and may have multiple and variable sizable indentations. Blinded ob-servers
(n = 3) scoring the nuclear morphology from SBF-SEM images in-cluded
a research student, a research fellow and a neuroscientist. Observers
were trained on the same nuclear examples in each category and practiced
using 20 nuclei comprising all shapes before scoring the nuclei. Kappa test
showed good pairwise agreement rates among observers (0.8) and the data
from the neuroscientist are used. Quantifications were done in 3 individual
mice from 3 EAE inductions including 28–35 cells from two separate lesions
from each mouse in the assay.
by demonstrating and characterizing differential responses of
infiltrating monocytes and resident microglia in a relevant dis-ease
model at a prespecified time point, at which point patho-genic
events are taking place. Therefore, we focused our analysis
on the day of EAE onset rather than subsequent events to
challenge our overall hypothesis that infiltrating monocytes
versus resident microglia respond very differently to acute in-flammatory
stimuli.
Activated myeloid cells are the proximate effectors of a
bewildering array of acute and chronic disorders (Wynn et al.,
2013). The technical and conceptual approach taken in this
study may be applicable to other tissues and disease processes.
In many pathological conditions, tissues harbor a mixed pop-ulation
of activated resident and recruited monocytes. The
therapeutic strategy will differ conclusively based on the spe-cific
effector properties of each cell type and the stage of dis-ease.
In particular, if monocytes are pathogenic, then their
trafficking should be blocked using a peripherally active agent.
The optimal application of agents that regulate leukocyte mi-gration
and intracellular signaling will be promoted by de-tailed
examination of each individual myeloid population.
MATERIALS AND METHODS
Mice. C57BL/6 mice were obtained from the National Cancer Institute.
Ccr2rfp/+::Cx3cr1gfp/+ mice were generated by crossbreeding Ccr2rfp/rfp::C57BL/6
mice (Saederup et al., 2010) with Cx3cr1gfp/gfp::C57BL/6 mice ( Jung et al.,
2000). Ccr2rfp/rfp::Cx3cr1gfp/gfp mice were generated by breeding Ccr2rfp/+
::Cx3cr1gfp/+ mice. Ccr2rfp/rfp::Cx3cr1gfp/+ mice were generated by crossbreed-ing
Ccr2rfp/rfp::C57BL/6 mice with Ccr2rfp/rfp::Cx3cr1gfp/gfp mice. Animal ex-periments
were performed according to the protocols approved by the
Institutional Animal Care and Use Committee at the Cleveland Clinic fol-lowing
the National Institutes of Health guidelines for animal care.
EAE induction and clinical evaluation. EAE was induced in Ccr2rfp/+
::Cx3cr1gfp/+ mice and Ccr2rfp/rfp::Cx3cr1gfp/+ mice of 24–28 wk of age using
myelin-oligodendrocyte-glycoprotein peptide 35–55 (MOG) as previously
described (Huang et al., 2006). All mice were weighed and graded daily for
clinical stages as previously reported (Saederup et al., 2010). We defined clinical
stage of EAE as follows: pre-onset was the day sudden weight loss for 8–10%
occurred; onset was the day EAE signs appeared; peak was the second day score
didn’t increase after sustained daily worsening; and recovery was the second
day score didn’t decrease after a period of sustained daily improvement.
To address our research questions, we integrated flow cytometry, immunohistochemistry
with quantitative morphometry, cell sorting for expression
profiling, and serial block-face scanning electronic microscopy. In all, we per-formed
12 EAE immunizations in Ccr2rfp/+::Cx3cr1gfp/+ mice and 19 immu-nizations
in Ccr2rfp/rfp::Cx3cr1gfp/+ mice for this project, with 8–10 mice in
each immunization. We selected EAE mice at onset, peak or recovery de-pending
on the specific studies underway at that time, with the majority of
mice coming from the onset stage of EAE. Each experiment incorporated
samples from at least three separate immunizations. Details of mouse numbers
and how they were selected for each experiment were included in the figure
legends as requested.
Cell isolation and flow cytometry. Brains and spinal cords were removed
and homogenized. Mononuclear cells were separated with a 30%/70% Per-coll
(GE Healthcare) gradient as previously reported (Pino and Cardona,
2011). Single-cell suspensions from CNS were stained with anti–F4/80-APC
(BM8; eBioscience) and anti–CD45-PerCP (30-F11; BioLegend). Cells were
either analyzed on a LSR-II (BD) or sorted on a FACSAria II (BD) running
Diva6. Data were analyzed with FlowJo software (Tree Star).](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-29-320.jpg)
![Figure 4. Blockade of IL-21 signaling before or after tMCAO reduces infarct size in WT mice. (a) Infarct volumes 24 h after tMCAO in WT mice
treated with 500 μg recombinant mIL-21R.Fc or PBS 1 h before (pretreatment) or 2 h after (posttreatment) surgery. Representative TTC-stained brain
slices shown on left (n = 3–4 mice per group). (b) Still image from Video 1 depicting behavioral differences between WT mice posttreated with IL-21R.Fc
or PBS. (c) IL-21R.Fc protein levels in the indicated organs 20–24 h after tMCAO in WT mice injected with 500 μg IL-21R.Fc 2 h after start of reperfusion
(n = 2–4 mice per group). N.D., not detected. Data are representative of two independent experiments. **, P 0.01; ***, P 0.001, by Student’s t test. Error
bars indicate SEM. Representative images of postmortem paraffin-embedded human acute stroke lesions stained with control sera (d), or primary anti-bodies
against CD4 (e and g [ii-iii]), IL-21 (f and g [iii]), or eosin (g [i]) visualized with Fast Red (d, e, and g) and/or DAB (d, f, and g [iii]) and counterstained
with hematoxylin. High magnification images are shown on right. Arrows indicate positive staining. Bars, 50 μm.
600 IL-21 promotes brain injury after stroke | Clarkson et al.](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-52-320.jpg)
![Br ief Def ini t ive Repor t
Statistical analyses and quality standards. All surgeries were performed
in a blinded manner by a third party and measurements masked where possi-ble.
Infarct volume measurements from TTC stained sections were averaged
from two to three independent blinded observers. Based on power calcula-tions,
n = 3–10 sex- and age-matched mice were used for each experiment
and group assignment was randomized. Among animals receiving MCAO
procedure, 86.5% of WT mice, 93.5% of IL-21KO mice, and 100% of
RAG2KO mice were included in analysis. Mice were excluded due to prema-ture
death (13.5% of WT mice, 3.2% of IL-21 KO mice) or vessel variation
(3.2% of IL-21KO mice). Results are given as means ±1 SD. Multiple com-parisons
were made using one-way ANOVA. Where appropriate, two-tailed
Student’s t test analysis was used for comparing measures made between two
groups. For comparison of RT-PCR data, nonparametric Mann-Whitney
rank sum analysis was used. P-values 0.05 were considered significant.
Online supplemental material. Video 1 shows groups of WT C57BL6
mice treated with 500 μg IL-21R.Fc or PBS control via i.p. injection. On-line
supplemental material is available at http://www.jem.org/cgi/content/
full/jem.20131377/DC1.
We thank Satoshi Kinoshita for expert histopathology services, Guoqing Song for
assisting in the surgical procedures, Dr. Wenda Gao for providing reagents and
protocols for the purification of IL-21R.Fc protein, and members of our laboratory
for helpful discussions and constructive criticisms of this work. We also thank Khen
Macvilay and Sinarack Macvilay for their expertise provided for cytofluorimetry and
immunohistochemistry studies and Samuel (Joe) Ollar for assisting in the OGD procedure.
This work was supported by awards from the American Heart Association (pre-doctoral
fellowship #12PRE12060020 to B.D.S. Clarkson) and the National Institutes
of Health (NS037570 and NS076946 to ZF, AI048087 to M.S. Salamat, and
AI068730 to J.D. Lambris).
The authors have no competing financial interests.
Submitted: 1 July 2013
Accepted: 24 February 2014
REFERENCES
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Steinbach, E. Orthey, T.V. Arumugam, F. Leypoldt, O. Simova, et al.
2012. Neutralization of the IL-17 axis diminishes neutrophil invasion
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.org/10.1182/blood-2012-02-412726
Hecker, A., A. Kaufmann, M. Hecker, W. Padberg, and V. Grau. 2009. Expression
of interleukin-21, interleukin-21 receptor alpha and related type I
cytokines by intravascular graft leukocytes during acute renal allograft
forepaw(s); 2, hangs on with forepaws and moves laterally on string; 3, hangs
onto string with forepaws and hindpaw(s); 4, hangs onto string with forepaws,
hindpaw(s) and tail; 5, escape to supports. Mice were allowed to rest between
trials. Scores for each mouse were determined by averaging 5–10 trials (each
lasting 15 s). Global neurological deficit was determined by a modified Bed-erson
scoring system: 0, no deficit; 1, forelimb flexion; 2, unidirectional circling
after being lifted by tail; 3, spontaneous unidirectional circling; 4, longitudinal
rolling upon being lifted by tail; 5, spontaneous longitudinal rolling.
Generation of IL-21 receptor Fc fusion protein. Chinese hamster ovary
cell line (Korn et al., 2007) expressing the extracellular domain (aa 20–236)
of mouse IL-21R fused to the fragment crystallizable (Fc) portion of human
IgG4 (IL-21R.Fc) were maintained in UltraCHO (BioWhittaker). IL-21R.
Ig was purified from the culture supernatant by passage through a protein
G–Sepharose column and concentrated by ultrafiltration. Concentration was
determined spectrophotometrically. Purity and molecular weight were con-firmed
by sodium dodecyl-sulfate PAGE and human-IgG4 ELISA (eBiosci-ence)
following the manufacturer’s instructions. The IL-21R.Fc reagent was
tested in vitro for its ability to suppress IL-21–induced T cell proliferation.
Immunohistochemistry. Paraffin-embedded postmortem brain tissue sec-tions
from individuals with acute and chronic stroke lesions were obtained
from the Neuropathology Laboratory of the University of Wisconsin De-partment
of Pathology. After rehydration and deparaffinization, sections un-derwent
heat-induced antigen retrieval in 10 mM sodium citrate, pH 6.0, for
surface antigens or Tris-EDTA (10 mM/1 mM) with 0.05% Tween-20 for
intracellular antigens. Sections were blocked for 30 min with secondary
serum (10% in Tris-buffered saline) and then stained with primary antibodies,
0.5% chicken anti–IL-21 (Lifespan Biosciences) or prediluted mouse anti-
CD4 ([1F6]; ab17131; Abcam) for 1–2 h at 37°C or overnight at 4°C. Nor-mal
primary sera (5–10%) were used for negative control. After several washes,
secondary antibodies (biotin-labeled goat anti–chicken or biotin-labeled
goat anti–mouse; Vector Laboratories) were applied to sections and incubated
for 2 h at room temperature. Staining was developed using the VECTA-STAIN
ABC-HRP kit (Vector Laboratories) with diaminobenzidine sub-strate
(BD) or streptavidin-alkaline phosphatase with Fast Red substrate
(Laboratory Vision), following the manufacturer’s instructions. Slides were
lightly counterstained with hematoxylin, rinsed with running tap water, and
mounted. For frozen mouse sections WT and IL-21 KO mice underwent 1-h
tMCAO and 1–7-d reperfusion. Brains were perfused with PBS and 3% for-malin,
embedded in OCT, and cut into 8-μm frozen sections for immunohistochemistry.
Frozen sections were thawed for 10 min at room temperature
and blocked with 5% goat serum solution in PBS for 15 min. Sections were
stained with rabbit polyclonal antibodies for ATG6 for 1 h at room tempera-ture,
followed by phycoerythrin-labeled goat anti–rabbit IgG (Santa Cruz
Biotechnology, Inc.). Images were acquired on a BX40 microscope equipped
with a Q-Color 3 camera using Q-Capture software (Olympus). Digital
images were processed and analyzed using Photoshop CS4 software (Adobe).
Color balance, brightness, and contrast settings were manipulated to generate
final images. All changes were applied equally to entire image.
Neuronal cell oxygen glucose deprivation. Primary neuronal cultures
derived from embryonic day 14–18 mouse cortices were grown to 80% con-fluency
in neural basal media supplemented with B27 (2%) and penicillin/
streptomycin (1%), as previously described (Kintner et al., 2010). Astrocytic
and microglial contamination was excluded based on the absence of GFAP+
and CD11b+ cells when stained by immunocytochemistry. For OGD, media
was replaced with neural basal media with or without glucose and placed in
a hypoxic chamber or under normoxic conditions for 2 h at 37°C. Afterward,
cells were lysed and mRNA isolated using RNeasy mini kit (QIAGEN). For
XTT viability assay, Neuro2A underwent OGD and were treated with dose
curve of IL-21 immediately after return to normoxic media and incubated
for 4 h at 37°C. XTT labeling mixture (50 μl per well; Roche) was added ac-cording
to manufacturer’s instructions, and at18 h fluorescence was read on
a GENious Microplate reader (Tecan). Cell number was calculated using a
standard curve of known untreated cells kept under normoxic conditions.
JEM Vol. 211, No. 4 603](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-55-320.jpg)
![TDP-43 levels correlated with those of nuclear Ran (Fig. 3 C).
Moreover, in the inferior frontal gyrus of three patients with
FTLD-TDP due to GRN mutations, we found a significant
correlation between nuclear depletion of TDP-43 and Ran
(Fig. 3, D and E).
To understand the mechanisms underlying the intimate
correlation between nuclear TDP-43 and Ran, we next as-sessed
whether Ran mRNA is altered in the brains of FTLD-TDP-
43 patients, in which TDP-43 is mislocalized. By
mining an existing mRNA-expression database comparing
healthy control versus GRN-mutation-carrying FTD sub-jects
(Chen-Plotkin et al., 2008), we found that cortical Ran
expression was reduced by 60% in human subjects carrying a
GRN mutation (P = 0.04). As TDP-43 regulates the expression
of thousands of genes, in many cases by binding directly to
mRNAs and altering their stability (Polymenidou et al., 2011),
we explored the possibility that Ran mRNA is a substrate of
TDP-43. Indeed, analyses of a published unbiased screen of the
TDP-43–RNA interactions (Sephton et al., 2011) revealed
that TDP-43 binds to the 3 UTR of Ran mRNA (Fig. 4 A).
Moreover, inhibiting TDP-43 expression by shRNA-mediated
knockdown significantly reduced levels of Ran mRNA
(Fig. 4 B) and protein (Fig. 4, C–D) in N2A cells. Ran mRNA
levels were also reduced in retinas of aged Grn KO mouse
(Fig. 4 E), consistent with our observations of nuclear depletion
were strikingly reduced in Grn-KO retinal GCL neurons,
whereas levels of cytoplasmic TDP-43 were unchanged (Fig. 2,
B and C). Depletion of nuclear TDP-43 also occurred in
12-mo-old Grn-KO mice, before significant GCL neuron
loss (Fig. 2 D). Interestingly, neither nuclear nor cytoplasmic
TDP-43 inclusions were found in the 100 Grn-KO GCL
neurons we examined (Fig. 2 B). Thus, in progranulin-deficient
FTLD-TDP, nuclear depletion of TDP-43 and neurodegeneration
can occur independent of cytoplasmic TDP-43
accumulation/aggregation. These results are consistent with
observations that TDP-43, especially nuclear TDP-43, is re-quired
for neuron survival (Wegorzewska and Baloh, 2011;
Igaz et al., 2011; Arnold et al., 2013).
TDP-43 regulates Ran mRNA levels
and requires Ran for nuclear localization
We then explored how nuclear clearing of TDP-43 occurs in
FTLD. The small GTPase Ran is a master regulator of nuclear
transport (Melchior et al., 1995), and Ran accessory proteins
are necessary for nuclear TDP-43 localization (Nishimura
et al., 2010). We hypothesized that Ran expression might be
altered in our retinal FTLD model and contribute to nuclear
TDP-43 depletion. Indeed, nuclear Ran was significantly de-pleted
in Grn-KO GCL neurons (Fig. 3, A and B), and nuclear
Figure 3. Nuclear clearing of TDP-43
and Ran are pathologically associated in
FTLD-TDP. (A) 18-mo-old GCL neurons from
WT and Grn-KO retinas were co-stained for
TDP-43 and Ran. Nuclei were labeled with
DAPI. (B) Nuclear Ran levels in 18-mo-old GCL
neurons. n = 165–278 cells from 6 mice/gen-otype;
*, P = 0.019, linear regression model;
2 independent experiments. Scatter plot of
individual cell intensities with medians shown.
(C) Nuclear Ran and TDP-43 intensities are
correlated in Grn-KO GCL neurons. Each dot
represents a single cell. n = 165 cells from 6
Grn-KO mice; r = 0.8963; P 0.001, Spear-man’s
rho; 2 independent experiments.
(D) Immunofluorescence co-staining of GRN
mutant human cortex shows depletion of Ran
and TDP-43 in the same neuron (noted with
an arrow; compare to neurons with high lev-els
of TDP-43 and Ran [arrowhead]). (E) TDP-43
and Ran levels correlate in cortical neurons
from human GRN-mutation carriers. Shown
are the correlation analyses of nuclear Ran
and TDP-43 intensities of individual neurons
from post-mortem brain. n = 111–141 cells
from each of 3 subjects;, r = 0.56; P 0.001.
The serum progranulin levels were 19.3–21.2
ng/ml for R493X carrier (control patients: 41.3 ±
15.5 ng/ml). Spearman’s rho. Bars: 2 μm (A),
10 μm (D).
1940 Retinal thinning and TDP-43 mislocalization in FTLD | Ward et al.](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-60-320.jpg)
![the background AlphaLISA signal. Therefore, the actual in-hibitory
efficacy of these compounds could be higher than
the results from dose–response experiments of AlphaLISA.
In addition, avidity effects may cause higher IC50 values in
AlphaLISA than FP. There could be multiple A–fibrinogen
interactions between acceptor bead and donor bead in Alph-aLISA
(Fig. 1 B), and therefore blocking one interaction may
not reduce the signal.
Because both AlphaLISA and the FP assay are based on
optical measurements, colored compounds could significantly
modify the measurement through inner filter effects. Thus, we
confirmed the potency of our candidates using a pull-down
assay. All five compounds showed inhibitory effects, whereas
RU-505 had significant inhibitory efficacy (Fig. 2 A). These
combined experiments show that the compounds identified
are inhibitors of the A–fibrinogen interaction.
Soluble oligomeric A has been hypothesized to be the
primary toxic species in AD (Cleary et al., 2005). Therefore, we
tested which form of A, monomer or oligomer (prepared as
in Stine et al. [2011]), interacts with fibrinogen and whether
RU-505 can selectively inhibit the interaction of one or the
other. Using the AlphaLISA assay, we found that both A42
monomer and oligomer interact with fibrinogen, but the affin-ity
of oligomer for fibrinogen binding is 4 times higher than
that of the monomer (Fig. 2 B). RU-505 inhibits the interaction
of both monomer and oligomer with fibrinogen, but
has higher inhibitory efficacy against the monomer–fibrinogen
interaction than the oligomer (Fig. 2 C).
Validation of hit compounds using in vitro clotting assay
Because the interaction between A42 and fibrinogen in-duces
a structurally abnormal fibrin clot and delays fibrin clot
degradation during fibrinolysis (Ahn et al., 2010; Cortes-
Canteli et al., 2010; Zamolodchikov and Strickland, 2012),
one of the main objectives of our study was to identify com-pounds
that restore A-induced delayed fibrinolysis. When
fibrinogen associates into a fibrin meshwork after cleavage by
thrombin, the fine structure of this fibrin clot scatters light
and the solution increases in turbidity. Thus, the kinetics of
turbidity can be used as a read-out to analyze fibrin network
formation and degradation. We tested whether our hits re-stored
A-induced altered thrombosis and fibrinolysis in vitro.
Each hit compound (20 μM) or vehicle (0.4% DMSO) was
incubated for 10 min with purified human fibrinogen and
plasminogen in the presence or absence of A42. Fibrin clot
formation and degradation were analyzed by measuring tur-bidity
immediately after adding thrombin and tissue plasmin-ogen
activator (tPA) to the mixture. In the presence of A42,
the maximum turbidity of the fibrin clot was decreased be-cause
A altered fibrin clot structure and the dissolution of
the fibrin clot was delayed (Fig. 2 D; red). RU-505 restored
the A-induced decrease in turbidity during fibrin clot for-mation
(Fig. 2 D; green) and significantly reduced the delay in
fibrin degradation in the presence of A (Fig. 2 E). We also
tested other hit compounds, including RU-965, using the
turbidity assay, but none had significant effects (Fig. 2 F and
Cortes-Canteli et al., 2010); 3) A binds specifically to fibrino-gen;
and 4) fibrin clots formed in the presence of A have an
abnormal structure, making them resistant to degradation by
fibrinolytic enzymes (Ahn et al., 2010; Cortes-Canteli et al.,
2010). Overall, these results indicate that in the presence of A,
any fibrin clots formed might be more persistent and may ex-acerbate
neurovascular damage and cognitive impairment.
Therefore, molecules that block this interaction without affect-ing
clotting in general could restore altered thrombosis and fi-brinolysis
and protect against vascular damage in AD patients,
and could be used as therapeutic agents.
RESULTS
Hit identification and optimization
using high-throughput screening
To investigate this idea, we designed a high-throughput screen
(HTS) to identify small molecules that inhibit the interaction
between A and fibrinogen. Low molecular weight compounds
were screened using fluorescence polarization (FP) and Alpha-
LISA assays in a complementary fashion to cross check the
activity of the hit compounds and to ensure the removal of
false-positive artifacts. Primarily, 93,000 compounds were
screened using FP, which measured the changes in the anisotro-phy
induced by binding of a 5-carboxy-tetramethylrhodamine
(TAMRA)–labeled A peptide to fibrinogen (Fig. 1 A). Then,
hits from FP were screened using AlphaLISA to independently
confirm the activity of the inhibitors identified in the FP assay
(Fig. 1 B). After both steps, we selected only drug-like com-pounds
using Lipinski’s Rule of Five, which allowed us to de-termine
which chemical compounds have pharmacological
properties that would make them likely orally active drugs in
humans (Lipinski et al., 2001). We also filtered out artifactual
compounds using a quenching assay, which identifies insoluble
compounds, singlet oxygen quenchers, and biotin mimetics in-terfering
with the AlphaLISA signal. We identified several candi-date
compounds with half-maximal inhibitions (IC50) between
10 and 50 μM from the dose-response assays using both FP and
AlphaLISA assays (Table 1).
To expand and improve our candidate compounds, we
purchased a focused analogues compound library, based on
combinatorial variations of scaffolds from the primary hit
compounds. These analogues were screened at three different
concentrations (5, 10, and 20 μM) using AlphaLISA. Next, we
selected only drug-like compounds using Lipinski’s Rule of
Five and also included the quenching assay. If inhibition by
quenching was 30%, the compounds were removed from fur-ther
analyses because these compounds were more likely to be
false positives. Finally, we screened the active nonquenching
compounds in concentration-response experiments with freshly
dissolved powders using both FP and AlphaLISA assays. We
identified five drug-like compounds with IC50 3 μM by FP
and IC50 10 μM by AlphaLISA (Fig. 1 C and Table 2).
In some cases, the maximum inhibition of several com-pounds
in AlphaLISA was lower than that of FP. There are
several possible reasons for these differences. First, some hit
compounds showed negative quenching values, which increases
1050 A-fibrin interaction inhibitor as AD treatment | Ahn et al.](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-68-320.jpg)
![JEM Vol. 211, No. 6
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to the sensor chip surface, and RU-505 was injected for 2 min
at 30 μl/min. Sulindac sulfide was used as positive control, and
sulindac was used as negative control (Richter et al., 2010). To
analyze the correlation between HTS and SPR, we used an
analogue of RU-505, RU-4180 (Fig. 2 H), which did not in-hibit
the A–fibrinogen interaction in AlphaLISA assay. Al-though
RU-4180 weakly binds to A42 (green; Fig. 2 G),
RU-505 showed strong binding to A42 (blue; Fig. 2 G). Fur-thermore,
because it is known that sulindac sulfide binds A,
we tested whether it could inhibit the A–fibrinogen interac-tion
by AlphaLISA and found that it had no effect. These results
suggest that RU-505 inhibits the A–fibrinogen interaction
1051
not depicted). Moreover, RU-505 did not have any effect
on fibrin clot formation and degradation in the absence of
A (Fig. 2 D, purple). This result suggests that RU-505 could
effectively restore A-induced altered fibrin clot structure
and delayed degradation without affecting normal clot for-mation
and fibrinolysis.
The interaction between A and RU-505
To elucidate how RU-505 inhibits the A–fibrinogen inter-action,
surface plasmon resonance (SPR) was used to analyze
the binding characteristics of RU-505 (Fig. 2 G). Hexaflu-oroisopropanol-
treated monomerized A42 was immobilized
Figure 1. The chemical structure and
dose–response curve of A–fibrinogen
interaction inhibitors. (A) TAMRA–labeled
A peptide was bound to fibrinogen and the
test compound, and the anisotropy of
TAMRA–A–fibrinogen binding was deter-mined
by FP. (B) Biotin-labeled A42, which
binds a streptavidin donor, was incubated
with fibrinogen, which binds a protein A ac-ceptor
bead coated with antifibrinogen anti-body.
A42 and fibrinogen interactions bring
the beads in close proximity, resulting in the
excitation of the donor beads and release of
singlet oxygen molecules that triggers light
emission in acceptor beads (AlphaLISA [AL]).
(C) The half-maximal inhibitory concentration
(IC50) values of the indicated compounds were
determined by dose–response FP and AL ex-periments
and are indicated inside the panel
(red, FP; blue, AL). A quenching test was also
performed to calculate how much each hit
compound interfered with the AL signal at
10 μM concentration. Quenching values are
indicated below the dose–response curve.
n = 3–4 repeats per assay and all error bars
indicate SEM. Data are representative of at
least three independent experiments.](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-69-320.jpg)
![Figure 5. RU-505 restored cognitive function in Tg6799 mice. (A) Freezing behavior was measured before electric foot shock during the training
day to assess the basal freezing tendency of each group of mice. (n = 8–10 mice per group). (B) Contextual memory was assessed by measuring freezing
behavior upon reexposure to the training chamber 24 h after fear conditioning training. (*, P 0.05; **, P 0.01; n = 8–10 mice per group). Results are
from two independent experiments. (C–E) Spatial learning and memory retention of WT and Tg6799 mice was assessed using the Barnes maze after 3 mo
of treatment with RU-505 or vehicle. One target hole was connected to a hidden escape chamber. (C) During training trials, latency to poke the target
hole was measured. Significance was assessed using two-way ANOVA analysis with repeated measure (WT/vehicle vs. Tg6799/vehicle: F[1,120] = 40.47;
P 0.001; Tg6799/vehicle vs. Tg6799/RU-505: F[1,108] = 11.97; P 0.01; n = 10–14 mice per group). Differences in latency were assessed by Bonferroni post hoc
analysis. (D–F) During the Barnes maze probe trial, latency to reach the closed target hole (D), number of visits to the target hole (E), and total traveled
distance (F) were measured ([E] *, P 0.05; **, P 0.01; ***, P 0.001; n = 10–14 mice per group; [F] ***, P 0.001; n = 10–14 mice per group). All results
of the Barnes maze are from three independent experiments.
deposition (green; Fig. 7 A) outside the endothelial cells of
blood vessels that were labeled using CD31 (red; Fig. 7 A), and
the area of activated microglia that were labeled using CD11b
(red; Fig. 7 B). The levels of infiltrated fibrinogen and micro-gliosis
were highly increased in the cortex of Tg6799 com-pared
with WT mice (Fig. 7, C and D), and these increases
were significantly decreased by RU-505 (Fig. 7, C and D).
in AD patients and mouse models of AD, and an increase of
inflammation in the brain is correlated with memory im-pairment
(Bayer et al., 1999; Dhawan and Combs, 2012;
Vom Berg et al., 2012).
Therefore, we measured the level of infiltrated fibrinogen
and microgliosis in the cortex of Tg6799 or WT littermate
mice after RU-505 treatment. We quantified fibrinogen
Figure 6. RU-505 restored spatial retention memory
in TgCRND8 mice without affecting motor behavior.
(A) The spatial memory of vehicle- or RU-505–treated WT
and TgCRND8 mice was assessed using Barnes maze.
(B and C) Spatial memory of RU-505–treated WT and
TgCRND8 mice was tested using the Barnes maze probe
trials. Time to reach the target hole (B), the number of visits
to the closed target hole (C), and total distance traveled (D)
were assessed (n = 7–11 mice per group). The results cor-roborate
those in Fig. 5 and are from one experiment. All
values are means and SEM.
1056 A-fibrin interaction inhibitor as AD treatment | Ahn et al.](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-74-320.jpg)
![over 3 mo, which minimized this issue. Our future direction
would modify RU-505, and find less toxic analogues with sim-ilar
or better efficacy.
For more than a decade, A has been the major target for
developing AD therapies. Most of these efforts focused on using
antibodies to lower A levels, preventing A aggregation, or re-ducing
A production. However, none of these methods were
successful as the treatments did not show clinical efficacy or
caused serious adverse side effects such as aseptic meningoen-cephalitis
(Gilman et al., 2005; Mangialasche et al., 2010). How-ever,
numerous studies still support the hypothesis that A plays
an important role in the pathogenesis of AD (Tanzi and Bertram,
2005; Jonsson et al., 2012). Therefore, new strategies for anti-A
therapy are necessary for developing novel treatments for AD. In-hibiting
the interaction between A and its binding proteins
could be an alternative therapeutic approach, and our study
shows that a small molecule, bioavailable inhibitor of the A–
fibrinogen interaction, RU-505, significantly restored altered
thrombosis and improved cognitive deficits observed in AD
transgenic mouse models. Therefore, treatment of the neurovas-cular
pathology observed in AD using an inhibitor of the
A–fibrinogen interaction may be a valuable strategy for devel-oping
novel AD therapeutics.
MATERIALS AND METHODS
Animals
Tg6799 mice (The Jackson Laboratory) are double transgenic mice for APP/
Presenilin 1 that coexpress five early onset familial AD mutations on a mixed
background C57BL/6 x SJL (Oakley et al., 2006). TgCRND8 mice (pro-vided
by A. Chishti and D. Westaway, University of Toronto, Canada) have
three APP mutations (K670N, M671L, and V717F) driven by the human
prion protein promoter on a mixed background C57 x C3H/C57 (Chishti
et al., 2001). RU-505 was prepared in 2.5% EtOH, 4.5% Cremophor RH40
(Sigma-Aldrich), and 14% D5W (5% dextrose in water) in saline. We admin-istered
35 mg/kg dose of RU-505 or vehicle to Tg6799 mice and 25 mg/kg
dose or vehicle to TgCRND8 mice subcutaneously every other day. Non-transgenic
(WT) littermates were used in all experiments. The assigned geno-type
of all the mice used in the experiments throughout the paper was
double-checked by taking tail tissue the day of sacrifice. Only male mice
were used in experiments, and all animals were maintained in The Rocke-feller
University Comparative Biosciences Center and treated in accordance
with protocols approved by The Rockefeller University Institutional Animal
Care and Use Committee.
Primary compound screening
Approximately 93,000 compounds were screened using HTS. Compound
screening libraries that include known off-patent drugs, natural products, and
combinatorially elaborated active pharmacophores were purchased from
several vendors listed in Table 3. The primary assay used FP to measure the
changes in the anisotropy induced by binding of TAMRA-labeled A42
(Anaspec) to fibrinogen. TAMRA-A42 (2 nM) was mixed with 300 nM
fibrinogen (EMD Millipore) and 20 μM compounds (dissolved in 1% DMSO
[final]) in 50 mM PBS, pH 7.4, 0.001% Tween 20, and 0.001% BSA as 50 μl
final volume in black 384-well plates (Greiner) at RT. After binding reached
equilibrium, polarization measurements were recorded with a Perkin-Elmer
EnVision plate reader with excitation at 490 nm and emission at 535 nm. The
FP response was monitored and plotted as milli-Polarization (mP) units.
Compounds that showed 75% inhibition of the A–fibrinogen inter-action
in the FP assay were selected for screening by AlphaLISA as a second-ary
assay. Compounds (12.5 μM) were plated in white 384-well plates
affinity is 10 times less than A42, and RU-505 has a strong
inhibitory efficacy on A40–fibrinogen interaction (unpub-lished
data). Therefore, RU-505 could reduce CAA through
inhibition of both A42- and A40–fibrinogen interaction.
Second, despite the higher levels of A40 in vascular amy-loid,
A42 is also essential for vascular amyloid deposition
in transgenic mice overexpressing human APP (Van Dorpe
et al., 2000; McGowan et al., 2005) and AD human patients
(Roher et al., 1993; Shinkai et al., 1995). A42 could act as
a nucleation seed of amyloid deposit in the vessel walls and
accelerate deposition of A40 (Van Dorpe et al., 2000; Yoshiike
et al., 2003; McGowan et al., 2005). Therefore, even though
the ratio of A40 to A42 is higher in vascular amyloid, the
A42–fibrinogen interaction could be critically involved in
CAA formation.
Fibrinogen is a proinflammatory mediator in several dis-eases
and induces the activation of microglia in the nervous
system (Adams et al., 2007; Davalos and Akassoglou, 2012).
Our study showed that RU-505 treatment reduced the level
of infiltrated fibrinogen and activated microglia in the brain
of AD transgenic mice. One possible mechanism for this re-duction
is that RU-505 binds A, inhibits the A–fibrinogen
interaction, and facilitates fibrinogen degradation. The decreased
level of infiltrated fibrinogen could result in the decrease of
microgliosis. The other possible mechanism is that long-term
RU-505 treatment reduced vascular amyloid deposits and
prevented BBB leakage. This recovery of a healthy BBB could
reduce fibrinogen infiltration and inflammation in the paren-chyma
of Tg6799 mice.
Increased levels of plasma fibrinogen are associated with
cognitive deficits (Xu et al., 2008), AD risk (van Oijen et al.,
2005), and brain atrophy (Thambisetty et al., 2011), and in-creased
levels of fibrinogen have also been found in the CSF
of AD patients (Craig-Schapiro et al., 2011; Vafadar-Isfahani
et al., 2012). Moreover, several studies have shown that anti-coagulant
treatment improves cognition in mouse models of
AD and dementia patients (Ratner et al., 1972; Walsh et al.,
1978; Cortes-Canteli et al., 2010; Timmer et al., 2010). How-ever,
anticoagulant therapy can cause severe problems in
elderly patients who have a more fragile vasculature because
it may increase the incidence of major systemic bleeding. There-fore,
drugs should specifically block the A–fibrinogen inter-action
so that A-induced altered blood clot formation and
degradation can be restored without affecting general hemo-stasis.
RU-505 successfully targeted only A-induced altered
blood clot formation and did not affect general clot forma-tion
and degradation (Fig. 2 D and Fig. 3).
The maximum tolerated dose of RU-505 after single in-travenous
dose in mice was between 100 and 200 mg/kg.
When we treated Tg6799 mice and WT littermates with two
doses (100 and 50 mg/kg) of RU-505 every other day for 3 mo,
we found that 100 mg/kg for long-term treatment was toxic
to the AD mice, but 50 mg/kg showed no clinical signs of
toxicity except local chronic inflammation at the injection
site. To address the issue of local inflammation, we lowered the
dose to 35 mg/kg for Tg6799 or 25 mg/kg for TgCRND8
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![JEM Vol. 211, No. 6
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biotinylated A42 (Anaspec) and 5 nM fibrinogen (EMD Millipore) for 1 h
at room temperature in 500 μl of binding buffer (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 0.1% Nonidet P-40, 0.1% BSA, and protease inhibitor mix-ture).
The samples were gently rotated for 1 h at room temperature with
30 μl streptavidin–Sepharose high performance beads (GE Healthcare). After
incubation, the beads were washed five times with binding buffer, and non-reducing
sample buffer was added to the beads for elution. Western blots
were performed using antifibrinogen antibody (Dako). Dot blots were per-formed
using anti-A antibody 4G8 (Covance) to show comparable amounts
1059
of A were also being pulled down.
The binding assay between fibrinogen
and monomeric or oligomeric A42
Biotin-A42 monomers and oligomers were prepared as in (Stine et al.,
2011). In brief, biotin-LC-A42 (Anaspec) was monomerized by treatment
with hexafluoroisopropanol, dissolved to 5 mM with dimethyl sulfoxide,
then diluted to 100 μM with cold PBS, and sonicated. Monomeric biotin-
LC-A42 was incubated at 4°C for 24 h for oligomeric preparation. 1 nM
fibrinogen was mixed with increasing concentrations of monomeric or
oligomeric biotin-LC-A42 (0.5–20 nM) for 30 min at room temperature
and the binding affinity was measured using AlphaLISA assay. The inhibitory
efficacy of RU-505 on the interaction between fibrinogen and monomeric
or oligomeric biotin-LC-A42 was accessed in dose–response experiments
using AlphaLISA assay.
In vitro thrombosis and fibrinolysis assay
To test whether hit compound have an effect on fibrin clot formation and
lysis, 20 μM of each compound (dissolved in 0.4% DMSO [final]) or DMSO
control was incubated with fibrinogen (1.5 μM) in the presence or absence
of A42 (3 μM) for 10 min and then mixed with plasminogen (0.25 μM) in
20 mM Hepes buffer (pH 7.4) with 137 mM NaCl. Fibrin clot formation
and degradation was analyzed measuring turbidity right after adding throm-bin
(0.5 U/ml), tPA (0.15 nM), and CaCl2 (5 mM) in a final volume of
150 μl. Assays were performed at RT in High Binding 96-well plates
(Thermo Fisher Scientific) in triplicate and were measured at 450 nm using
a Spectramax Plus384 reader (Molecular Devices).
SPR
SPR experiments were performed to test whether our lead compounds bind
to A42 as described previously (Richter et al., 2010). Biacore 3000 instru-ment
and CM5 sensor chips (GE Healthcare) were used for this assay. Hexa-fluoroisopropanol-
treated monomerized A42 was immobilized to the
sensor chip surface by amine coupling. Compounds were diluted to 40 μM
from DMSO stock solutions in PBS as running buffer (final 2% DMSO) and
injected for 2 min at a flow rate of 30 μl/min using the KINJECT command.
After the dissociation phase the chip was rinsed with 20 mM HCl. Corre-sponding
DMSO dilutions were used as a buffer blank, and a solvent correc-tion
assay was performed to correct the difference of DMSO response
between empty reference surface and protein-immobilized surface. Sulindac
sulfide and sulindac were used as positive control and negative controls, re-spectively
(Richter et al., 2010).
In vivo toxicity study
Maximum tolerated dose studies were performed to determine the toxicity
of RU-505 (AMRI) and to identify the optimal dose for in vivo assays. Sin-gle
injection toxicity was performed at Absorption Systems LP (Exton, PA),
and four different doses (200, 100, 50, and 20 mg/kg mouse) of RU-505,
along with saline and vehicle, were injected into male and female CD-1
mice intravenously. Mortality and overt clinical signs of toxicity were moni-tored
for 2 d. All animals dosed with 200 mg/kg were found dead after single
intravenous injection, and no clinical signs of toxicity were observed after
single dose of 20, 50, or 100 mg/kg for 2 d after injection. Therefore, the
maximum tolerated dose of RU-505 after single intravenous dose in mice
was established as 100 mg/kg.
(Greiner) and were incubated with 10 nM biotinylated A42 (Anaspec) and
1 nM fibrinogen for 30 min at RT in final volume of 10 μl assay buffer
(25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 0.1%
BSA). The mixture was incubated with anti–fibrinogen antibody (Dako),
20 μg/ml streptavidin-conjugated donor, and protein A–conjugated accep-tor
beads (PerkinElmer) for 90 min at RT. Samples were read by a PerkinElmer
EnVision plate reader.
Hit compounds from the secondary assay were evaluated using Lipinski’s
Rule of Five to determine whether each chemical compound has properties
that make it a potential usable drug. If compounds violated more than one of
Lipinski’s Rule of Five, those compounds were removed from our list. The
AlphaScreen TruHits kit (PerkinElmer) was used to detect those compounds
that react with singlet oxygen and thus unspecifically quench the assay. The
AlphaScreen TruHits kit also allows for the identification of color quenchers,
light scatterers (insoluble compounds), and biotin mimetics interfering with
the AlphaLISA signal. If inhibition by quenching was more than 30% at 10 μM
compound, those compounds were removed from our list. After completing
the quenching test, we screened hit compounds in a dose–response experiment
with various compound concentrations (0.01–20 μM) using FP and Alpha-
LISA. The data were fitted to sigmoidal dose–response equation (Y = Bottom +
(Top – Bottom)/1 + 10(logIC50 X) × Hill coefficient)) using GraphPad Prism 4 to
calculate half-maximal inhibition (IC50) of each compound. Compounds with
IC50 50 μM in both FP and AlphaLISA were purchased as powder and were
retested in dose–response experiments using both assays.
Analogue compound screening
To improve our candidate compounds, we had access to the ChemNavigator
database, which has 50 million commercially available compounds and software
for Tanimoto-based similarity searching. We purchased 2,000 analogue com-pounds
through ChemNavigator or directly from Albany Molecular Research
Inc. These analogues were tested using AlphaLISA at 5, 10, and 20 μM. We se-lected
compounds which have 50% inhibition at 10 μM and a proportional in-hibitory
effect at 5 or 20 μM. Drug-like compounds were evaluated using
Lipinski’s Rule of Five, and false-positive compounds were filtered out using the
AlphaScreen TruHits kit (PerkinElmer) as described above. Compounds with
IC50 10 μM in both FP and AlphaLISA were selected using dose–response ex-periments.
Selected compounds were purchased as powder and were retested in
dose–response experiments using both assays. Finally, we identified hit com-pounds
of with IC50 3 μM in FP and IC50 10 μM AlphaLISA assay.
Pull-down assay
Hit compounds were tested using a pull-down assay as described previously
(Ahn et al., 2010). In brief, compounds at 10 μM were incubated with 100 nM
Table 3. Vendor list for primary screening library
Provider No. of compound
from each provider
ChemDiv 21,986
Prestwick 1,110
Cerep 4,000
ChemBridge 5,000
Microsource 2,000
AMRI 50,000
Biofocus 7,750
GreenPharma 240
Sigma LOPAC 1,280
Prof. Derek Tan (Memorial Sloan-
Kettering Cancer Center, New
York, NY)
350
Total 93,716](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-77-320.jpg)
![Ar t icle
Acid sphingomyelinase modulates
the autophagic process by controlling
lysosomal biogenesis in Alzheimer’s disease
Jong Kil Lee,1,2,3 Hee Kyung Jin,1,4 Min Hee Park,1,2,3 Bo-ra Kim,1,2,3
Phil Hyu Lee,5 Hiromitsu Nakauchi,6 Janet E. Carter,7 Xingxuan He,8
Edward H. Schuchman,8 and Jae-sung Bae1,2,3
1Stem Cell Neuroplasticity Research Group, 2Department of Physiology, Cell and Matrix Research Institute, School of Medicine,
3Department of Biomedical Science, BK21 Plus KNU Biomedical Convergence Program, 4Department of Laboratory Animal
Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Korea
5Department of Neurology and Brain Research Institute, Yonsei University College of Medicine, Seoul 120-752, Korea
6Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science,
University of Tokyo, Tokyo 108-8639, Japan
7Mental Health Sciences Unit, Faculty of Brain Sciences, University College London, London WC1E 6DE, England, UK
8Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029
In Alzheimer’s disease (AD), abnormal sphingolipid metabolism has been reported, although
the pathogenic consequences of these changes have not been fully characterized. We show
that acid sphingomyelinase (ASM) is increased in fibroblasts, brain, and/or plasma from
patients with AD and in AD mice, leading to defective autophagic degradation due to
lysosomal depletion. Partial genetic inhibition of ASM (ASM+/) in a mouse model of famil-ial
pathological findings, including reduction of amyloid- (A) deposition and improvement
of memory impairment. Similar effects were noted after pharmacologic restoration of ASM
to the normal range in APP/PS1 mice. Autophagic dysfunction in neurons derived from FAD
patient induced pluripotent stem cells (iPSCs) was restored by partial ASM inhibition.
Overall, these results reveal a novel mechanism of ASM pathogenesis in AD that leads to
defective autophagy due to impaired lysosomal biogenesis and suggests that partial ASM
inhibition is a potential new therapeutic intervention for the disease.
The Rockefeller University Press $30.00
J. Exp. Med. 2014 Vol. 211 No. 8 1551-1570
www.jem.org/cgi/doi/10.1084/jem.20132451
AD (FAD; amyloid precursor protein [APP]/presenilin 1 [PS1]) ameliorated the autopha-gocytic
1551
defect by restoring lysosomal biogenesis, resulting in improved AD clinical and
Alzheimer’s disease (AD) is the most common
form of dementia. It is characterized clinically
by progressive loss of memory, and pathologi-cally
by the presence of neuritic plaques and
neurofibrillary tangles (Selkoe, 2001). There are
profound biochemical alterations in multiple
pathways in the AD brain, including changes in
amyloid- (A) metabolism, tau phosphoryla-tion,
and lipid regulation, although to date the
underlying mechanisms leading to these complex
abnormalities, as well as the downstream conse-quences,
remain largely unknown (Yankner et al.,
2008; He et al., 2010; Mielke et al., 2012).
Sphingolipid metabolism is an important pro-cess
for tissue homeostasis that regulates the
formation of several bioactive lipids and second
messengers that are critical in cellular signaling
(Lahiri and Futerman, 2007; Wymann and
Schneiter, 2008). In the brain, the proper bal-ance
of sphingolipid metabolites is essential for
normal neuronal function, and subtle changes in
sphingolipid homeostasis may be intimately in-volved
in neurodegenerative diseases including
AD (Cutler et al., 2004; Grimm et al., 2005;
Hartmann et al., 2007; Grösgen et al., 2010;
Haughey et al., 2010; Mielke and Lyketsos, 2010;
Di Paolo and Kim, 2011; Tamboli et al., 2011).
Recently, our studies and those of others
(Katsel et al., 2007; He et al., 2010) have shown
that the activity of several sphingolipid metabolizing
enzymes, including acid sphingomyelinase
CORRESPONDENCE
Jae-sung Bae:
jsbae@knu.ac.kr
Abbreviations used: A, amyloid-;
AC, acid ceramidase; AD,
Alzheimer’s disease; ALP,
autophagy–lysosome pathway;
AMI, amitriptyline-hydrochloride;
AP, alkaline phosphatase;
ApoE4, apolipoprotein E4;
APP, amyloid precursor protein;
ASM, acid sphingomyelinase;
AV, autophagic vacuole; CM,
conditioned medium; EM,
electron microscope; FAD,
familial AD; i.c., intracerebral;
iPSC, induced pluripotent stem
cell; Lamp1, lysosomal-associated
membrane protein 1; LBPA,
lysobisphosphatidic acid; LC3,
microtubule-associated protein 1
light chain 3; M6P, mannose-6-
phosphate; NPD, Niemann-
Pick disease; PD, Parkinson’s
disease; PS1, presenilin 1;
SA--gal, senescence-associated-
-galactosidase; TFEB, tran-scription
factor EB. J.K. Lee and H.K. Jin contributed equally to this paper.
© 2014 Lee et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months
after the publication date (see http://www.rupress.org/terms). After six months
it is available under a Creative Commons License (Attribution–Noncommercial–
Share Alike 3.0 Unported license, as described at http://creativecommons.org/
licenses/by-nc-sa/3.0/).](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-81-320.jpg)
![ASM elevation causes defective autophagic
degradation by lysosomal depletion
To gain more direct insights into the relationship of ASM and
autophagic dysfunction, we treated human fibroblasts and neu-rons
with recombinant 1–10 μM ASM and determined the
LC3-II and p62 levels. ASM strongly accelerated LC3-II and
Figure 5. Partial genetic inhibition of
ASM reverses defective autophagy in
APP/PS1 mice. (A) Western blot analysis of
LC-3 and beclin-1 levels in controls, PS1-FAD,
and ApoE4 fibroblasts. (B) LC3-II and beclin-1
levels were quantified (n = 4 per group).
(C) Immunocytochemistry for LC3 in controls,
PS1-FAD, and ApoE4 fibroblast (n = 4–5 per
group; bars, 20 μm). (D) Degradation of long-lived
proteins was measured in controls, PS1-
FAD, and ApoE4 fibroblasts (n = 6 per group).
(E) Representative images and quantification
of LBPA in control, PS1-FAD, and ApoE4
fibroblast (n = 4 per group; bars, 50 μm).
(F) Western blot analyses for LC3, beclin-1,
p62, and cathepsin D in tail fibroblast derived
from WT, APP/PS1, ASM+/, and APP/PS1/
ASM+/ mice. (G) Densitometric analysis of
LC-3-II, beclin-1, p62, and cathepsin D (n =
7–8 per group). (H) Cathepsin D activity in
mice tail fibroblast (n = 4 per group). (I) Rates
of proteolysis of long-lived proteins in fibro-blasts
(n = 6 per group). (J) Representative
images and quantification data of SA--gal
staining in the mice tail fibroblasts (n = 5 per
group; bars, 50 μm). (K) Western blot analy-ses
for LC3, beclin-1, p62, and cathepsin D in
the brains of 9-mo-old WT, APP/PS1, ASM+/,
and APP/PS1/ASM+/ mice. (L) Densitometric
quantification of LC-3-II, beclin-1, p62, and
cathepsin D (n = 6–8 per group). (M) Cathep-sin
D activity in brain extracts of WT, APP/
PS1, ASM+/, and APP/PS1/ASM+/ mice (n = 4
per group). (N) EM images and quantifica-tion
data of cortical region. Higher magnifi-cation
of boxed area shows detail of AVs
(arrow; n = 5 per group; bars: [low magnifi-cation]
2 μm, [high magnification] 1 μm).
(O) Western blot analysis of Rab5 and Rab7
levels in the brain lysates (n = 5 per group).
Data are representative of two (A–E and N) or
three (F–M and O) independent experiments.
B–O, one-way ANOVA, Tukey’s post hoc test.
*, P 0.05. All error bars indicate SEM.
p62 levels in human fibroblasts and neurons in a concentration-dependent
manner (Fig. 6, A–C). The level of beclin-1 ex-pression
was not affected by ASM (Fig. 6, A and C), indicating
that the accumulation of autophagosomes was not due to the
biogenesis pathway. ASM is found in a secretory and a lysosomal
form ( Jenkins et al., 2009), and the mannose-6-phosphate (M6P)
1558 Role of ASM in the pathogenesis of AD | Lee et al.](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-88-320.jpg)
![To examine whether partial genetic inhibition of ASM
affected the ALP in APP/PS1 mice, we also analyzed TFEB
and Lamp1 levels in the brain samples derived from WT,
APP/PS1, ASM+/, and APP/PS1/ASM+/ mice. Compared
with WT, APP/PS1 mice showed significantly decreased TFEB
and Lamp1 expression, which were increased in APP/PS1/
ASM+/ mice (Fig. 8 H). Together, these findings show for
the first time a direct correlation of lysosomal ASM and the
function of the ALP, and suggest that abnormal autophagic
degradation in AD may be due to the effects of elevated ASM
expression on this pathway.
Pharmacological restoration of ASM to the
normal range improves pathology in AD mice
The ASM-mediated lysosomal/autophagic dysfunction in AD
prompted us to examine possible therapeutic implications of
this pathway. To decrease ASM in APP/PS1 mice, we undertook
pharmacological inhibition using amitriptyline-hydrochloride
(AMI) for 4 mo (Fig. 9 A). AMI is a known inhibitor of ASM
that can cross the blood–brain barrier. At 9 mo of age, AMI-treated
APP/PS1 mice exhibited decreased ASM activity
compared with vehicle-treated mice (Fig. 9 B). Other sphingo-lipid
metabolites were not changed (Fig. 9 C). A levels were
decreased in the AMI-treated APP/PS1 mice compared with
the nontreated littermates (Fig. 9, D–G). The levels of LC3-II,
p62, and cathepsin D were decreased in the AMI-treated APP/
PS1 mice (Fig. 9, H and I). Actual activity of cathepsin D was
not changed by AMI treatment (Fig. 9 J). AMI treatment sig-nificantly
increased TFEB and Lamp1 protein levels in APP/
PS1 mice (Fig. 9, H and I). Similarly, APP/PS1 mice treated
with AMI showed recovery of memory function (Fig. 9, K–P).
Overall, these positive but relatively moderate results (e.g., A
levels) in AMI-treated APP/PS1 mice might be due to under
dosing of the animals. We speculate that this may be improved
in the future by adjusting the dose or using modified, more
potent drugs of a similar class.
Restoration of ASM ameliorates autophagic
dysfunction in the AD patient–specific cells
To further validate our observation made by partial ASM in-hibition
in AD mice, we studied possible changes in autophagy
dysfunction in human AD fibroblast after ASM inhibition.
Elevated ASM levels in human AD fibroblasts (PS1–familial
AD [FAD] and ApoE4) were restored to normal range by ASM
siRNA treatment (Fig. 10 A). ASM siRNA-treated human
AD fibroblasts (PS1-FAD and ApoE4) showed decreased
LC3-II and p62 accumulation compared with control siRNA-treated
cells (Fig. 10 B). Also, ASM siRNA was able to increase
lysosome levels (as judged by Lamp1 expression) by activating
TFEB in the human AD fibroblasts (Fig. 10 C).
Many insights into the pathogenesis in neurodegenerative
disease have come from investigating postmortem brain tis-sues
due to the difficulty of invasive access to living human
CNS. The recent developments in induced pluripotent stem
cells (iPSCs) and induced neurons have allowed investigation
of pathogenesis of neurological diseases in vitro (Kondo et al.,
2013). To explore whether the observed effects of ASM in
previous results are paralleled by similar alterations in AD human
neurons, we first established iPSCs with PS1 mutation (PS1
iPSC-2, -4, and -21) by transduction of human fibroblast with
retroviruses encoding OCT4, SOX2, KLF4, and c-Myc. The
PS1-iPSC cell line was shown to be fully reprogrammed
to pluripotency, as judged by colony morphology, alkaline
phosphatase (AP) staining, expression of pluripotency-associated
transcription factors and surface markers, karyotype stability,
and generation of teratomas (Fig. 10, D–G). To establish
whether the PS1 mutation may affect neuronal differentia-tion,
PS1 iPSC and control iPSC lines were induced to dif-ferentiate
into neurons for 10 d. Consistent with previous
results (Kondo et al., 2013), no obvious differences in the abil-ity
to generate neurons were observed between control and
PS1-iPSCs (Fig. 10 H). A42 secretion level was increased in
PS1 iPSC-derived neurons compared with control iPSC-derived
neuron (Fig. 10 I).
Next, we investigated whether elevated ASM in fibroblasts
was also evident increased in PS1 iPSC and iPSC-derived
neurons. The ASM activity in PS1 iPSC was not changed ex-cept
for PS1-4 iPSC in comparison to those in control iPSC,
but the activity of ASM was significantly higher in PS1 iPSC-derived
neurons compared with control iPSC-derived neu-ron
(Fig. 10 J). Elevated ASM levels in PS1 iPSC-derived
neurons were restored to normal range by ASM siRNA treat-ment
(Fig. 10 J). Neurons from PS1-4 iPSCs also had signifi-cantly
higher abnormal autophagic markers than neurons
from control iPSC (Fig. 10 K). ASM siRNA treatment significantly
decreased the protein level of abnormal autophagic
markers in PS1 iPSC-derived neurons (Fig. 10 K). To corrob-orate
the immunoblotting results, we performed EM analysis
using control and PS1 iPSC-derived neurons. As expected,
PS1 iPSC-derived neurons exhibited increased AV accumula-tion,
whereas ASM siRNA-treated PS1 iPSC-derived neu-rons
showed a reduced number of these vesicles (Fig. 10 L).
and A42 in the brains of AMI treated or nontreated APP/PS1 mice were assessed using immunofluorescence staining (E and F; n = 8 per group; bars,
200 μm) and ELISA kits (G; n = 6 per group). (H and I) Western blot analyses and quantification for LC3, Beclin-1, p62, cathepsin D, TFEB, and Lamp1 in
the brains of APP/PS1 mice treated with AMI or control (n = 6–8 per group). (J) Cathepsin D activity in the brain extracts of AMI-treated or nontreated
APP/PS1 mice (n = 4 per group). (K) Escape latencies of APP/PS1 mice treated with AMI or control over 10 d (WT, n = 14; nontreated APP/PS1, n = 10; and
AMI-treated APP/PS1, n = 12). (L–O) Probe trial day 11. (L and M) Path length (L) and swim speed (M) were recorded and analyzed. (N) Time spent in target
platform and other quadrants was measured. (O) The number of times each animal entered the small target zone during the 60-s probe trial. (P) Repre-sentative
swimming paths at day 10 of training. Data are representative three independent experiments. B–J and N, Student’s t test; K–M and O, one-way
ANOVA, Tukey’s post hoc test. *, P 0.05; **, P 0.01. All error bars indicate SEM.
1564 Role of ASM in the pathogenesis of AD | Lee et al.](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-94-320.jpg)
![2007). Moreover, it has been shown that the abnormal au-tophagic
flux in AD may be due to dysfunction at the late au-tophagy
stage associated with the lysosome (Lai and McLaurin,
2012; Zhou et al., 2012). Similar to previous results (Lee et al.,
2010b; Lai and McLaurin, 2012), we found that the impaired
autophagic flux in AD was associated with reduced autopha-gic
degradation due to decreased ALP function. In addition,
we show for the first time that this is directly linked to ele-vated
ASM activity. Suppression of ASM expression or inhibi-tion
of its uptake and delivery to lysosomes using M6P reversed
abnormal autophagic degradation. These findings indicate
that increased lysosomal ASM plays a negative role in AD by
causing autophagic dysfunction, suggesting that therapeutic
strategies to restoring ASM activity to the normal range may
be beneficial for AD pathology.
This was further studied in the AD mice by finding that
partial genetic or systemic inhibition of ASM activities in these
animals largely reversed autophagic pathology by restoring
ALP function, as well as reducing the accumulation of incom-pletely
digested substrates within the autophagic-lysosomal
compartments (e.g., LC3-II and p62). A accumulation also
was reduced in response to ASM inhibition, as was cathepsin D
expression. There are several challenges associated with inter-pretation
of cathepsin D levels in AD. Although some reports
have shown that cathepsin D activities were decreased in AD
(Lee et al., 2010b), many studies indicate that cathepsin D is
elevated in AD and contributes to the pathogenesis, such as A
formation (Cataldo et al., 2004; Lai and McLaurin, 2012; Zhou
et al., 2012). A recent paper suggested that COP9 signalosome
deficiency increased cathepsin D levels but reduced the au-tophagic
degradation. They suggested that these results were
associated with a failure of lysosomal assembly of cathepsin D
because only a lysosomal cathepsin D could affect autophagic
degradation (Su et al., 2011). In this study, we have found that
maturation of cathepsin D was increased in AD mice, but the
actual enzyme activity was not changed between the groups.
This result indicated that the elevated levels of cathepsin D did
not ultimately translate into a significant increase of enzyme
activity. Based on these papers and our data, a plausible inter-pretation
of increased cathepsin D in our AD mice is that AD
microenvironment attempts to increase cathepsin D synthesis,
but this does not have a direct impact on lysosomal function
because the activity of the enzyme is unchanged. Therefore,
Decreased TFEB target genes in PS1 iPSC-derived neurons
were also significantly increased by ASM siRNA treatment
(Fig. 10 M). These results confirm that abnormal autophagy
observed in AD mice and human fibroblasts by ASM also
occur in AD patient neurons, and restoration of ASM back to
normal levels is able to ameliorate autophagic dysfunction by
restoring lysosomal biogenesis in AD patient cells.
DISCUSSION
Although the exact causes of AD are unknown, the complex
interactions of genetic and environmental factors are likely play
important roles in the pathogenesis. ASM activity is known to
be increased by environmental stress and in various diseases,
and is elevated in AD patients (He and Schuchman, 2012).
One downstream consequence of increased ASM is elevated
ceramide, contributing to cell death, inflammation, and other
common disease findings. Although elevated ASM is known
to occur in AD, the cellular mechanisms that link ASM and
AD have not been fully characterized. The data presented
here suggest a previously unknown role of ASM in the down-regulation
of lysosomal biogenesis and inhibition of lysosome-dependent
autophagic proteolysis. The findings also establish
proof of concept for ASM inhibitor therapy in AD.
Our previous study showed that sphingolipid metabolism
was severely impaired in the human AD brain, and that ASM
activity was positively correlated with the A levels (He et al.,
2010). Consistent with our previous study, we found that ASM
was significantly increased in fibroblasts, brain, and/or plasma
from patients with AD and in AD mice, although other sphin-golipid
factors were unaltered. There are some differences be-tween
the previous and this study. For example, the previous
results showed increased ceramide level in AD, but we could
not found significant changes of sphingolipid factors includ-ing
ceramide in AD compared with normal samples. These
differences might be related to the fact that once formed, ce-ramide
can rapidly enter several metabolic pathways. It may
be used for either the biosynthesis of complex lipids or bro-ken
down into sphingosine, which itself is rapidly converted
to sphingosine 1 phosphate.
Accumulation of abnormal AVs has been observed in AD
(Lee et al., 2010b; Nixon and Yang, 2011), and the autophagy
pathway is increasingly regarded as an important contributor
to A-mediated pathogenesis in AD (Yu et al., 2005; Nixon,
fibroblast after ASM inhibition (n = 5–6 per group). (D–G) Generation of PS1 iPSC lines from patient fibroblast. (D) Established iPSCs showed embryonic
stem cell–like morphology (Phase; bar, 1 mm), AP activity (bar, 200 μm), and expressed pluripotent stem cell markers SSEA4 (bar 100 μm), TRA1-60 (bar
100 μm), and TRA1-81 (bar 100 μm). (E) Normal karyotype of PS1 iPSC. (F) Quantitative real-time PCR analysis of hESC marker gene of PS1 iPSC (n = 3 per
group). (G) Gross morphology and hematoxylin-eosin staining of representative teratomas generated from PS1-4 iPSCs (bars, 50 μm). (H) Estimation of
neural differentiation from control and PS1-4 iPSCs. Representative images of immunocytochemical staining the -III tubulin after neural differentiation
(bars, 50 μm). (I) The amount of A42 secreted from control iPSC-derived neuron and PS1 iPSC-derived neuron (n = 5 per group). (J) Characterization of
ASM activity in the control and PS1 iPSC and iPSC-derived neurons (n = 6 per group). (K) Western blot analyses for LC3, beclin-1, p62, TFEB, and Lamp1 in
the control and PS1-4 iPSC–derived neuron after ASM siRNA treatment (n = 5–6 per group). (L) EM images and quantification data of control and PS1
iPSC-derived neurons. Higher magnification of boxed area shows detail of AVs (arrow; n = 4 per group; bars: [low magnification] 1 μm, [high magnifica-tion]
500 nm). (M) Quantitative real-time PCR analysis of TFEB-target gene expression in iPSC-derived neurons after ASM siRNA treatment (n = 5–6 per
group). Data are representative of two (A, D–G, I, and L), or three (B, C, H, J, K, and M) independent experiments. A, C, F, I, J, and M, Student’s t test. B, K,
and L, one-way ANOVA, Tukey’s post hoc test. *, P 0.05; **, P 0.01; ***, P 0.001. A–K, error bars indicate SEM. L and M, Error bars indicate SD.
1566 Role of ASM in the pathogenesis of AD | Lee et al.](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-96-320.jpg)
![JEM Vol. 211, No. 8
Ar t icle
1567
enhanced cathepsin D level in our AD mice induced by in-creased
ASM is more likely a compensatory response to an
impaired lysosome system. The present study also provides the
first evidence of increased ASM activity and autophagic dys-function
in living human (iPSC-derived) neurons derived
from AD patients and that restoring normal levels of ASM in
AD neurons effectively blocks abnormal autophagy.
Overall, the data presented here show that increased ASM
activity in AD contributes to the abnormal lysosomal/au-tophagic
process by leading to dysfunction of ALP. This results
in an inability to break down appropriate substrates during
the autophagy process. Restoration of ASM effectively blocks
AD progression by increasing autophagic degradation. Al-though
the involvement of other ASM-related mechanisms in
AD remains to be explored, the data in this study demonstrate
that inhibition of ASM improves A clearance and rescues
impaired memory in a validated mouse model of AD, suggest-ing
this as a potential therapy for AD patients in the future.
MATERIALS AND METHODS
Mice. Transgenic mouse lines overexpressing the hAPP695swe (APP) and
presenilin-1M146V (PS1) mutations, respectively, were generated at GlaxoSmithKline
by standard techniques as previously described (Howlett et al.,
2004). In brief, a Thy-1–APP transgene was generated by inserting the 695 aa
isoform of human cDNA (APP695) harboring the Swedish double familial
mutation (K670N; M671L) into a vector containing the murine Thy-1 gene.
The Thy-1–PS-1 transgene was generated by inserting the coding sequence
of human PS-1 cDNA harboring the M146V familial mutation into a vector
containing the murine Thy-1 gene. Transgenic lines were generated by pro-nuclear
microinjection into fertilized oocytes from either C57BL/6xC3H
mice in the case of Thy-1-APP transgene, or into fertilized oocytes from pure
C57BL/6 mice in the case of Thy-1–PS-1 transgene. Thy-1 APPswe mice were
generated and backcrossed onto a pure C57BL/6 background before crossing
with TPM (PS-1 M146V) mice to produce heterozygote double mutant mice.
ASM+/ mice (C57BL/6 background; Horinouchi et al., 1995) were bred
with APP/PS1 mice to generate APP/PS1/ASM+/ mice. Because APP/PS1
mice show sex difference in disease progression, we used only male mice. Data
analysis of APP/PS1 mice was done at 5 or 9 mo old. Block randomization
method was used to allocate the animals to experimental groups. To eliminate
the bias, we were blinded in experimental progress such as data collection and
data analysis. SCID Beige mice (Charles River) were used for teratoma forma-tion
assay. Mice were housed at a 12 h day/12 h night cycle with free access to
tap water and food pellets. Mouse studies were approved by the Kyungpook
National University Institutional Animal Care and Use Committee (IACUC).
Plasma collection. Human plasma samples were obtained from individuals
with AD, PD, and age-matched, non-AD controls from Yonsei University
Severance Hospital (Table S1). Informed consent was obtained from all sub-jects
according to the ethics committee guidelines at the Yonsei University
Severance Hospital.
Cell culture. Human fibroblast lines (normal, PS1, ApoE4, and PD) ac-quired
from the Coriell Institute were maintained in DMEM with 15% FBS.
The human cortical neuronal cell line HCN-2 was acquired from ATCC.
Cells with a passage number 10–15 were used in this study. To obtain CM
containing ASM, 5 × 105 Chinese hamster ovary cells overexpressing human
ASM (He et al., 1999) were cultured in DMEM for 2 d. Cells were washed
with PBS and changed with new media. 24 h later, the CM was collected,
centrifuged, and filtered using a 0.22 μm filter. We isolated WT, APP/PS1,
APP/PS1/ASM+/, and ASM+/ mouse tail fibroblasts as previously de-scribed
(Takahashi et al., 2007a) from 9-mo-old mice. For some experiments,
cells were treated with purified, recombinant ASM or ASM siRNA to mea-sure
autophagy regulation. NH4Cl was used to inhibit the autophagic flux.
For the inhibition of lysosomal ASM uptake, 10 mM M6P or M6P receptor
siRNA were added to the fibroblast culture media at the same time as ASM.
Drug or CM treatments. 4-mo-old APP/PS1 mice received 100 μg/g body
weight AMI (Sigma-Aldrich) per os in their drinking water for 4 mo, and a
control group received water without drug. 3-mo-old C57BL/6 mice were treated
with ASM-CM via i.v. (100 μl) or i.c. (3 μl) injections on 10 consecutive days.
Immunofluorescence. Thioflavin S staining was done according to previ-ously
described procedures (Lee et al., 2012). We used anti-20G10 (mouse,
1:1,000, provided by D.R. Howlett, GlaxoSmithKline, Harlow, Essex, UK)
for A 42, anti-G30 (rabbit, 1:1,000, provided by D.R. Howlett) for A40,
rabbit anti–Iba-1 (1:500; Wako), rabbit anti-GFAP (1:500, Dako), mouse
anti–-SMA (1:400; Sigma-Aldrich), rabbit anti-AT8 (1:500; Thermo Fisher
Scientific), and rabbit anti–active caspase3 (1:50; EMD Millipore). The sec-tions
were analyzed with a laser-scanning confocal microscope (FV1000;
Olympus) or with a BX51 microscope (Olympus). MetaMorph software
(Molecular Devices) was used to quantification.
Ab ELISA. For measurement of A40 and A42, we used commercially
available ELISA kits (BioSource). Hemispheres of mice were homogenized
in buffer containing 0.02M guanidine. ELISA was then performed for A40
and A42 according to the manufacturer’s instructions.
Behavioral studies. We performed behavioral studies to assess spatial learn-ing
and memory in the Morris water maze as previously described (Lee et al.,
2012). Animals were given four trials per day for 10 d to learn the task. At 11 d,
animals were given a probe trial in which the platform was removed. Fear
conditioning was conducted as previously described techniques (Kojima et al.,
2005). On the conditioning day, mice were individually placed into the con-ditioning
chamber. After a 60-s exploratory period, a tone (10 kHz, 70 dB)
was delivered for 10 s; this served as the conditioned stimulus (CS). The CS co-terminated
with the unconditioned stimulus (US), a scrambled electrical foot-shock
(0.3 mA, 1 s). The CS-US pairing was delivered twice at a 20-s intertrial
interval. On day 2, each mouse was placed in the fear-conditioning chamber
containing the same exact context, but with no administration of a CS or foot
shock. Freezing was analyzed for 5 min. On day 3, a mouse was placed in a test
chamber that was different from the conditioning chamber. After a 60-s ex-ploratory
period, the tone was presented for 60 s without the footshock. The
rate of freezing response of mice was used to measure the fear memory.
Quantitative real-time PCR. RNA was extracted from the brain homog-enates
and cell lysates using the RNeasy Lipid Tissue Mini kit and RNeasy
Plus Mini kit (QIAGEN) according to the manufacturer’s instructions.
cDNA was synthesized from 5 μg of total RNA using a commercially avail-able
kit (Takara Bio Inc.). Quantitative real-time PCR was performed using
a Corbett research RG-6000 real-time PCR instrument. Used primers are
described in Table S2.
EM. Brain tissues and cells were fixed in 3% glutaraldehyde/0.1 M phosphate
buffer, pH 7.4, and postfixed in 1% osmium tetroxide in Sorensen’s phosphate
buffer. After dehydration in ethyl alcohol, the tissue and cells were embedded
in epon (Electron Microscopy Sciences). Samples were cut serially and placed
on copper grids and analyzed using a transmission EM (Tecnai). Images were
captured on a digital camera and Xplore3D tomography software.
Intracellular protein degradation measurement. Total protein degrada-tion
in cultured cells was measured by pulse-chase experiments with 48 h
pulse with 2 μCi/ml [3H]-leucine for 48 h to preferentially label long-lived
proteins (Lee et al., 2010b).
Western blotting. Samples were immunoblotted as previously described
(Settembre et al., 2011; Lee et al., 2012). Primary antibodies to the following](https://image.slidesharecdn.com/jemneurosciencespecialissue-141202045722-conversion-gate02/85/Jem-neuroscience-special_issue-97-320.jpg)
Uploaded byElsa von Licy
Jem neuroscience special_issue
The document summarizes recent publications from The Journal of Experimental Medicine focusing on neuroscience. It highlights several studies: one finding that monocyte-derived macrophages initiate myelin destruction in multiple sclerosis, while microglia-derived macrophages clear debris; another activating B-RAF kinase to promote axon regeneration after nerve injury; and one showing that blocking interleukin-21 reduces brain injury following stroke in mice. It also summarizes studies on retinal thinning in frontotemporal dementia and targeting neurovascular pathology in Alzheimer's disease models.
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- 2. Behind good imagesare great protocols Everyone loves beautiful images, but the challenge is to generate clean data. Selecting the right IHC/ICC protocol does not have to be a daunting task. Receive an IHC/ICC Protocol Guide with your next purchase of performance-validated reagents. This easy-to-use laboratory reference is suitable for basic to intermediate skill levels and contains: Direct and indirect protocols Colorimetric and immunofluorescence staining techniques At-a-glance workflow diagrams Required material lists Troubleshooting guide See how you can get your IHC/ICC Protocol Guide. www.ebioscience.com/ihc-jem Great protocols found here Biology for a better world. NORTH AMERICA: 888.999.1371 EUROPE: +43 1 796 40 40-305 JAPAN: +81 (0)3 6430 4020 INQUIRIES: info@ebioscience.com ©Affymetrix, Inc. All rights reserved. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
- 3. Welcome Neuroscience TheJournal of Experimental Medicine now prints topic-specific mini collections to showcase a handful of our recent publications. In this installment, we highlight papers focusing on the mechanisms and models of neurological disease. Myelin destruction in multiple sclerosis (MS) is mediated by inflammatory macrophages, but the origin of these cells has been unclear. Our collection begins with an Insight from Michael Heneka discussing findings from Yamasaki et al. who use a mouse model of MS to distinguish tissue-resident microglial cells from infiltrating monocytes. Using double chemokine reporter mice, the authors find that monocyte-derived macrophages initiate myelin destruction, mainly in the nodes of Ranvier, while microglia-derived macrophages are involved with clearing debris. In a different type of central nervous system (CNS) injury, O’Donovan et al. demonstrate that activation of intraneuronal B-RAF kinase is sufficient to drive axon regeneration after nerve crush injury. An Insight from Valeria Cavalli and David Holtzman discusses how reactivation of the B-RAF pathway, which appears to be quiescent in central axons, can be utilized for regrowth. Together, these studies offer new strategies to treat inflammatory pathologies and promote repair. Acute cerebral ischemia reperfusion injury is mediated in part by T cells. Clarkson et al. show that brain-infiltrating CD4+ T cells sustain neuroinflammation after stroke in mice by producing interleukin (IL)-21 and increasing neuronal death. Treatment with an IL-21 decoy receptor or genetic lack of IL-21 protected animals from brain injury following stroke, offering a potential target for immunotherapy. Additionally, analysis of postmortem human brain tissue confirmed that IL-21 localizes to CD4+ T cells surrounding acute stroke lesions. Loss of cells in the retina is one of the earliest signs of frontotemporal dementia (FTD), occurring even before behavioral changes appear. Ward et al. show that mislocalization of CNS protein TDP-43 in eye neurons is associated with retinal thinning and occurs before the neurologic symptoms of FTD develop. Misplaced TDP-43 appears to be due to low expression of the TDP-43 regulating protein Ran, as boosting Ran expression corrects TDP-43 localization and increases neuron survival in the eye. Understanding the mechanisms underlying FTD could lead to novel therapeutic targets. Cerebral vascular abnormalities in Alzheimer’s disease (AD) have been shown to correlate with the degree of cognitive impairment. Strickland and colleagues describe a small molecule, RU-505, that inhibits the interaction between amyloid-b (Ab) and the blood clotting protein fibrinogen, reducing vascular pathologies and ameliorating cognitive impairment in mouse models of AD. Thus targeting neurovascular pathology may offer new a therapeutic strategy for treating AD. In AD, various biochemical functions of brain cells can also go awry, leading to progressive neuronal damage and eventual memory loss. Impaired autophagy causes the accumulation of toxic protein plaques characteristic of the disease. Bae and colleagues find elevated levels of acid sphingomyelinase (ASM), which breaks down cell membrane lipids in the myelin sheath that coat nerve endings. Reducing levels of ASM in mice with AD-like disease restored autophagy, lessened brain pathology, and improved learning and memory in the mice. Together these studies provide new insights into the biology and mechanisms of neurologic diseases and offer insight into therapeutics. We hope you enjoy this complimentary copy of our Neuroscience collection. We invite you to explore additional collections at www.jem.org and to follow JEM on Facebook, Google+, and Twitter. Selected Articles November 2014 Macrophages derived from infiltrating monocytes mediate autoimmune myelin destruction Michael T. Heneka Differential roles of microglia and monocytes in the inflamed central nervous system Ryo Yamasaki, Haiyan Lu, Oleg Butovsky, Nobuhiko Ohno, Anna M. Rietsch, Ron Cialic, Pauline M. Wu, Camille E. Doykan, Jessica Lin, Anne C. Cotleur, Grahame Kidd, Musab M. Zorlu, Nathan Sun, Weiwei Hu, LiPing Liu, Jar-Chi Lee, Sarah E. Taylor, Lindsey Uehlein, Debra Dixon, Jinyu Gu, Crina M. Floruta, Min Zhu, Israel F. Charo, Howard L. Weiner, and Richard M. Ransohoff
- 4. AVANTI’S NEW PROBES PACFA pacFA is a new technology that Avanti is making available to probe cellular protein-lipid interactions in vivo. The pacFA lipid contains a photoactivable diazirine ring with a clickable alkyne group and was developed by Dr. Per Haberkant at the European Molecular Biology Laboratory. To screen for protein-lipid interactions cells are fed pacFA as a pre-cursor for the biosynthesis of bifunctional lipids. Proteins in contact with the bifunctional lipids are then cross-linked by UV irradiation of the diazirine ring. Finally, click chemistry is used to label the alkyne with a reporter molecule. Labeling with a biotinylated azide allows for the affinity purification and profiling of cross-linked proteins with mass spectrometry; la-beling with a fluorescent azide allows visualization of cross-linked proteins with microscopy. Haberkant, P., R. Raijmakers, M. Wildwater, T. Sachsenheimer, B. Brugger, K. Maeda, M. Houweling, A.C. Gavin, C. Schultz, G. van Meer, A.J. Heck, and J.C. Holthuis. (2013). In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids. Angew Chem Int Ed Engl 52:4033-8. AVANTI® IS THE WORLD’S FIRST CHOICE FOR - • PHOSPHOLIPIDS, SPHINGOLIPIDS, DETERGENTS & STEROLS • CGMP LIPIDS FOR PHARMACEUTICAL PRODUCTION • LIPID ANALYSIS VISIT AVANTILIPIDS.COM pacFA Avanti Number 900401 pacFA-18:1 PC Avanti Number 900408 16:0-pacFA PC Avanti Number 900407 pacFA Galactosyl Ceramide Avanti Number 900406 pacFA Glucosyl Ceramide Avanti Number 900405 pacFA Ceramide Avanti Number 900404
- 5. B-RAF unlocks axonregeneration Valeria Cavalli and David M. Holtzman B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS Kevin J. O’Donovan, Kaijie Ma, Hengchang Guo, Chen Wang, Fang Sun, Seung Baek Han, Hyukmin Kim, Jamie K. Wong, Jean Charron, Hongyan Zou, Young-Jin Son, Zhigang He, and Jian Zhong T cell–derived interleukin (IL)-21 promotes brain injury following stroke in mice Benjamin D.S. Clarkson, Changying Ling, Yejie Shi, Melissa G. Harris, Aditya Rayasam, Dandan Sun, M. Shahriar Salamat, Vijay Kuchroo, John D. Lambris, Matyas Sandor, and Zsuzsanna Fabry Early retinal neurodegeneration and impaired Ran-mediated nuclear import of TDP-43 in progranulin-deficient FTLD Michael E. Ward, Alice Taubes, Robert Chen, Bruce L. Miller, Chantelle F. Sephton, Jeffrey M. Gelfand, Sakura Minami, John Boscardin, Lauren Herl Martens, William W. Seeley, Gang Yu, Joachim Herz, Anthony J. Filiano, Andrew E. Arrant, Erik D. Roberson, Timothy W. Kraft, Robert V. Farese, Jr., Ari Green, and Li Gan A novel Ab-fibrinogen interaction inhibitor rescues altered thrombosis and cognitive decline in Alzheimer’s disease mice Hyung Jin Ahn, J. Fraser Glickman, Ka Lai Poon, Daria Zamolodchikov, Odella C. Jno-Charles, Erin H. Norris, and Sidney Strickland Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease Jong Kil Lee, Hee Kyung Jin, Min Hee Park, Bo-ra Kim, Phil Hyu Lee, Hiromitsu Nakauchi, Janet E. Carter, Xingxuan He, Edward H. Schuchman, and Jae-sung Bae
- 6. NEW SIXTH EDITION MOLECULAR BIOLOGY OF THE CELL BRUCE ALBERTS, University of California, San Francisco, USA ALEXANDER JOHNSON, University of California, San Francisco, USA JULIAN LEWIS, Formerly of Cancer Research, UK DAVID MORGAN, University of California, San Francisco, USA MARTIN RAFF, University College London, UK KEITH ROBERTS, Emeritus, University of East Anglia, UK PETER WALTER, University of California, San Francisco, USA As the amount of informaƟ on in biology expands dramaƟ cally, it becomes increasingly important for textbooks to disƟ ll the vast amount of scienƟ Į c knowledge into concise principles and enduring concepts. As with previous ediƟ ons, Molecular Biol-ogy of the Cell, Sixth EdiƟ on accomplishes this goal with clear wriƟ ng and beauƟ ful illustraƟ ons. The Sixth EdiƟ on has been extensively revised and updated with the latest research in the Į eld of cell biology, and it provides an excepƟ onal framework for teaching and learning. December 2014 1,464 pages • 1,492 illustraƟ ons Hardback • 978-0-8153-4432-2 • $169 Loose-leaf • 978-0-8153-4524-4 • $125 E-books available, including rentals FÊÙ ÃÊÙ› ®Ä¥ÊÙÃã®ÊÄ ƒÄ— ãÊ ÖçÙ‘«ƒÝ› ƒã ƒ 25% —®Ý‘ÊçÄã, ò®Ý®ã www.garlandscience.com/mboc6 ƒÄ— ƒÖÖ½ù PÙÊÃÊ CÊ—› AGL90 ƒã ‘«›‘»Êçã. CONTENTS INTRODUCTION TO THE CELL 1. Cells and Genomes 2. Cell Chemistry and BioenergeƟ cs 3. Proteins BASIC GENETIC MECHANISMS 4. DNA, Chromosomes, and Genomes 5. DNA ReplicaƟ on, Repair, and RecombinaƟ on 6. How Cells Read the Genome: From DNA to Protein 7. Control of Gene Expression WAYS OF WORKING WITH CELLS 8. Analyzing Cells, Molecules, and Systems 9. Visualizing Cells INTERNAL ORGANIZATION OF THE CELL 10. Membrane Structure 11. Membrane Transport of Small Molecules and the Electrical ProperƟ es of Membranes 12. Intracellular Compartments and Protein SorƟ ng 13. Intracellular Membrane Traĸ c 14. Energy Conversion: Mitochondria and Chloroplasts 15. Cell Signaling 16. The Cytoskeleton 17. The Cell Cycle 18. Cell Death CELLS IN THEIR SOCIAL CONTEXT 19. Cell JuncƟ ons and the Extracellular Matrix 20. Cancer 21. Development of MulƟ cellular Organisms 22. Stem Cells and Tissue Renewal 23. Pathogens and InfecƟ on 24. The Innate and AdapƟ ve Immune Systems www.garlandscience.com
- 7. THE JOURNAL OF EXPERIMENTAL MEDICINE Executive Editor Marlowe S. Tessmer phone (212) 327-8575 fax (212) 327-8511 email: jem@rockefeller.edu Senior Editor Heather L. Van Epps Scientific Editors Teodoro Pulvirenti Catarina Sacristán Editors Jean-Laurent Casanova Adolfo Garcia-Sastre David Holtzman Lewis L. Lanier William A. Muller Carl Nathan Michel Nussenzweig Anne O’Garra Alexander Rudensky Alan Sher Sasha Tarakhovsky Andreas Trumpp David Tuveson Editor Emeritus Alan N. Houghton Manuscript Coordinator Sylvia F. Cuadrado phone (212) 327-8575 fax (212) 327-8511 email: jem@rockefeller.edu Preflight Editor Rochelle Ritacco Assistant Production Editors Brianna Caszatt and Maya Frank-Levine Production Editor Shauna O’Garro Production Manager Camille Clowery Production Designer Erinn A. Grady Advisory Editors Shizuo Akira Kari Alitalo Frederick W. Alt K. Frank Austen Albert Bendelac Michael J. Bevan Christine A. Biron Christian Bogdan Hal E. Broxmeyer Meinrad Busslinger Arturo Casadevall Ajay Chawla Yongwon Choi Robert L. Coffman Daniel J. Cua Myron I. Cybulsky Riccardo Dalla Favera Glenn Dranoff Michael Dustin Douglas T. Fearon Vincent A. Fischetti Richard A. Flavell Patricia Gearhart Ronald N. Germain Christopher Goodnow Siamon Gordon Or Gozani Sergio Grinstein Philippe Gros Kristian Helin Chyi Hsieh Christopher A. Hunter Kayo Inaba Gerard Karsenty Jay Kolls Paul Kubes Vijay K. Kuchroo Ralf Kuppers Tomohiro Kurosaki Bart N. Lambrecht Klaus F. Ley Yong-Jun Liu Clare Lloyd Tak Mak Bernard Malissen James S. Malter Philippa Marrack Diane Mathis Ira Mellman Matthias Merkenschlager Sean J. Morrison Muriel Moser Christian Münz Cornelis Murre Benjamin G. Neel Michael Neuberger Victor Nussenzweig John J. O’Shea Paul H. Patterson Fiona Powrie Lluis Quintana-Murci Klaus Rajewsky Gwendalyn J. Randolph Jeffrey Ravetch Sergio Romagnani Nikolaus Romani David L. Sacks Shimon Sakaguchi Matthew D. Scharff Olaf Schneewind Stephen P. Schoenberger Hans Schreiber Gerold Schuler Robert A. Seder Rafick-P. Sékaly Charles N. Serhan Nilabh Shastri Ethan M. Shevach Roy L. Silverstein Jonathan Sprent Janet Stavnezer Andreas Strasser Stuart Tangye Steven L. Teitelbaum Thomas J. Templeton Kevin J. Tracey Giorgio Trinchieri Shannon Turley Marcel R.M. van den Brink Ulrich von Andrian Harald von Boehmer Christopher M. Walker Raymond M. Welsh E. John Wherry Linda S. Wicker Ian Wilson Thomas Wynn Monitoring Editors Marco Colonna Jason Cyster Stephen Hedrick Kristin A. Hogquist Andrew McMichael Luigi Notarangelo Anjana Rao Federica Sallusto Louis M. Staudt Toshio Suda Consulting Biostatistics Editors Glenn Heller Madhu Mazumdar Copyright to articles published in this journal is held by the authors. Articles are published by The Rockefeller University Press under license from the authors. Conditions for reuse of the articles by third parties are listed at http://www.rupress.org/terms Print ISSN 0022-1007 Online ISSN 1540-9538
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- 14. INSIGHTS doi10.1084/jem.218insight1/doiaidjem.218insight1/aidauMichael T.Heneka/auAF1University of Bon/AF1cormichael.heneka@ukb.uni-bon.de/cordocheadInsights/docheaddoctopicNews/Macrophages derived from infiltrating monocytes mediate autoimmune myelin destruction IDjem.218insight1fig1.jpeg/IDMacrophages mediate myelin destruction in multiple sclerosis (MS), but the origin of these cells (whether de-rived from tissue-resident microglial cells or infiltrating monocytes) has been widely debated. Now, Yamasaki and colleagues distinguish these cells in a mouse model of MS and show that monocyte-derived macrophages (MDMs) mediate myelin destruction, whereas microglia-derived macrophages (MiDMs) clear up the debris. Previous attempts to decipher the nature and role of cells involved in autoimmune demyelination have proven challenging. Although ontogenetically distinct, it has not been possible to distinguish macrophages derived from tissue-resident or -infiltrating cells based on morphological features (by light microscopy) or surface phenotype. Previous attempts to address this problem include parabiosis and bone marrow trans-plantation after irradiation, both strategies with substantial technical problems and limitations. IDjem.218insight1fig2.eps/IDYamasaki et al. studied double chemokine receptor (CCR2-RFP+; CX3CR1-GFP+) mice in the experimental autoimmune encephalo-myelitis (EAE) mouse model of MS. Inflammatory lesions were filled with both MDMs and MiDMs. Confocal immunohistochemistry, serial block-face scanning electron microscopy (SBF-SEM), and subsequent 3D reconstruction revealed that myelin destruction was initiated by MDMs, often at the nodes of Ranvier, whereas MiDMs were not detected at this site. Disruption of MDM infiltration by CCR2 deficiency completely abolished the presence of macrophages at the nodes of Ranvier. Gene expression profiling of both cell types at disease onset revealed substantial differences, which correlated well with the observations obtained by SBF-SEM. MDMs expressed genes attributable to effector functions, including those involved in phagocytosis and cell clearance. In contrast, MiMD gene expression patterns at disease onset were characteristic of a repressed metabolic state. This paper sets a new standard for further studies in the field. For the first time, MDMs and MiDMs have been clearly differentiated and their morphological relation-ship to axoglial structures has been analyzed. The finding that MDMs rather than MiDMs initiate myelin destruction at disease onset should enable this cell population to be targeted more effectively in future. The next stage is to verify these findings in human tissue. Future research should also assess further time points over the entire disease course, in particular to exclude that MiDMs do not join MDMs at the node of Ranvier at later stages of disease. A precise distinction between local and infiltrating cell popula-tions may also contribute to a better understanding of pathogenesis in other CNS disor-ders such as stroke and brain trauma and will hopefully lead to the development of new therapeutic strategies. Yamasaki, R., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20132477. Insight from Michael Heneka Nodes of Ranvier represent a prime site of attack for MDMs at the onset of EAE. This 3D reconstruction of SBF-SEM images shows a monocyte-derived macrophage encircling the node of Ranvier, as shown by the two primary processes (white and black arrows). Michael T. Heneka, University of Bonn: michael.heneka@ukb.uni-bonn.de
- 15. Ar t icle The Rockefeller University Press $30.00 J. Exp. Med. 2014 Vol. 211 No. 8 1533-1549 www.jem.org/cgi/doi/10.1084/jem.20132477 1533 Blood-derived monocytes and resident microglia can both give rise to macrophages in the cen-tral nervous system (CNS). In tissue sections, macrophages derived from these two distinct precursors are indistinguishable at the light microscopic level both morphologically and by surface markers. Using flow cytometry, microglia-and monocyte-derived macrophages can be isolated separately from CNS tissue lysates and expression profiling suggests distinct functional capacities (Gautier et al., 2012; Chiu et al., 2013; Butovsky et al., 2014). Microglia and monocytes are ontogeneti-cally distinct: microglia derive from yolk-sac pro-genitors during embryogenesis (Ginhoux et al., 2010; Schulz et al., 2012), whereas monocytes continuously differentiate throughout postnatal life from bone marrow hematopoietic stem cells CORRESPONDENCE Richard M. Ransohoff: ransohr@ccf.org Abbreviations used: CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; MDM, monocyte-derived macrophage; MiDM, microglia-derived mac-rophage; MS, multiple sclerosis; SBF-SEM, serial block-face scanning electron microscopy. Differential roles of microglia and monocytes in the inflamed central nervous system Ryo Yamasaki,1 Haiyan Lu,1 Oleg Butovsky,5 Nobuhiko Ohno,2 Anna M. Rietsch,1 Ron Cialic,5 Pauline M. Wu,2 Camille E. Doykan,2 Jessica Lin,1,6 Anne C. Cotleur,1 Grahame Kidd,2 Musab M. Zorlu,1,7 Nathan Sun,8 Weiwei Hu,2,9 LiPing Liu,1 Jar-Chi Lee,3 Sarah E. Taylor,10 Lindsey Uehlein,1,6 Debra Dixon,1,11 Jinyu Gu,1 Crina M. Floruta,1,12 Min Zhu,1 Israel F. Charo,13 Howard L. Weiner,5 and Richard M. Ransohoff 1,4,11 1Neuroinflammation Research Center and 2Department of Neurosciences, Lerner Research Institute; 3Department of Quantitative Health Sciences; and 4Mellen Center for Multiple Sclerosis Treatment and Research, Neurological Institute, Cleveland Clinic, Cleveland, OH 44106 5Center for Neurological Diseases, Brigham and Women’s Hospital, Harvard Institutes of Medicine, Boston, MA 02115 6Ohio State University College of Medicine, Columbus, OH 43210 7Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey 8Vanderbilt University, Nashville, TN 37235 9Department of Pharmacology, School of Basic Medical Sciences, Zhejiang University, Hangzhou, 310058 Zhejiang, China 10Case Western Reserve University, School of Medicine, Cleveland, OH 44106 11Cleveland Clinic Lerner College of Medicine, Cleveland, OH 44106 12Baylor University, Waco, TX 77030 13Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, San Francisco, CA 94158 In the human disorder multiple sclerosis (MS) and in the model experimental autoimmune encephalomyelitis (EAE), macrophages predominate in demyelinated areas and their num-bers correlate to tissue damage. Macrophages may be derived from infiltrating monocytes or resident microglia, yet are indistinguishable by light microscopy and surface phenotype. It is axiomatic that T cell–mediated macrophage activation is critical for inflammatory demyelination in EAE, yet the precise details by which tissue injury takes place remain poorly understood. In the present study, we addressed the cellular basis of autoimmune demyelination by discriminating microglial versus monocyte origins of effector macro-phages. Using serial block-face scanning electron microscopy (SBF-SEM), we show that monocyte-derived macrophages associate with nodes of Ranvier and initiate demyelination, whereas microglia appear to clear debris. Gene expression profiles confirm that monocyte-derived macrophages are highly phagocytic and inflammatory, whereas those arising from microglia demonstrate an unexpected signature of globally suppressed cellular metabolism at disease onset. Distinguishing tissue-resident macrophages from infiltrating monocytes will point toward new strategies to treat disease and promote repair in diverse inflamma-tory pathologies in varied organs. © 2014 Yamasaki et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial– Share Alike 3.0 Unported license, as described at http://creativecommons.org/ licenses/by-nc-sa/3.0/). R. Yamasaki, H. Lu, and O. Butovsky contributed equally to this paper.
- 16. were isolated fromCNS and analyzed by flow cytometry using cells from double-heterozygous Ccr2rfp::Cx3cr1gfp mice with EAE, GFP was expressed by CD45dim/Ly6C microglia, whereas RFP was restricted to CD45high/Ly6C+ monocytes (Saederup et al., 2010; Mizutani et al., 2012). These findings suggested an approach to clarifying distinct roles of MDMs and MiDMs in EAE based on differential expression of GFP and RFP reporters. Here, we use that strategy to extend pre-vious findings and address the hypothesis that MDMs and MiDMs exert different functions in neuroinflammation. We detected detailed ultrastructural characterization of MDMs and MiDMs at EAE onset. Unexpectedly, this approach provided insight into the cel-lular basis for autoimmune demyelination, which has remained obscure despite 80 yr of study in the EAE model. Here we provide evidence that MDMs initiate demyelination, often at nodes of Ranvier. In contrast, phagocytic microglia appear rela-tively inert at disease onset. Results from expression profiling provided insight into mechanisms and signaling pathways un-derlying the disparate effector properties of MDMs and MiDMs in EAE. The distinct functions of tissue-resident myeloid cells as compared with infiltrating macrophages broadly underlie disease pathogenesis in manifold circumstances and also hold promise for innovative treatment strategies. RESULTS In the CNS of mice of EAE, MDMs and MiDMs exhibit different accumulation kinetics The histological strategy in this study is shown in Table 1. At onset of EAE, two pools of CD11b+ mononuclear phagocytic cells (putative red MDMs and green MiDMs) predominated in spinal cord (Fig. 1 A), indicating that fluorochrome mark-ers could be distinguished at this time point. Using cells iso-lated from Ccr2rfp/+::Cx3cr1gfp/+ spinal cords at disease onset, flow cytometry demonstrated distinct expression of RFP and (HSCs), which require the transcription factor Myb. Microg-lial precursors are Myb independent, and microglia self-renew independently of bone marrow HSCs (Gomez Perdiguero et al., 2013). Distinct developmental origin and renewal mech-anisms imply that monocyte-derived macrophages (MDMs) and microglia-derived macrophages (MiDMs) might exert dif-ferent functions in pathological processes. Microglia represent one instance of tissue-resident macrophages, which reside in all organs. Studying the CNS as compared with other organs may carry advantages for distinguishing tissue-resident my-eloid cells from infiltrating monocytes during disease, as there is virtually no background trafficking of monocytes in the CNS parenchyma of healthy animals. In EAE, which models inflammatory aspects of MS (Williams et al., 1994; Ransohoff, 2012), macrophages dominate the inflammatory infiltrates and their numbers correlate to EAE severity (Huitinga et al., 1990, 1993; Ajami et al., 2011). However the cellular mechanisms by which macrophages promote disease progression are uncertain. Whether MiDMs or MDMs are functionally distinct and whether the two cell types differentially initiate demyelination or promote repair (Steinman et al., 2002) also remains elusive (Bauer et al., 1995). In MS autopsy tissues, prominent macrophage accumulation correlates with active demyelination (Ferguson et al., 1997; Trapp et al., 1998). Based on kinetics of cell accumulation and differential marker expression, it’s estimated that 30–50% of activated macrophages in active MS lesions derive from mi-croglia (Brück et al., 1995; Trebst et al., 2001). Therefore, dif-ferential functions of MDMs and MiDMs are relevant for human demyelinating disease. To date, no research techniques have permitted distinction between monocytes and microglia in CNS tissue without ir-radiation chimerism or parabiosis, techniques that confound interpretation or impose practical limitations (Ajami et al., 2007, 2011; Ransohoff, 2007). When F4/80+ macrophages Table 1. Histology analysis strategy Method Purpose Finding Confocal analysis of 0.2 mm optical To distinguish MDMs (RFP+) from sections (n = 104 cells). MiDMs (GFP+). MDMs and MiDMs can be distinguished by cell volume and primary processes. SBF-SEM inspection in 0.2 mm sections from 14 lesions, 7 mice at EAE onset. To detect MDMs and MiDMs in SBF-SEM using cell volume and process criteria. Using criteria detected in the previous step, it is possible to distinguish MDMs and MiDMs in SBF-SEM images. SBF-SEM inspection of ultrastructure of MDMs and MiDMs. To detect ultrastructural characteristics of MDMs and MiDMs. MDMs and MiDMs show characteristic ultrastructural differences regarding their mitochondria, nuclei, osmiophilic granules and microvilli. Quantification of relation of MDMs (n = 169) and MiDMs (n = 86) to axoglial units (n = 29 intact axons, 46 demyelinated axons). To determine relationship of MDMs and MiDMs to axoglial units and characterize presence of myelin debris. Most (55/75; 73%) axoglial units are contacted in limited fashion by MDMs and MiDMs. If one cell type is present (20/75 cells), it’s nearly always (18/20 segments) MDMs. Reconstruction of 3D shape of four representative MDMs at axoglial units. To detect relationship of MDMs with axoglial units at EAE onset. In all, 49 MDMs interacting with axoglial units in absence of nearby MiDMs, 2-3 MDMs were attached to each (n = 18) axoglial unit. MDMs have close relationship with nodes of Ranvier (7 MDMs/75 axoglial units). 3D reconstructions showed four representative MDMs at axoglial units: show one carrying out active demyelination, three at nodes of Ranvier. 1534 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
- 17. JEM Vol. 211,No. 8 Ar t icle of MDMs and MiDMs could be assayed and early events in the demyelinating disorder could be explored. Morphological features distinguish MDMs and MiDMs at EAE onset Immunofluorescence staining for RFP and GFP in spinal cord at EAE onset showed that red MDMs exhibited elongated or spindle shape, whereas green MiDMs showed a process-bearing morphology (Fig. 1 C). Quantification in 3D reconstructions from 0.2-μm confocal z-stack images showed that MiDMs exhibited much larger size than MDMs along with multiple primary processes, which were sparse in MDMs (Fig. 1 D). Several 3D shape parameters also discriminated between MDMs 1535 GFP by F4/80+/CD45high MDMs and F4/80+/CD45dim MiDMs, respectively (Fig. 1 B). Enumeration of cells recovered from cell sorting using F4/80+RFP+ as MDMs gate and F4/ 80+GFP+ as MiDMs gate indicated that MDMs and MiDMs showed equal numbers at disease onset when explosive MDM accumulation occurred. MiDM expansion began at peak (Fig. 1 B). At recovery, MiDMs were found near preonset numbers as MDM frequency continued to decline, which is compatible with previous studies (Ajami et al., 2011). There-fore, there were unequal numbers of MiDMs/MDMs before and after disease onset (Fig. 1 B). Morphological analyses and definitions of relations between myeloid cells and axoglial units were conducted at disease onset so that equal numbers Figure 1. MDMs and MiDMs exhibit different time courses of accumulation in the CNS of mice with EAE and morphological characteristics can distinguish them. (A) Immunohistochemistry shows expression of CD11b for red RFP+ MDMs and green GFP+ MiDMs in the spinal cords of Cx3cr1gfp/+::Ccr2rfp/+ mice at EAE onset. Bars: 25 μm. We studied 6 mice at EAE onset from 3 EAE inductions. In each EAE induction, 8–10 mice were used and 2 mice were selected from each induction. (B) Flow cytometric analysis of CCR2-RFP+ and CX3CR1-GFP+ populations in cells gated for F4/80 expression (top); CD45 expression of F4/80+RFP+ MDMs and F4/80+GFP+ MiDMs populations (middle); and MDMs and MiDMs numbers at EAE onset, peak, and recovery (bottom). We studied 3 mice for naive groups; 12 for onset; 15 for peak; 13 for recovery from 5 EAE inductions. For each induction, 8–10 mice were used and 2–3 mice were selected at each time point (onset, peak, and recovery) for analysis. (C) Confocal microscopy assessment of myeloid cell morphology in lumbar spinal cord from mice at EAE onset. We studied 54 MDMs and 51 MiDMs of 5 EAE onset mice from 3 EAE inductions for (C–E); 2 sections/mouse; 4–6 cells/section; 8–12 cells/mouse. In each EAE induction, 8–10 mice were induced and 1–2 EAE onset mice were selected from each experiment. Bars, 25 μm. (D) Cell volumes of 500 μm3; surface areas of 1,000 μm2; primary process numbers ≤3 or ≥5; solidity3D of 0.4; and Formfactor3D of 0.3 discrimi-nate between MDMs and MiDMs. (E) Model plot of cell volume against primary process number to distinguish MDMs (red symbols and pink area) from MiDMs (green symbols and green area).
- 18. in MDMs andMiDMs (unpublished data). MDMs had bi-lobulated or irregular nuclei, whereas MiDMs had round nu-clei (unpublished data). MDMs, but not MiDMs, frequently contained osmiophilic granules and microvilli (unpublished data). Collectively, these ultrastructural features provided con-firmatory ultrastructural characteristics to distinguish MDMs from MiDMs. MDMs initiated demyelination at EAE onset Results from confocal and EM analysis yielded a secure basis for examining the relationships of MDMs and MiDMs to ax-oglial units at EAE onset (n = 7 mice; 14 lesions) using serial block-face scanning electron microscopy (SBF-SEM), as pre-sented diagrammatically (Table 1). We quantified contacts made by MDMs (n = 169) and MiDMs (n = 86) with axoglial units (n = 75; Fig. 2), and observed that most (55/75; 73%) of all segments (both intact and demyelinated) contacted both MDMs and MiDMs (Fig. 2). Where only one myeloid cell type was present (20/75; 27%), nearly all axoglial units made contacts to MDMs (Fig. 2). In particular, 8/29 intact and 10/46 and MiDMs (Fig. 1 D). We observed scant overlap of several values between MDMs and MiDMs (Fig. 1 D), and entirely nonoverlapping distributions for cell volume and primary processes (Fig. 1 E). MDMs and MiDMs exhibit differentiating ultrastructural characteristics at EAE onset We used confocal microscopy in 0.2-μm optical sections to correlate structural features of MDMs and MiDMs with RFP or GFP fluorescence, as a bridge to characterizing cells in 0.2 μm SBF-SEM images (Table 1). Using this approach (Fig. 1 E), MDMs and MiDMs were identified by estimating volume and counting primary processes. Volume estimations came from multiplying the midcell area by the number of sections in which the cell was identified. In electromagnetic (EM) images, quantitative analysis also demonstrated differentiating ultrastruc-tural characteristics for mitochondria, nuclei, cytoplasmic os-miophilic granules and microvilli (unpublished data). MDMs had shorter, thicker mitochondria than MiDMs (unpublished data). Total mitochondrial numbers and volumes were equal Figure 2. SBF-SEM shows MDMs initiating demyelination at EAE onset. Quantitation of MiDMs and MDMs interacting with axoglial units in SBF-SEM images of CNS at EAE onset. Intact (69%) and demyelinated (76%) segments interacted with MDMs and MiDMs. Red and pink, MDMs; green and light green, MiDMs; yellow, both MDMs and MiDMs. We studied 29 intact axon segments, 46 demyelinated axon segments, 86 MiDMs, and 169 MDMs in 14 lesions of 7 EAE onset mice from 3 EAE inductions as follows: 8–10 mice were immunized at each experiment and 2–3 onset mice were selected from each induction. 1536 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
- 19. JEM Vol. 211,No. 8 Ar t icle inside the MDMs (Fig. 3 A). Remaining myelin was undergoing vesicular breakdown (Fig. 3 A). In contrast, a nearby MiDM encompassed a large fragment of myelin debris (Fig. 3 B) and contacted the nearby MDMs (Fig. 3 B), but made minimal connection to the axoglial unit (Fig. 3 B). In our SBF-SEM data, only MDMs seemed to be implicated in active damage to myelin. These observations suggested that MDMs initiated demyelination at the onset of EAE. MDMs surrounded apposed and invaded nodes of Ranvier at EAE onset We analyzed axoglial units to examine the nature of contacts with myeloid cells. Unexpectedly, 7/75 (9%) of axoglial units demonstrated MDMs attached to nodes of Ranvier. In each case, the contact between MDMs and node appeared to be pathogenic. One representative monocyte surrounded a node of Ranvier with two microvilli interposed between myelin 1537 demyelinated axoglial units were contacted solely by MDMs. We found 2–3 MDMs attached to each of the 18/20 (90%) axoglial units where only MDMs were present (Fig. 2). More than half of all analyzed MDM and MiDM cells (n = 255 total) contained myelin debris, regardless of whether axon segments were intact or demyelinated (Fig. 2). Of the MDMs found in sole contact with axoglial units, virtually all (90%) MDMs contained myelin when found in sole contact with a demyelinated axon (Fig. 2). These findings motivated evalua-tion of relationships of MDMs to axoglial units by 3D recon-struction of SBF-SEM image stacks. MDMs frequently exhibited morphological characteristics suggesting an involvement in active demyelination. Reconstruc-tion of one representative image stack shows MDMs with large intracellular myelin inclusions tightly encircling a partially demyelinated axon (Fig. 3 A). The myelin peeled away from the axon remained in continuity with a large myelin inclusion Figure 3. SBF-SEM shows example of MDM-initiating demyelination at EAE onset. (A) Representative MDMs encircles the axoglial unit. A myelin ovoid within an intra-cellular phagolysosome shows physical conti-nuity with myelin remaining attached to an axoglial unit which is undergoing active de-myelination. In serial images, disrupted myelin shows continuity from outside to inside the MDM. (B) Rotated view from B demonstrating MDM-extensive attachment to axoglial unit and MiDM nearby with limited attachment to axon. A, axon; m, myelin; c, cytosol; n, nucleus. Red, MDM cytosol; green, MiDM cytosol; yellow: nuclei; blue, myelin and myelin debris; gray, axoplasm; red line, MDM plasma mem-brane. We studied 14 lesions from 7 EAE on-set mice from 3 EAE inductions as follows: 8–10 mice were immunized at each experi-ment and 2–3 EAE onset mice were selected from each induction. Bar, 2 μm.
- 20. address the roleof MDMs in demyelination at EAE onset, we investigated clinical characteristics in relation to node pathology and demyelination in Ccr2rfp/rfp::Cx3cr1gfp/+ mice in which MDMs were virtually absent from inflamed EAE tissues and replaced in large part by neutrophils (Saederup et al., 2010). We observed equivalent magnitude of weight loss in Ccr2rfp/+:: Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and onset stages of EAE, showing that CCR2 deficiency did not affect systemic inflammation in this model (Fig. 5 A). There was a moderate delay in disease onset (Fig. 5 B) and slight reduction in EAE onset severity (Fig. 5 A) in Ccr2rfp/rfp::Cx3cr1gfp/+ mice. SBF-SEM was used to evaluate nodal pathology, myeloid cell relations to axoglial units and demyelination at and before EAE onset. In three distinct tissues from individual Ccr2rfp/+ ::Cx3cr1gfp/+ mice with EAE preonset, we found five MDMs attached to disrupted nodes of Ranvier. In an equivalent sam-ple of EAE tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ mice, only one MDM was found in contact with a node of Ranvier, despite the presence of disrupted nodes in proximity to neutrophils. One representative MDM from Ccr2rfp/+::Cx3cr1gfp/+ tissue and axolemma near the paranode complex (Fig. 4 A). The ax-oglial unit appeared otherwise healthy and no myelin debris was found in the MDM cytosol. This observation suggested that initial MDM–axoglial contacts might occur at nodes of Ranvier. Further, we detected an intratubal (Stoll et al., 1989) MDMs with myelin debris interposed between compact my-elin and axolemma near a node of Ranvier (Fig. 4 B). Addi-tionally we identified an MDM apposed to a node of Ranvier and actively phagocytizing myelin (Fig. 4 C). At this node, paranode loops were disrupted and surrounded by MDM cy-tosol (Fig. 4 C), indicating likely involvement in damaging my-elin near the node. No MiDMs contacted nodes of Ranvier. Nodal pathology without demyelination at EAE onset in Ccr2rfp/rfp::Cx3cr1gfp/+ mice We interpreted our ultrastructural findings to indicate that MDMs recognized altered nodal structure and initiated demy-elination at EAE onset. CCR2 is essential for monocyte recruit-ment to CNS tissues during immune-mediated inflammation (Fife et al., 2000; Izikson et al., 2000; Savarin et al., 2010). To Figure 4. MDMs surrounded, apposed, and invaded nodes of Ranvier at EAE onset. (A) SBF-SEM images and 3D reconstruc-tion of SBF-SEM images of MDMs with a node of Ranvier. White and black arrow: microvil-lus. (B) SBF-SEM images and 3D construction of intratubal MDMs with demyelinated axon and node of Ranvier. (C) SBF-SEM images and 3D reconstruction of an MDM with intracel-lular myelin debris apposed to a node of Ran-vier. Red, MDM cytosol; yellow, nucleus; blue, myelin; gray, axoplasm. M, myelin; c, cytosol. red line, MDM plasma membrane. We studied 14 lesions from 7 EAE onset mice collected as follows: 8–10 mice were immunized at each induction and 2–3 EAE onset mice were selected from each immunization. Bar, 2 μm. 1538 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
- 21. Figure 5. Nodalpathology without demyelination at EAE onset in Ccr2rfp/rfp::Cx3cr1gfp/+ mice. (A) Magnitude of weight loss in Ccr2rfp/+::Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and onset stages of EAE. Clinical score in Ccr2rfp/+::Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at EAE onset stage. (B) Days at disease preonset and onset stages of EAE. We studied 28 Ccr2rfp/+::Cx3cr1gfp/+ mice and 26 Ccr2rfp/rfp::Cx3cr1gfp/+ mice for A and B. Data were collected from 12 EAE inductions in Ccr2rfp/+::Cx3cr1gfp/+ mice and 19 EAE inductions in Ccr2rfp/rfp::Cx3cr1gfp/+ mice as follows: 8–10 mice were immunized at each induction and 1–3 EAE recovery mice were selected from each immunization. **, P 0.01; ***, P 0.001. (C) SBF-SEM imaging of MDMs with nodes of Ranvier phagocytosis in Ccr2rfp/+::Cx3cr1gfp/+ mice at EAE preonset. Pink, MDM cytosol; red arrow, myelin inclusion of MDM connecting to the node of Ranvier. We studied 3 tissues from 3 Ccr2rfp/+::Cx3cr1gfp/+ EAE mice at preonset stage from 3 EAE inductions: 8–10 mice were immunized at each experiment and one EAE preonset mouse was selected from each induction. Bar, 2 μm. (D) SBF-SEM of disrupted nodes (black arrows) in preonset spinal cord tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice. Bar, 2 μm. (E) SBF-SEM JEM Vol. 211, No. 8 Ar t icle 1539 of neutrophil is with myelin phagocytosis from internode at preonset stage of Ccr2rfp/rfp::Cx3cr1gfp/+ mouse. Blue, neutrophil cytosol. For D–E, we studied three tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ EAE mice at preonset stage from 3 EAE inductions: 8–10 mice were immunized at each experiment and one EAE preonset mouse was selected from each induction. Bar: 2 μm. (F) Histochemical staining and with aurohalophosphate complexes (black gold staining) and quanti-fication of demyelinated area of Ccr2rfp/+::Cx3cr1gfp/+ mice and Ccr2rfp/+::Cx3cr1gfp/+ mice. We studied 5 naive Ccr2rfp/+::Cx3cr1gfp/+ mice, 5 naive Ccr2rfp/rfp ::Cx3cr1gfp/+ mice, 5 onset Ccr2rfp/+::Cx3cr1gfp/+ mice, and 5 onset Ccr2rfp/rfp::Cx3cr1gfp/+ mice from 3 EAE inductions as follows: 8–10 mice were immunized at each experiment and 1–2 onset mice were selected from each induction. **, P 0.01. Bar: 250 μm.
- 22. Figure 6. Inflammatorysignature in MiDMs versus MDMs in the CNS of Cx3cr1gfp/+::Ccr2rfp/+ mice with EAE. (A) Quantitative nCounter expres-sion profiling of 179 inflammation related genes was performed in CNS-derived GFP+ microglia and RFP+ recruited monocytes from naive and EAE mice at onset, peak and recovery stages. Each row of the heat map represents an individual gene and each column an individual group from pool of 5 mice at each time point. The relative abundance of transcripts is indicated by a color (red, higher; green, median; blue, low). For A–H, we studied five mice in each time point (onset, peak, recovery) from 3 EAE inductions; 8–10 mice were immunized in each induction. (B) Heat map of differentially expressed microglia 1540 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
- 23. JEM Vol. 211,No. 8 Ar t icle We noted a subset of genes that were expressed in microglia and highly regulated in MiDMs during EAE, but not ex-pressed at all in monocytes or MDMs (Fig. 6, A and B). Con-versely, a subset of MDM-enriched genes were dynamically regulated in monocytes and MDMs but not in microglia (Fig. 6, A and B). MDM-enriched genes were sharply up-regulated from naive monocytes to onset and peak-stage MDMs (Fig. 6 B), descending toward naive levels during recovery (Fig. 6 B). In contrast, MiDM-enriched genes were strongly expressed in naive cells, almost uniformly si-lenced at onset, and began a return toward naive levels at peak and recovery (Fig. 6 B). Comparing MDM-enriched genes with MiDM-enriched genes showed that MDMs were more likely to express effector functions, including secreted factors and surface molecules (18/28; 64.3% of MDM-enriched genes encoded effector functions; Fig. 6 C: and Table S1, purple genes). In contrast, only 18/48 (37.5%) of MiDM-enriched genes encoded effector functions (Fig. 6 D, Table S1, purple genes). These observations indicated that MiDMs and MDMs exhibited markedly distinct expression profiles during EAE. Differential expression of macrophage effector functions by MiDMs and MDMs Our ultrastructural analysis of myeloid cells in EAE focused on myeloid cell relationships to tissue elements. Expression profiling also addressed the cytokine and growth factor output of MiDMs and MDMs, potentially providing insight into disease pathogenesis. We used k-means clustering to discriminate five distinct patterns of MiDM gene expression during the course of EAE (Fig. 6, E and F). The red, blue and green groups in-creased in MiDMs at onset, peak, and recovery, respectively. Red group genes involved several surface molecules. Green group genes, up-regulated at onset and transiently further up-regulated at peak, were comprised mainly of complement-system elements (C3aR1; C4a, C1qa, C1qa, C3, and Cfb); mononuclear cell–specific chemokines (CCl2, 3, 4, 5, 7, and CXCL9); proliferation related genes ( fos, jun, myc, and CSF1); and acute inflammation–related genes (IL1a, IL1b, TNF, CEBP, STAT1). Cell growth–related genes expressed at this time point correlated to reported patterns of microglial pro-liferation during EAE (Ajami et al., 2011). Blue group genes up-regulated at recovery included heterogeneous cytokines (IFN-, IFN-, TGFB3, IL2, IL3, IL4, IL12, IL12, PDGFA, CSF2, and CXCL2). Both yellow group and golden group genes were strongly expressed in naive microglia, re-duced drastically at onset, and either returned to preEAE lev-els during recovery (yellow) or failed to do so (golden). These 1541 having concave nucleus (Fig. 5 C, left) had multiple intracellu-lar myelin inclusions, one of which (Fig. 5 C, left middle) was physically connected to a myelin sheath (Fig. 5 C, right mid-dle) at a paranode (Fig. 5 C, right), indicating active ongoing demyelination at a node of Ranvier. By distinct contrast, EAE onset tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice were characterized by nodal pathology often without cellular infiltrates (Fig. 5 D). In one instance, we detected a neutrophil abstracting myelin from the myelin internode (Fig. 5, left and right) despite a nearby disrupted node (Fig. 5, left) in tissues from a Ccr2rfp/rfp:: Cx3cr1gfp/+ mouse. Importantly, there was no evidence for neutrophil recognition of disrupted nodes of Ranvier. We in-terpreted these observations to suggest that MDMs specifically recognized nodal components to initiate demyelination, and that absence of MDMs at disrupted nodes of Ccr2rfp/rfp::Cx3cr1gfp/+ mice with EAE was caused by the virtual absence of infiltrat-ing monocytes (Saederup et al., 2010). To quantify the outcome of these ultrastructural differences, we monitored demyelination using histochemical staining with aurohalophosphate complexes at disease onset in Ccr2rfp/rfp:: Cx3cr1gfp/+ and Ccr2rfp/+::Cx3cr1gfp/+ mice. Demyelination was significantly reduced at EAE onset in CCR2-deficient mice (Fig. 5 F), indicating the importance of MDM recognition of disrupted nodes for efficient inflammatory demyelination. Fur-thermore, as nodal pathology was equivalent in Ccr2rfp/rfp:: Cx3cr1gfp/+ (Fig. 5 D) and Ccr2rfp/+::Cx3cr1gfp/+ mice at the preonset stage of EAE, the results suggested that inflammatory nodal disruption could be reversible if MDMs were prevented from initiating demyelination at those sites. Expression profiling demonstrates differential MiDMs and MDMs gene expression across the time course of an EAE attack We reasoned that different phenotypes (Fig. 1) and effector properties (Figs. 2–4) of MDMs and MiDMs should be re-flected in distinct gene expression profiles in the dynamic CNS microenvironment during EAE. To address this hypothesis, nCounter digital multiplexed gene expression analysis (Kulkarni, 2011) was performed using directly ex vivo naive microglia and splenic F4/80+ macrophages (here termed monocytes and considered similar to microglia by expression profiling; Gautier et al., 2012), as well as flow-sorted MiDMs or MDMs across the time course of an EAE attack. Microglia and MiDMs clus-tered together during unsupervised hierarchical clustering, as did monocytes and MDMs (Fig. 6, A and B). In both MiDMs and MDMs, naive and recovery-stage expression profiles were more alike than were onset and peak-stage profiles (Fig. 6, A and B) suggesting a return to homeostasis at EAE recovery. and monocyte genes. (C) Enriched monocyte genes as compared with resident microglia. Bars represent fold changes of gene expression across naive and all disease stages versus resident microglia. (D) Enriched microglia genes as compared with recruited monocytes. Bars represent fold changes of gene expression across naive and all disease stages versus recruited monocytes. (E–H) K-means clustering of inflammation genes in resident microglia and recruited monocytes. K-means clustering was used to generate 5 disease stage–related clusters in MiDMs. Heat map (E) and expression profile (F) of in-flammation genes in MiDMs are shown by generated clusters. MDM expression matrix overlaid on microglial based clusters shows (G) heat map and (H) expression profile.
- 24. other time pointsas well. However, a substantial minority of genes both for MDMs and MiDMs showed some dissonant time points, at which a previously down-regulated gene might show up-regulation (unpublished data). We show this subset of recovered genes in Fig. 8. In virtually every case (Fig. 8, A–D), these dissonant compensatory changes took place during recov-ery and almost always showed an increase in a gene that had been down-regulated during onset and peak. Both MiDMs (Fig. 8 B) and MDMs (Fig. 8 D) demonstrated this pattern of gene-expression kinetics. Convergent and divergent responses to upstream regulatory signaling by MiDMs and MDMs Translation of observations made using expression profiles can be enabled through identification of upstream regulators. We used Ingenuity IPA software to identify putative upstream reg-ulators of the gene expression alterations demonstrated by MiDMs and MDMs at disease onset. Putative regulatory ele-ments were then grouped in signaling modules and subjected to pathway analysis. Cell motility pathways were clearly differ-ent in MiDMs and MDMs (unpublished data). Core elements such as RhoA (Xu et al., 2009) were regulated divergently and associated signaling components were predicted to be enhanced in MDMs but depressed in MiDMs, consistent with our pheno-typic characterization using SBF-SEM. Both HIF-1 (Fig. 9 A) and TNF pathways (not depicted) were also differentially reg-ulated in MiDMs and MDMs. By contrast, type I IFN pathway (Fig. 9 B) was regulated virtually identically in MiDMs and MDMs. Collectively, these data suggest that HIF-1 and TNF signaling may partly drive pathogenic properties of MDMs. Additionally, these data indicated that the separate ontogeny of microglia and monocytes will lead, probably by epigenetic influences, to divergent responses to some but not all environ-mental stimuli, with phenotypic consequences according to the CNS microenvironment. DISCUSSION In this study, we developed a novel strategy to discriminate MDMs from MiDMs. We used SBF-SEM to address the de-tailed relationships of MiDM and MDM to axoglial units in the spinal cords of mice at EAE onset and expression profiling to examine potential mechanisms. Selection of the EAE disease model ensured that both recruited monocytes and resident mi-croglia were exposed to the same intensely inflammatory en-vironment to increase the likelihood that ambient conditions could activate these two myeloid cell types toward a convergent inflammatory phenotype. Instead, we found strikingly diver-gent relationships of MDMs and MiDMs to axoglial units, by quantitative and qualitative ultrastructural analysis. Results from expression profiling supported this interpretation by show-ing that MiDM metabolism was severely down-regulated, whereas expression profiles of MDMs reflected the activated phagocytic phenotype observed through SBF-SEM. Several salient new observations emerged from these ex-periments. First, we showed that MDMs initiate demyelination at EAE onset, as MDMs were the overwhelmingly dominant genes included a large spectrum of intracellular signaling com-ponents from the MAP-kinase pathways, as well as TGF and receptor, both of which are implicated in the naïve microglial phenotype (Butovsky et al., 2014). These five gene groups were also analyzed for MDM ex-pression patterns during EAE (Fig. 6, G and H). None of the gene groups showed coordinate regulation patterns in MDMs, as were observed in MiDMs (Fig. 6 H). This observation un-derscored disparate responses of MiDMs and MDMs to the inflammatory CNS microenvironment of EAE, despite their being present in close proximity (Fig. 1 A). Expression patterns at EAE onset in relation to MiDM and MDM function To determine whether gene expression patterns could be in-formative for understanding the relationships of cells to ax-oglial elements in tissues at EAE onset, we interrogated naive versus onset MDM and MiDM gene expression related to cellular functions (Fig. 7). MiDMs showed highly significant up-regulation of functions associated with cell movement, che-moattraction, and migration (Fig. 7 B). In the Ingenuity IPA database, the terms cell movement, chemoattraction, and mi-gration indicated production of chemokines such as CCL2, CCL3, CCL4, CCL5, and CCL7, which are up-regulated at onset and further increased at peak (Fig. 6, E and F, green group and genes). In other respects, MiDMs exhibited a re-pressed metabolic and activation phenotype by comparison to naive microglia (Fig. 7 B) including proliferation, RNA metabolism, cytoskeletal organization, microtubule dynamics, extension of processes, phagocytosis and generation of reac-tive oxygen species. MDMs showed up-regulation of functions associated to macrophages, including phagocytosis, calcium signaling, pro-duction of prostanoids, adhesion, autophagy, and cell clearance (Fig. 7 B). This pathway analysis corresponded well to effector properties displayed by MDMs in our SBF-SEM analysis (Figs. 2–4). No functions were reported to be down-regulated in MDMs at EAE onset as compared with naive monocytes. A comprehensive listing (Table S1) of all genes regulated by at least twofold in MiDMs or MDMs as compared with expression levels in naive mice affirmed and extended these interpretations. At EAE onset when SBF-SEM analyses were performed, MiDMs predominantly suppressed the distinctive gene expression pattern which correlates to their unique phe-notype (Chiu et al., 2013), reflected by the observation that MiDMs down-regulated far more genes than were up-regulated (Table S1). In contrast, MDMs up-regulated far more genes than did MiDMs and up-regulated more transcripts than were down-regulated. Additionally, the extent of gene up-regulation in MDMs exceeded that seen in MiDMs. MiDM and MDM gene expression kinetics reflected return toward homeostasis in recovery stage These expression profiles showed consonant changes for the vast majority of genes analyzed: if a gene was up-regulated at any time point, then its expression level showed an increase at 1542 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
- 25. JEM Vol. 211,No. 8 Ar t icle importance of MDMs for this mechanism of demyelination. In particular, neutrophils in inflamed CNS of Ccr2rfp/rfp::Cx3cr1gfp/+ mice did not recognize disrupted nodes. These observations are clinically pertinent: our detection of MDMs at nodes of Ranvier is consistent with recent reports of nodal pathology in clinical demyelinated tissues (Fu et al., 2011; Desmazières et al., 2012). The present observations extend this concept and provide a cellular basis for nodal pathology at the earliest stages of demyelination. Given the presence of potential phagocytic signals at nodes (antibodies to paranodal proteins such as contac-tin and neurofascins; Meinl et al., 2011); complement-derived 1543 cells found in isolation attached to axoglial units and demon-strated destructive interactions with myelinated axons in 3D reconstructions. Second, MDMs were unexpectedly observed at nodes of Ranvier in 9% of axoglial units and showed remark-ably invasive behavior, including extension of microvilli (Fig. 4 A) or localization of cell soma (Fig. 4 B) between axolemma and myelin sheath. Our observed frequency of MDM–nodal inter-action represents a minimum estimate as MDMs found at hemi-nodes adjacent to a demyelinated segment (Fig. 3 B) were not scored. Comparison of Ccr2rfp/rfp::Cx3cr1gfp/+ and Ccr2rfp/+:: Cx3cr1gfp/+ mice at and before EAE onset emphasized the Figure 7. Affected functions in MiDMs and MDMs at EAE onset. nCounter inflammatory gene expression data were uploaded to IPA. Genes with fold change (EAE onset vs. Naive) ≥1.5 or ≤1.5 were included in downstream effects analysis. (A) MDMs up-regulated functions, sorted by activation z-score. (B) MiDMs up-regulated (left) and down-regulated (right) functions, sorted by activation z score. The bias term indicates imbalanced numbers of up- and down-regulated genes associated with a distal function requiring significance at the P 0.01 level. We studied pooled samples from 5 mice in each time point (onset, peak, recovery) from 3 EAE inductions; 8–10 mice were immunized in each induction.
- 26. Figure 8. Restorationof affected inflammatory genes in resident microglia and recruited monocytes at recovery stage. For each gene, fold change of all different disease stages versus naive state were calculated. Genes that contained at least one fold change 2 or 0.5 and average fold change for peak and onset 2 or 0.5 were presented. (A) Microglial up-regulated; (B) Microglial down-regulated; (C) monocyte up-regulated; (D) monocyte down-regulated. We studied pooled samples from five mice in each time point (onset, peak, recovery) from three EAE inductions; 8–10 mice were immu-nized in each induction. FC, fold change. 1544 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
- 27. Figure 9. Functionnetworks in MiDMs and MDMs. nCounter inflammatory gene expression data were uploaded to IPA. Genes with fold change (EAE onset vs. Naive) ≥1.5 or ≤1.5 were included in upstream regulators analysis. Predicted upstream regulators were manually curated to form func-tional clusters. Clusters were uploaded to IPA using Z scores as reference value for each gene. Networks were generated for each cluster consisting of uploaded genes and additional predicted molecules. (A) Typical example of functions with dissimilar activation pattern in MiDMs and MDMs: HIF1A. (B) Function with similar activation pattern in MiDMs and MDMs: Type I IFN. Red object denotes positive (2) z score and green object denotes negative JEM Vol. 211, No. 8 Ar t icle 1545
- 28. a gradual returntoward a homeostatic expression profile, as suggested by MiDM up-regulation of fos, jun, myc, and CSF1 (Wei et al., 2010) at disease onset. By striking contrast, MDMs up-regulated a large suite of inflammation-associated genes at EAE onset, with subsequent regression to the expression phe-notype of circulating monocytes. Blood monocytes and resident microglia were exposed to the same inflammatory environment. However, their preEAE states were extremely distinct, with monocytes being gener-ated from a bone marrow progenitor within weeks of entry into CNS, whereas microglia originated during early embryo-genesis and had inhabited a serum-free unique environment from midgestation. In a recent study, we characterized resident microglia by profiling mRNA, miRNA, and protein in com-parison with infiltrated brain macrophages, nonmicroglial resi-dent brain cells, and peripheral macrophages (Butovsky et al., 2014). The detailed profiling after separating cells via CD45dim status showed distinct mRNA, miRNA, and protein expres-sion by microglia as compared with infiltrating monocytes or neuroepithelial brain cells (Butovsky et al., 2014). The study described transcription factors and miRNAs characteristic of microglia in healthy brain but not in peripheral monocytes. These findings partially explain a divergent response of these two cell types to the same stimuli (Butovsky et al., 2014). The strength of the study is that the dual reporter system is sufficient to accurately distinguish monocyte versus mi-croglial cells and thus to address the general concept that monocytes and microglia can exert differential functions in a CNS disease process. At the onset of EAE, the time point at which our imaging studies were focused, we are able to make an unequivocal distinction between resident microglia (CX3CR1gfp) and infiltrating monocytes (CCR2rfp). Two em-pirical observations underline this discrimination: microglia are uniformly CX3CR1+ from early embryonic time points through adulthood (Cardona et al., 2006; Ginhoux et al., 2010; Schulz et al., 2012), and CCR2+Ly6C+ cells constitute the vast majority of infiltrating monocytes at EAE onset (Saederup et al., 2010; Mizutani et al., 2012). There were unavoidable limitations of our research; spe-cifically, to address how monocytes and microglia respond to a shared microenvironment, we focused on a single, patho-genically relevant time point: onset of EAE. For this reason, it was beyond the scope of our study to decipher the phenotypic fate of infiltrated monocytes. In peripheral models of inflam-mation, Ly6Chi/CCR2rfp monocytes down-regulate the re-porter over time and show phenotypic evolution. Furthermore, our conclusions should not be generalized beyond the present disease paradigm: in other models, such as spinal cord contusion, the inflammatory infiltrate includes Ly6Clow/CX3CR1gfp monocytes, which are highly pathogenic (Donnelly et al., 2011). Our findings carry biological and medical significance opsonins (Nauta et al., 2004); and stress-induced eat-me sig-nals (Hochreiter-Hufford and Ravichandran, 2013), it may be feasible to identify a direct molecular pathway for initiating de-myelination in this model. Third, we characterized a molecu-lar signature for resident microglia at EAE onset. Grouping of regulated genes into functional categories demonstrated a remarkable down-regulation of microglial metabolism at the nuclear, cytoplasmic and cytoskeletal levels. In our initial experiments we found that the presence of myelin debris at the peak of EAE did not discriminate MDMs from MiDMs. We considered that SBF-SEM would exhibit advantages for spatial resolution (Denk and Horstmann, 2004) required for characterizing relationships of myeloid cells to axoglial units during the inflammatory demyelinating process at EAE onset. To take advantage of this technique we developed methods based on cell volume and process number (Fig. 1 E), to distinguish MDMs from MiDMs in 0.2-μm confocal optical sections, and translated this approach directly to SBF-SEM image sets at 0.2-μm intervals. We also noted differential nuclear morphology, mitochondrial shape, and osmiophilic granule content between MDMs and MiDMs. These characteristics of MDMs and MiDMs may not be universally present in other pathological circumstances but demonstrate an approach to ultrastructural distinction of myeloid cell populations in tissue sections. Gene expression profiling across the time course of EAE yielded intriguing kinetics as analyzed by k-means clustering. Five patterns were observed. Red group genes (increased at onset) comprised the smallest number and involved several sur-face molecules: CCR1, CCR7, CXCR2, and CD40. Of these, CCR7 and CD40 have been reported on activated microglia, including those observed in MS tissue sections (Kivisäkk et al., 2004; Serafini et al., 2006). GAPDH was up-regulated in MiDMs at onset. Although often regarded as a housekeeping gene, GAPDH is found in complexes that limit the translation of inflammatory gene transcripts in activated mouse macro-phages (Mukhopadhyay et al., 2009; Arif et al., 2012). As pre-viously reported (Chiu et al., 2013), MiDM gene expression during the course of EAE did not correspond to the M1/M2 pattern of peripheral macrophage responses to infection or tis-sue injury. Microglial morphological transformation can be relatively uniform regardless of the inflammatory process that provokes it. Despite this apparent uniformity, gene expression by morphologically identical microglia can differ drastically contingent on context (Perry et al., 2007). Unsupervised hierarchical clustering provided insight into gene expression patterns of MDMs and MiDMs. Naive and recovery patterns were similar for both cell types. At disease onset, microglia showed drastic down-regulation of the expres-sion profile observed in cells from healthy brain. Brisk mi-croglial proliferation (Ajami et al., 2011) may have accelerated (2) z-score. Orange object denotes predicted activation of the network object. Blue object denotes predicted inhibition of the network object. Predicted relationships (connecting lines): orange, leads to activation; blue, leads to inhibition; yellow, finding inconsistent with state of downstream molecule; gray, effect not predicted. 1546 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
- 29. JEM Vol. 211,No. 8 Ar t icle Histological and immunohistochemical analysis. Spinal columns were removed after mice were perfused with 4% paraformaldehyde (PFA). For im-munofluorescence assay, free floating sections of the lumbar spinal cord were prepared as previously described (Huang et al., 2006). For immunofluores-cence assay, sections were blocked with 10% normal serum for 2 h and stained with primary antibodies at 4°C for 24–48 h. After washing with PBS-T (PBS with 0.1% Triton X-100; Sigma-Aldrich) three times, the sections were incu-bated with secondary antibodies at room temperature for 2 h and mounted in ProLong Gold antifade reagent (Invitrogen). Antibodies used include rat anti-CD11b (BD), mouse anti-GFP (Abcam), rabbit anti-RFP (Abcam), Alexa Fluor 488 goat anti–mouse IgG (Invitrogen), Alexa Fluor 594 goat anti–rabbit IgG (Invitrogen), and Alexa Fluor 647 goat anti–rat IgG (Invitrogen). Nuclei were labeled by DAPI. Images were collected by confocal laser-scanning mi-croscope 1547 (SP5; Leica). Quantitative 3D morphology. Quantitative 3D morphology of MDMs and MiDMs was analyzed in confocal images from spinal cord of mice at EAE onset. Free floating sections of the lumbar spinal cord were stained with RFP for MDMs, GFP for MiDMs, and DAPI for nuclei. Stack images were taken at 0.2-μm step size along the z-direction with a 63× objective (numer-ical aperture [NA] = 1.4) and zoom factor 2. A square (1,024 × 1,024 pixels) corresponding to 123 × 123 μm2 was used for the analysis. Cells were 3D re-constructed by ImageJ software and all analyses were performed using ImageJ with 3D Convex Hull plugin. The parameters analyzed include voxel (volu-metric pixel), convex voxel, volume, convex volume, surface, and convex sur-face area. Other calculated parameters were: Solidity3D = volume/convex volume; Convexity3D = convex surface area/surface area; Formfactor3D = 36 3 π × volume2 surface area3. The number of primary processes was esti-mated visually. We included 5 mice, 54 MDMs; 51 MiDMs in this assay with 2 sections/mouse, 4–6 cells/section and 8–12 cells/mouse. Those mice came from three EAE inductions. SBF-SEM. Spinal cords were removed after mice were perfusion-fixed using 4% PFA with 1% glutaraldehyde. Lumbar spinal cord sections were made on a vibratome (Leica). Sections were stained with 0.4% OsO4, uranyl acetate and lead aspartate, then embedded in epon resin (Electronic Micros-copy Sciences). SBF-SEM images were acquired using a Sigma VP SEM (Carl Zeiss) with 3View (Gatan). Serial image stacks of images at 100-nm steps were obtained by sectioning 48 × 48 × 20 μm3 tissue blocks (length × width × depth) at a resolution of 8192 × 8192 pixels. Image stacks were pro-cessed for 3D reconstruction by TrakEM2 in FIJI software (National Institutes of Health). Alternating sections from the same stacked images were chosen to make stacks for 3D reconstructions which matched the 0.2-μm step size used for acquiring confocal stacked images. In SBF-SEM images, we discriminated MDMs and MiDMs using the volume/primary processes model (Fig. 1 E) generated from analyzing confocal images. Quantifications of myeloid-cell spatial relationships to axoglial units, including myelin incorporation, were done in SBF-SEM images. Quantification of nuclei and mitochondria. Characterizations of nu-clear shapes were conducted in SBF-SEM images. Nuclei were categorized as follows: round, round shape and smooth surface with ratio of length/ width ≤1.5; elongated, elongated or oval shape with length/width 1.5, and may have small indentations; Bilobulated: two connected lobes with single intervening large indentation; Irregular: complicated shape with corrugated surface, and may have multiple and variable sizable indentations. Blinded ob-servers (n = 3) scoring the nuclear morphology from SBF-SEM images in-cluded a research student, a research fellow and a neuroscientist. Observers were trained on the same nuclear examples in each category and practiced using 20 nuclei comprising all shapes before scoring the nuclei. Kappa test showed good pairwise agreement rates among observers (0.8) and the data from the neuroscientist are used. Quantifications were done in 3 individual mice from 3 EAE inductions including 28–35 cells from two separate lesions from each mouse in the assay. by demonstrating and characterizing differential responses of infiltrating monocytes and resident microglia in a relevant dis-ease model at a prespecified time point, at which point patho-genic events are taking place. Therefore, we focused our analysis on the day of EAE onset rather than subsequent events to challenge our overall hypothesis that infiltrating monocytes versus resident microglia respond very differently to acute in-flammatory stimuli. Activated myeloid cells are the proximate effectors of a bewildering array of acute and chronic disorders (Wynn et al., 2013). The technical and conceptual approach taken in this study may be applicable to other tissues and disease processes. In many pathological conditions, tissues harbor a mixed pop-ulation of activated resident and recruited monocytes. The therapeutic strategy will differ conclusively based on the spe-cific effector properties of each cell type and the stage of dis-ease. In particular, if monocytes are pathogenic, then their trafficking should be blocked using a peripherally active agent. The optimal application of agents that regulate leukocyte mi-gration and intracellular signaling will be promoted by de-tailed examination of each individual myeloid population. MATERIALS AND METHODS Mice. C57BL/6 mice were obtained from the National Cancer Institute. Ccr2rfp/+::Cx3cr1gfp/+ mice were generated by crossbreeding Ccr2rfp/rfp::C57BL/6 mice (Saederup et al., 2010) with Cx3cr1gfp/gfp::C57BL/6 mice ( Jung et al., 2000). Ccr2rfp/rfp::Cx3cr1gfp/gfp mice were generated by breeding Ccr2rfp/+ ::Cx3cr1gfp/+ mice. Ccr2rfp/rfp::Cx3cr1gfp/+ mice were generated by crossbreed-ing Ccr2rfp/rfp::C57BL/6 mice with Ccr2rfp/rfp::Cx3cr1gfp/gfp mice. Animal ex-periments were performed according to the protocols approved by the Institutional Animal Care and Use Committee at the Cleveland Clinic fol-lowing the National Institutes of Health guidelines for animal care. EAE induction and clinical evaluation. EAE was induced in Ccr2rfp/+ ::Cx3cr1gfp/+ mice and Ccr2rfp/rfp::Cx3cr1gfp/+ mice of 24–28 wk of age using myelin-oligodendrocyte-glycoprotein peptide 35–55 (MOG) as previously described (Huang et al., 2006). All mice were weighed and graded daily for clinical stages as previously reported (Saederup et al., 2010). We defined clinical stage of EAE as follows: pre-onset was the day sudden weight loss for 8–10% occurred; onset was the day EAE signs appeared; peak was the second day score didn’t increase after sustained daily worsening; and recovery was the second day score didn’t decrease after a period of sustained daily improvement. To address our research questions, we integrated flow cytometry, immunohistochemistry with quantitative morphometry, cell sorting for expression profiling, and serial block-face scanning electronic microscopy. In all, we per-formed 12 EAE immunizations in Ccr2rfp/+::Cx3cr1gfp/+ mice and 19 immu-nizations in Ccr2rfp/rfp::Cx3cr1gfp/+ mice for this project, with 8–10 mice in each immunization. We selected EAE mice at onset, peak or recovery de-pending on the specific studies underway at that time, with the majority of mice coming from the onset stage of EAE. Each experiment incorporated samples from at least three separate immunizations. Details of mouse numbers and how they were selected for each experiment were included in the figure legends as requested. Cell isolation and flow cytometry. Brains and spinal cords were removed and homogenized. Mononuclear cells were separated with a 30%/70% Per-coll (GE Healthcare) gradient as previously reported (Pino and Cardona, 2011). Single-cell suspensions from CNS were stained with anti–F4/80-APC (BM8; eBioscience) and anti–CD45-PerCP (30-F11; BioLegend). Cells were either analyzed on a LSR-II (BD) or sorted on a FACSAria II (BD) running Diva6. Data were analyzed with FlowJo software (Tree Star).
- 30. nonrandom association. Thep value of overlap is calculated by the Fisher’s Exact Test. The activation z-score is a value calculated by the IPA z-score algorithm. The z-score predicts the direction of change for a function or the activation state of the upstream regulator using the uploaded gene expression pattern (upstream to the function and downstream to an upstream regulator). An ab-solute z-score of ≥2 is considered significant. A function is increased/upstream regulator is activated if the z-score is ≥2. A function is decreased/upstream regulator is inhibited if the z-score ≤-2. The bias term is the product of the dataset bias and the bias of target molecules involved in a particular function annotation or upstream regulator activity. A biased dataset is one where there is more up- than down-regulated genes or vice versa. The dataset bias is constant for any given analysis and the function/upstream regulator bias is unique for each upstream regulator/function. When the absolute value of this term is 0.25 or higher, then that function/up-stream regulator’s prediction is considered to be biased and the Fisher’s exact p-value must be 0.01 or lower for the analysis to be considered significant. We thank Dr. Bruce D. Trapp for invaluable suggestions. We thank Flow core in Cleveland Clinic Foundation for the flow cytometry experiments. We thank Aishwarya Yenepalli for help with quantification. This research was supported by grants from the US National Institutes of Health, the Charles A. Dana Foundation, the National Multiple Sclerosis Society, and the Williams Family Fund for MS Research, as well as a Postdoctoral Fellowship from National Multiple Sclerosis Society (to N. Ohno). The authors have no competing financial interests. Submitted: 28 November 2013 Accepted: 9 June 2014 REFERENCES Ajami, B., J.L. Bennett, C. Krieger, W. Tetzlaff, and F.M. Rossi. 2007. 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Uploaded dataset for analysis were filtered using cutoff definition of 1.5-fold change. Level of con-fidence for analysis was set to high-predicted and experimentally observed. Terms used in IPA analyses. The p-value is a measure of the likelihood that the association between a set of genes in the uploaded dataset and a re-lated function or upstream regulator is due to random association. The smaller the p-value, the less likely it is that the association is random and the more significant the association. In general, P 0.05 indicate a statistically significant, 1548 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
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- 32. INSIGHTS B-RAF unlocksaxon regeneration The mechanisms that drive axon regeneration after central nervous system (CNS) injury or disease are proposed to recapitulate, at least in part, the developmental axon growth pathways. This hypothesis is bolstered by a new study by O’Donovan et al. showing that activation of a B-RAF kinase signaling pathway is sufficient to promote robust axon growth not only during development but also after injury. B-RAF was previously shown to be essential for developmental axon growth but it was not known if additional signaling pathways are required. In this study, the authors demon-strate that activation of B-RAF alone is sufficient to promote sensory axon growth during development. Using a conditional B-RAF gain-of-function mouse model, the authors Insight from Valeria Cavalli (left) and David Holtzman elegantly prove that B-RAF has a cell-autonomous role in the developmental axon growth program. Notably, activated B-RAF promoted overgrowth of embryonic sensory axons projecting centrally in the spinal cord, suggesting that this pathway may normally be quiescent in central axons. Could activated B-RAF also enhance axon regeneration in the adult central nervous system? The authors found that activated B-RAF not only enabled sensory axon growth into the spinal cord after spinal injury, but also promoted regrowth of axons projecting in the optic nerve. Regeneration in the injured CNS is prevented by both the poor intrinsic regrowth capacity of axons and by inhibitory factors in the tissue environment. Importantly, the B-RAF–activated signaling growth program was insensitive to this repulsive environment. Interestingly, the authors find that B-RAF synergizes with the PI3- kinase–mTOR pathway, which also functions downstream of growth factors. This opens the possibility that combinatorial approaches that integrate these two pathways may heighten regenerative capacity. This in vivo study significantly advances the understanding of the role Activation of B-RAF signaling enables crushed sensory axons (green) to grow into the adult spinal cord in both white (white arrows) and gray (pink arrows) matter. of MAP kinases in axon growth and suggests that reactivation of the B-RAF pathway may be exploited to promote axon regeneration in the injured central nervous system. An exciting future avenue will be to determine the downstream mechanisms controlled by B-RAF. O’Donovan, K.J., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20131780. Valeria Cavalli and David M. Holtzman, Washington University School of Medicine in Saint Louis: cavalli@pcg.wustl.edu and holtzman@neuro.wustl.edu
- 33. Ar t icle B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS Kevin J. O’Donovan,1,2 Kaijie Ma,1,2 Hengchang Guo,1 Chen Wang,3,4 Fang Sun,3,4 Seung Baek Han,5,6 Hyukmin Kim,5,6 Jamie K. Wong,7 Jean Charron,9 Hongyan Zou,7,8 Young-Jin Son,5,6 Zhigang He,3,4 and Jian Zhong1,2 1Burke Medical Research Institute, Weill Cornell Medical College of Cornell University, White Plains, NY 10605 2Brain and Mind Research Institute, Weill Cornell Medical College of Cornell University, New York, NY 10065 3F.M. Kirby Neurobiology Center, Boston Children’s Hospital; and 4Department of Neurology; Harvard Medical School, Boston, MA 02115 5Shriners Hospitals Pediatric Research Center and 6Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA 19140 7Fishberg Department of Neuroscience and 8Department of Neurosurgery, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029 9Centre de Recherche en Cancérologie de l’Université Laval, Centre Hospitalier Universitaire de Québec, Québec, Québec G1R 2J6, Canada Activation of intrinsic growth programs that promote developmental axon growth may also facilitate axon regeneration in injured adult neurons. Here, we demonstrate that condi-tional the growth of long-range peripheral sensory axon projections in vivo in the absence of upstream neurotrophin signaling. We further show that activated B-RAF signaling enables robust regenerative growth of sensory axons into the spinal cord after a dorsal root crush as well as substantial axon regrowth in the crush-lesioned optic nerve. Finally, the combi-nation extension beyond what would be predicted for a simple additive effect. We conclude that cell-intrinsic RAF signaling is a crucial pathway promoting developmental and regenerative axon growth in the peripheral and central nervous systems. The Rockefeller University Press $30.00 J. Exp. Med. 2014 Vol. 211 No. 5 801-814 www.jem.org/cgi/doi/10.1084/jem.20131780 activation of B-RAF kinase alone in mouse embryonic neurons is sufficient to drive 801 of B-RAF gain-of-function and PTEN loss-of-function promotes optic nerve axon Axon growth is essential for the establishment of a functional nervous system as well as for the restoration of neuronal connectivity after injury or disease. It has long been hypothesized that re-activation of developmental growth mechanisms might help to achieve axon regeneration in the injured adult nervous system (Filbin, 2006). The role of MAP kinases in axon growth signaling has been much studied and discussed (Markus et al., 2002; Hanz and Fainzilber, 2006; Agthong et al., 2009; Hollis et al., 2009). However, depend-ing on the model systems used, the outcomes have been controversial or even contradictory (Pernet et al., 2005; Sapieha et al., 2006; Hollis et al., 2009). We have shown that RAF–MEK signaling robustly promotes axon growth in pri-mary sensory neurons in vitro (Markus et al., 2002). In vivo, conditional gene targeting studies have shown that RAF signaling is necessary for developing sensory neurons to arborize in their target fields in the skin (Zhong et al., 2007). However, it remains unknown whether RAF sig-naling is sufficient to enable axon growth in vivo or whether concomitant activation of other signaling pathways is necessary to drive long-range axon projections. Furthermore, it is un-clear whether this pathway can promote axon growth in neuronal populations beyond the sen-sory neurons and the extent to which it can be harnessed to promote regeneration in the in-jured central nervous system (CNS). To address these questions, we have used conditional B-RAF gain-of-function mouse models to show that CORRESPONDENCE Jian Zhong: jiz2010@med.cornell.edu Abbreviations used: CGRP, calcitonin gene-related peptide; CNS, central nervous system; DREZ, dorsal root entry zone; DRG, dorsal root ganglion; kaB-RAF, kinase-activated B-RAF; NGF, nerve growth factor; RGC, retinal ganglion cell; SCI, spinal cord injury. K.J. O’Donovan and K. Ma contributed equally to this paper. © 2014 O’Donovan et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ by-nc-sa/3.0/).
- 34. Figure 1. Conditionalexpression of kaB-RAF specifically activates MAP kinase signaling in the nervous system. (A) E18.5 mouse embryos expressing kaB-RAF develop grossly normally, except for substantial hy-drocephalus. (B) DRG size is unaffected by kaB-RAF expression. (C) Representative West-ern blots show increased levels of pMEK1/2 and pERK1/2 in the E12.5 neocortex and spi-nal cord. (D) Quantitation of Western blots as in C. Amounts are normalized to correspond-ing III-tubulin levels. n = 4 repeats with three animals per group for each bar. Stu-dent’s t test: *, P 0.05; **, P 0.01. (E–G) Analysis of the interaction of kaB-RAF with other signaling pathways in the E12.5 nervous system. Representative Western blots are from three independent experiments, each with two or three E12.5 embryos for each genotype. (G) B-RAF protein levels are not increased in E12.5 DRG and spinal cord of LSL-kaBraf:nesCre embryos and control littermates. Molecular mass is indicated in kilodaltons. (H) Representative images of nerve endings in trunk skin of E13.5 embryos. Bars: (B) 200 μm; (H) 40 μm. (I) The number of branches per nerve trunk was quantitated, blinded to the genotypes, for a defined skin section by counting the total number of branch points observed along each major nerve trunk entering the skin (Zhong et al., 2007). n = 3 for each group. (D and I) Error bars indicate SEM. peripheral axons into the epidermis. To examine whether RAF signaling alone is sufficient to promote long-range axon extension of TrkA-positive neurons, we set out to selectively activate RAF kinase signaling in these neurons in a TrkA-null background. To this end, we first conditionally activated RAF signaling in a WT background using a genetically modified loxP-STOP-loxP- BrafV600E (LSL-kaBraf) knock-in mouse line (Mercer et al., 2005), in which a kinase-activated B-RAF (kaB-RAF) mutant is expressed from the endogenous B-RAF locus upon Cre recombination. We next bred LSL-kaBraf mice with a neuronal activation of intraneuronal RAF–MEK signaling is sufficient to promote robust axon growth in developing and regenerating neurons in the peripheral nervous system and CNS. RESULTS Activation of B-RAF signaling alone is sufficient to promote sensory axon extension during early development In vivo, the neurotrophin nerve growth factor (NGF) signals through its receptor kinase TrkA to promote developmental extension of dorsal root ganglion (DRG) nociceptive TrkA+ 802 B-RAF drives axon growth and regeneration | O’Donovan et al.
- 35. Figure 2. Expressionof kaB-RAF substantially rescues sensory afferent growth in the absence of TrkA/NGF signaling. (A, left) Normal sensory cutaneous innervation at E16.5. (middle) Sensory cutaneous innervation is lost in embryos lacking the NGF receptor TrkA. (right) Expression of kaB-RAF restores cutaneous innervation. Arrowheads label the blue -gal–positive (presumptive TrkA+) sensory trajectories. (B) Visualization of axon growth pat-terns after tissue clearing. The thoracic somatosensory innervation driven by kaB-RAF in a TrkA/ embryo (bottom; compare with middle for TrkA/ alone) is similar to that seen in a control TrkAWT/ littermate (top). White arrowheads indicate the normal pathways of peripheral axons extending from thoracic DRGs. Red arrowheads indicate sensory projections rescued by kaB-RAF in the TrkA/ background. (C) Expression of kaB-RAF substantially res-cues trigeminal TrkA+ afferent growth in the absence of TrkA/NGF signaling. Presumptive TrkA+ trigeminal axon projections (top) are lost in TrkA-deficient mice (middle) and are rescued by kaB-RAF (bottom). Ga, great auricular nerve; Go, greater occipital nerve; Mn, mandibular branch; Mx, maxillary branch; Op, ophthalmical branch. Images show littermates and are representative of three embryos per genotype. Bars: (A) 2 mm; (B and C) 1 mm. JEM Vol. 211, No. 5 Ar t icle (Fig. 1, C and D). Note that compared with MEK1/2, ERK1/2 activation appears minor in the kaB-RAF–expressing DRGs; this is because of relatively high levels of pERK1/2 in the DRG at baseline. B-RAF activation did not affect mTOR phosphory-lation (Fig. 1 E). Levels of pAKT, pS6K, and pGSK3 were not changed significantly in the DRG of LSL-kaBraf:nes-Cre mice (Fig. 1 F), indicating minimal cross talk between the MAP kinase and PI3-kinase–AKT pathways. Because the expression of kaB-RAF is under the control of endogenous Braf pro-moter, the expression level of B-RAF protein is not changed in 803 nestin promoter-driven Cre deleter (nes-Cre; Tronche et al., 1999). In DRG neurons, nes-Cre–mediated recombination oc-curs as early as embryonic day (E) 11.5 (Galabova-Kovacs et al., 2008). Embryos heterozygously expressing kaB-RAF progres-sively developed macrocephaly from E13.5 onwards (Fig. 1 A) but appeared otherwise normal, including normally sized DRGs (Fig. 1 B). The known RAF effectors were activated in neuronal tissues expressing Cre recombinase, as indicated by elevated phospho-MEK1/2 (pMEK1/2) and pERK1/2 in the neocortex and spinal cord of E12.5 LSL-kaBraf:nes-Cre mice
- 36. Figure 3. Axonterminal innervation of E18.5 footpad. (A, top) Normal innervation. (middle) In the absence of TrkA, innervation is diminished overall, and the CGRP-positive nociceptor endings are completely absent. Red arrowheads indicate the CGRP-positive axon terminals in the epidermis. (bottom) kaB-RAF expression partially rescues nocicep-tive innervation in the TrkA/ background. The dashed lines indicate the dermal–epidermal border. Bar, 100 μm. (B) Quantification of axon innervation in footpad (Luo et al., 2007; Hancock et al., 2011). Data are from three fetuses per genotype. Error bars indicate SEM. One-way ANOVA with post-hoc Tukey’s HSD test: **, P 0.01. (which include LSL-kaBraf:TrkA/:Bax/, nes-Cre:TrkA/: Bax/, and TrkA/:Bax/ genotypes), in which we detected no LacZ-positive fibers in the skin at E16.5, expression of kaB-RAF in TrkA/:Bax/ DRG and trigeminal neurons sub-stantially restored cutaneous sensory axon projections (Fig. 2, A–C). The morphologies of the radial thoracic trajectories de-rived from spinal DRGs as well as those growing from tri-geminal ganglia induced by kaB-RAF in the TrkA/:Bax/ background were grossly similar to those seen in control mice. Epidermal innervation of the footpad was partially rescued by kaB-RAF (Fig. 3, A and B). Thus, sustained B-RAF kinase activity can, to a large extent, substitute for TrkA-mediated axon growth signaling in presumptive TrkA+ sensory neurons. B-RAF–mediated axon growth indirectly rescues calcitonin gene-related peptide (CGRP) expression in TrkA/:Bax/ neurons CGRP expression in the peptidergic subset of nociceptive DRG neurons is induced by skin-derived factors and there-fore indicates that sensory axon peripheral innervation into the skin is complete (Hall et al., 1997, 2001; Patel et al., 2000; Xu and Hall, 2007). In TrkA/:Bax/ mice, DRG neu-rons and their centrally projecting axons are devoid of CGRP because of the lack of cutaneous innervation as previously de-scribed (Patel et al., 2000), whereas CGRP expression in the spinal motor neurons remains unaffected (Fig. 4 A, middle). the DRG and spinal cord at E12.5 (Fig. 1 G). At E13.5, the branching pattern of sensory nerves in the skin was not changed by kaB-RAF expression (Fig. 1, H and I). B-RAF activation rescues nociceptor axon extension in embryos lacking TrkA To test whether kaB-RAF is sufficient to drive nociceptor axon growth in the absence of TrkA signaling, we next mated the LSL-kaBraf:nes-Cre line with available TrkAtaulacZ and Bax/ lines to generate LSL-kaBraf:TrkAtaulacZ/taulacZ:Bax/: nes-Cre mice. In TrkAtaulacZ mice, the WT TrkA gene is re-placed by a taulacZ expression cassette, such that the axonal morphology of putative TrkA+ neurons can be visualized by -gal staining (Moqrich et al., 2004). Because TrkA expres-sion is absent in homozygous TrkAtaulacZ/taulacZ mice, we refer to the “TrkAtaulacZ/taulacZ” as “TrkA/” in the text below. Re-moval of the Bax gene blocks apoptosis in embryonic DRG neurons, rescuing them from cell death that is otherwise ob-served in the absence of TrkA signaling. The Bax/ back-ground thus allows for the molecular dissection of signaling pathways that specifically affect axon growth (Knudson et al., 1995; Lentz et al., 1999; Patel et al., 2000; Markus et al., 2002; Kuruvilla et al., 2004; Moqrich et al., 2004). In TrkA/: Bax/ mice, DRG neurons survive, but sensory afferent in-nervation in the skin is completely abolished (Fig. 2, A and B, middle; Patel et al., 2000). Compared with control littermates 804 B-RAF drives axon growth and regeneration | O’Donovan et al.
- 37. JEM Vol. 211,No. 5 Ar t icle Specifically, nociceptive TrkA+ fibers terminate in the superfi-cial laminae I and II of the dorsal horn, and proprioceptive parv-albumin- positive afferents project to intermediate laminae or to 805 the ventral spinal cord. In B-RAF gain-of-function mice, we observed excessive growth of both nociceptive and proprioceptive afferents (Fig. 5). Nociceptive axons normally restricted to superficial dorsal horn extended ectopically into deeper layers of dorsal spinal cord, and many axons aberrantly crossed the midline (Fig. 5 A). This kaB-RAF–driven overgrowth was substantially rescued by concomitant elimination of MEK1/2, the canonical down-stream kinases of RAF (Fig. 5 C), suggesting that the effect of kaB-RAF expressed from the endogenous Braf locus depends strictly on canonical signaling. In WT mice, the central proprioceptive afferents enter the cord medially at tightly circumscribed dorsal root entry zones (DREZs; Fig. 5 B, left). kaB-RAF expression caused the pro-prioceptive sensory axons to enter the spinal cord all across its surface and to aberrantly terminate some branches in the superficial dorsal laminae (Fig. 5 B, right). Proprioceptive axons in the DREZs normally are subject to repulsive guid-ance from Semaphorin 6C/D (Sema6) expressed in the spi-nal cord, acting on PlexinA1 on the sensory axons (Yoshida et al., 2006). kaB-RAF expression did not detectably alter the protein (Fig. 5 D) or transcript levels (RNAseq; not depicted) B-RAF gain-of-function restored CGRP expression in the absence of TrkA (Fig. 4 A, right). Note also that in contrast to the projections in the periphery, the growth of presumptive TrkA-positive afferent projections in the dorsal horn is inde-pendent of TrkA signaling (Fig. 4 B, middle; Patel et al., 2000; Harrison et al., 2004) and that kaB-RAF expression caused overgrowth of these afferents (Fig. 4 B, right) but not of pe-ripheral projections (Fig. 1). In vitro, kaB-RAF alone did not induce CGRP expression in DRG neurons (Fig. 4 C, top); this required the addition of NGF and skin-conditioned me-dium as previously reported (Fig. 4 C, bottom; Hall et al., 1997; Patel et al., 2000; Xu and Hall, 2007). In addition, we have shown previously that loss of both B- and C-RAF in DRGs does not abrogate the CGRP expression (Zhong et al., 2007). These data together indicate that the restoration of CGRP expression in LSL-kaBraf:nes-Cre:TrkA/:Bax/ DRG neu-rons is not directly caused by the elevation of neuron-intrinsic B-RAF activity, but indirectly through the restoration of cu-taneous innervation and subsequent retrograde signaling from skin-derived factors. kaB-RAF causes overgrowth of nociceptive and proprioceptive afferent fibers in the spinal cord In WT mice, different subpopulations of sensory neurons project from the DRG to highly specific targets in the spinal cord. Figure 4. Activation of B-RAF indirectly rescues CGRP expression in TrkA/ noci-ceptive neurons. (A, left) Normal CGRP stain-ing in the DRG and superficial dorsal horn. Arrowhead indicates CGRP-expressing spinal motoneurons. (middle) CGRP expression is completely abolished in the DRG and its projections in TrkA/Bax double-null mice. CGRP staining in spinal motoneurons is not affected by loss of TrkA signaling (arrowhead). (right) CGRP expression in DRG is rescued by expression of kaB-RAF, in the absence of TrkA signaling (LSL-kaBraf:nes-Cre:TrkA/:Bax/). Arrowhead indicates the CGRP+ motor neu-rons. Dashed white lines outline the spinal cord and DRG. (B) The nociceptive projection into the dorsal horn (left) does not depend on TrkA (middle). Expression of kaB-RAF causes over-growth and ectopic targeting of these fibers (right). (A and B) Images are representative of three embryos each. (C) Activation of B-RAF does not directly induce CGRP expression in cultured DRG neurons. (top) No CGRP is ex-pressed in 7-d in vitro cultures of dissociated E12.5 LSL-kaBraf:Bax/:nes-Cre DRG neurons. (bottom) NGF and conditioned medium from skin cultures are necessary to induce CGRP expression in E12.5 LSL-kaBraf:Bax/: nes-Cre DRG neurons. Images are representa-tive of three independent experiments. This experiment has been repeated three times. Each experiment used two embryos per geno-type. Bars: (A and B) 100 μm; (C) 20 μm.
- 38. Figure 5. Activationof B-RAF drives overgrowth of centrally projecting nociceptive and proprioceptive DRG axons in the E18.5 spinal cord. (A) Nociceptive projections stained for TrkA. Yellow arrowheads indicate the different patterns of axon projections of WT and kaB-RAF–express-ing, nociceptive neurons. (B) Proprioceptive projections stained for parvalbumin. White arrowheads indicate the different patterns of axon projections of WT and kaB-RAF–expressing, proprioceptive neurons. Asterisks label the presumptive DREZs. (C) kaB-RAF–driven overgrowth of central nociceptive projections (left) is abolished in the absence of the downstream effectors MEK1/MEK2 (right). (A–C) n = 3 per genotype. (D) kaB-RAF does not affect the expression of known guidance cues PlexinA1 and Sema6D in the E12.5 DRG and spinal cord. Western blot is representative of three indepen-dent experiments, each with two embryos per genotype. Molecular mass is indicated in kilodaltons. (E–J) Cross sections of P0 spinal cord at cervical (E and H), thoracic (F and I), and lumbar (G and J) levels were stained for CGRP, parvalbumin (Parv), and Draq5 (blue). Bars: (A and B) 200 μm; (C) 100 μm; (E–J) 50 μm. phenotype suggested that reactivation of the B-RAF path-way in injured adult neurons might be exploited to pro-mote regeneration. of these factors in E12.5 DRG and spinal cord. The overgrowth phenotype for both nociceptive and proprioceptive afferents was observed at all levels of the spinal cord (Fig. 5, E–J). This 806 B-RAF drives axon growth and regeneration | O’Donovan et al.
- 39. Figure 6. Activationof B-RAF signaling in mature DRG neurons elevates their growth competency. (A, top) Schematic of the brn3a-CreERT2 construct used to generate the brn3a-CreERT2 deleter mouse line. (bottom) A cross section of the spinal cord of a 10-wk-old Rosa26-lacZ:brn3a- CreERT2 mouse treated with tamoxifen. Blue LacZ staining indicates CreERT2-medicated recombination in the DRG neurons. (B) Representative DRGs from adult LSL-kaBraf:TdTom:brn3a-CreERT2 mice without (top left) and with (bottom left) tamoxifen treatment. TdTom expression indicates recombination JEM Vol. 211, No. 5 Ar t icle 807 in DRG neurons. Cells were counterstained with Draq5 (Dq5) to label nuclei. (C) ATF3 is induced by preconditioning lesion. Blue shows nu-clear stain Draq5. (D) Representative images of adult DRG neurons derived from intact brn3a-CreERT2:TdTom (left), LSL-kaBraf:brn3aCreERT2:TdTom
- 40. Figure 7. Activationof B-RAF signaling enables crushed sensory axons to regenerate into adult spinal cord. (A–D) Confocal views of regenerating dorsal root axons in whole mounts (A and B) or transverse sections (C and D) 2 wk after root crush. Axons were labeled by AAV-GFP injected into C6 and C7 DRGs at the time of the crush injury. (A and C) Con-trol mice (brn3a-CreERT2). (B and D) Mice expressing activated B-RAF in DRGs (LSL-kaBraf:brn3a-CreERT2). Dashed yellow lines indicate the DREZ, dashed gray lines indicate the border between gray and white matter, and arrowheads indicate the extent of axon growth across the DREZ (B) and into gray matter (D). DH, dorsal horn; DR, dorsal root; PNS, peripheral ner-vous system; SC, spinal cord. n = 2 DRGs from each of three animals per genotype. Bars, 200 μm. kaB-RAF enables regeneration of injured adult DRG central axons across the DREZ To test whether activation of B-RAF signaling can drive ma-ture sensory axon regeneration, we generated LSL-kaBraf: TdTomato (TdTom):brn3a-CreERT2 mice to inducibly express kaB-RAF in adult DRG neurons. The brn3a-CreERT2 deleter mouse line was generated using a brn3a promoter (Eng et al., 2001), which mediates expression selectively in sensory neu-rons (Fig. 6 A). We first assessed B-RAF gain-of-function in cultured adult neurons. 12-wk-old mice were treated with tamoxifen for a consecutive 5 d to induce kaB-RAF expres-sion, as indicated by TdTom expression (Fig. 6 B). ATF3, a marker whose expression is triggered by conditioning lesion (Smith and Skene, 1997; Seijffers et al., 2007) was not induced (Fig. 6 C). DRG neurons were cultured for up to 24 h. kaB-RAF expression correlated with both greatly increased num-bers of axon-bearing neurons and increased total axon length with more branching compared with that of WT neurons (Fig. 6, D–G). Furthermore, the axonal morphology of kaB-RAF– expressing neurons differs from WT neurons subject to a preconditioning lesion, which exhibited single long axons (Fig. 6, D and H). Having thus ascertained the functionality of the LSL-kaBraf: TdTom:brn3a-CreERT2 mouse line, we next tested whether kaB-RAF can enable axon regeneration after dorsal root crush injury in vivo. 12-wk-old mice were again treated with tamoxifen for a consecutive 5 d. After 2-d rest, C5–8 cervical roots were crushed and AAV2-GFP was injected to C6 and C7 DRGs to label regenerating sensory axons. After 2 wk, regeneration in the C6 and C7 roots and spinal cord was examined in whole-mount preparations (Fig. 7, A and B) or in transverse sections (Fig. 7, C and D). As expected, in control brn3a-CreERT2 mice (Fig. 7, A and C), axons regener-ated along the roots but stopped at the DREZ. In contrast, in mice with kaB-RAF expression in DRG neurons, numerous axons penetrated the DREZ and grew deeply into the spinal cord, exhibiting dense collateral branches in the dorsal col-umn (Fig. 7 B) and reaching superficial laminae of the dorsal horn (Fig. 7 D). Thus, elevation of intrinsic B-RAF signaling is sufficient, both in vitro and in vivo, to induce robust axon regrowth of adult DRG neurons and, importantly, renders the axons capable of overcoming growth-inhibitory signals that are abundant at the DREZ and within the spinal cord. kaB-RAF enables regenerative axon growth in the injured optic nerve through an MEK-dependent pathway To test whether activation of B-RAF kinase signaling can pro-mote axon regeneration of injured mature CNS neurons, we used an optic nerve regeneration model (Fig. 8, A and B; Park et al., 2008; Benowitz and Yin, 2010). 8–12-wk-old LSL-kaBraf: Bax/ mice and Bax/ controls were injected intravitreally with AAV2-Cre to induce kaB-RAF expression in retinal gan-glion cells (RGCs) and then subjected to optic nerve crush. The Bax/ background was used to minimize apoptotic death of retinal ganglion neurons triggered by optic nerve injury, which may amount to 80% at 2 wk after optic nerve crush (Li et al., 2000). 2 wk after the injury, we observed robust regenerative axon growth up to 3 mm past the lesion site in the kaB-RAF– expressing optic nerve (Fig. 8, D and G), with very limited growth in the control Bax/ littermates (Fig. 8 C), consistent with previous observations that survival alone is not sufficient to promote growth of adult RGC axons (Goldberg et al., 2002). Combined deletion of the canonical RAF effector kinases MEK1 and MEK2 substantially suppressed the regenerative axon growth caused by kaB-RAF (Fig. 8 E), indicating that kaB-RAF drives axon growth through the canonical MEK effectors. Whereas the length of axon extension induced by (middle), and WT preconditioning lesioned mice (right) after 24 h in vitro. TdTom is shown in green to improve contrast. Bars: (A–C) 100 μm; (D) 20 μm. (E–H) Quantitation of axon extension in adult DRG cultures at 24 h in vitro. Data were collected from three independent experiments from three ani-mals per genotype or condition and analyzed as described previously (Parikh et al., 2011); 100 cells were counted per group. Error bars indicate SEM. One-way ANOVA with post-hoc Tukey’s HSD test: *, P 0.01; **, P 0.005. 808 B-RAF drives axon growth and regeneration | O’Donovan et al.
- 41. JEM Vol. 211,No. 5 Ar t icle Figure 8. Activation of B-RAF enables regenerative axon growth in the crush-lesioned optic nerve via the canonical effectors MEK1/2. (A) Schematic of experimen-tal time course. (B) Intravitreal injection of AAV2-Cre induces expression of TdTom in retinal ganglion neurons, labeling the entire optic nerve (red). (C, top) Whole-mount image of a crushed Bax/ optic nerve. Crush site is indicated by a red asterisk here and in all following panels. (bottom) Confocal fluorescence image of the same nerve. Green shows axons anterogradely labeled with CTB–Alexa Fluor 488. (D) Whole-mount confocal imaging shows strong regenerative growth in the lesioned kaB-RAF–expressing optic nerve. (inset) Axons at 3.5 mm from the crush site (magnified from the boxed area). Arrowheads indicate outgrowing axons. (E) Loss of MEK1 and MEK2 abolishes the regeneration driven by kaB-RAF. (F) Optic nerve regeneration in the absence of PTEN. Bar, 0.5 mm. (C–F) Images are representative of three optic nerves per genotype. (G) Quantitation of axon regenerative growth in the optic nerve 2 wk after nerve crush; genotypes as shown in B–E. At 1.6 mm from the crush site, the den-sity of regenerating axons is more than threefold greater in the LSL-kaBraf:Bax/ genotype than in the Ptenf/f:Bax/ genotype. Data are from three nerves per genotype. Optic densities were acquired from the whole-mount optic nerves using an LSM710NLO two-photon confocal microscope with the ZEN2009 software. Data were normalized by setting the baseline OD, as measured 0.2 cm proximal to the crush site in all nerves, to the same (arbitrary) level. 809 B-RAF gain-of-function is comparable with the maximal axon growth reported in PTEN deletion mice (Fig. 8 F; Park et al., 2008), in a direct comparison, we found up to a 3.9-fold higher density of regenerating axons in the LSL-kaBraf:Bax/ mice 1.5 mm distal to the crush site than is seen in the crushed Pten/:Bax/ optic nerve (Fig. 8, D, F, and G).
- 42. DISCUSSION An understandingof the mechanisms that drive axon growth is important, both to decipher how connectivity develops in the nervous system and to develop therapeutic strategies for nervous system repair after injury or disease. We show that the RAF–MEK axis plays a key role in axon growth signaling. Activation of B-RAF in neurons is sufficient to drive sensory axon growth in the embryo, to enable adult sensory axons to regenerate across the DREZ and further into the spinal cord, and to induce robust regeneration of adult retinal axons in the injured optic nerve. Both developmental DRG axon overgrowth in the spinal cord and mature RGC axon re-generation in the optic nerve were abrogated by concomitant ablation of MEK1 and MEK2. We thus establish classical cell-autonomous RAF–MEK signaling as a fundamental driver of axon growth. We should note that this pathway seems to be selective to axon growth signaling because we have never ob-served that B-RAF activation supports neuronal survival (un-published data). In vitro work has long suggested a potential role for RAF– MEK signaling in axon growth. Previous in vivo data, however, have been scarce and controversial. In the retina, for example, pharmaceutical inhibition of MEK–ERK signaling abrogated optic nerve regeneration supported by FGF2 (Sapieha et al., 2006). Two putative intracellular activators of RAF signaling, BAG1 and Mst3b, have been shown to promote regenerative axon growth in the optic nerve (Planchamp et al., 2008; Lorber et al., 2009), but the expression of a constitutively active MEK1 did not drive any regeneration in the optic nerve (Pernet et al., 2005). Others have concluded that ERK activity promotes RGC axon regeneration via an indirect mechanism dependent on glial cells (Müller et al., 2009). Although it is likely that multiple mechanisms, direct as well as indirect, will contribute to axon regeneration in the inhibitory environment of the CNS, the current cacophonic state of the field is likely caused by the mainly indirect approaches of incomplete penetrance that have been taken by various laboratories. When using small molecule inhibitors or transient viral overexpression of interfering or ac-tivating constructs, it is difficult to accurately titrate the dose for the entire duration of an experiment. We believe that we have applied a stringent approach toward activation of RAF signal-ing in RGCs, and our data argue strongly for a direct positive effect of RAF–MEK signaling on axon growth and regenera-tion of RGCs, as well as in DRG neurons. Possible downstream mechanisms beyond the MEK kinases remain speculative at this point. Stabilization of microtubules improves axon regen-eration in a spinal cord injury (SCI) model through both neu-ron- intrinsic and -extrinsic mechanisms (Hellal et al., 2011), and it is likely that activation of RAF–MEK signaling will di-rectly affect microtubule stability in injured axons via its effects on microtubule-regulating enzymes such as HDAC6 (Williams et al., 2013). Furthermore, B-RAF has been shown to directly interact with tubulin (Bonfiglio et al., 2011). Activation of B-RAF signaling is also likely to trigger the expression of axon growth–enhancing gene sets in injured neurons. The elucida-tion of exact mechanisms awaits further study. Figure 9. Combined B-RAF activation and PTEN deletion enables long-range axon regeneration. (A) Representative longitudinal sec-tions of regenerating optic nerve 2 wk after crush injury. Genotypes are as indicated. Crush site is indicated by a red asterisk. Bar, 200 μm. (B) Quan-titation of data as shown in A. Axons were counted as described in Park et al. (2008). n = 6 nerves per genotype. Error bars indicate SEM. Two-way ANOVA tests comparing LSL-kaBraf with Ptenf/f, LSL-kaBraf and Ptenf/f with WT, or LSL-kaBraf and Ptenf/f with LSL-kaBraf:Ptenf/f all re-sulted in p-values 0.001. kaB-RAF expression and PTEN deletion synergize to increase axon regenerative growth To test whether combined activation of the B-RAF and PI3- kinase–mTOR pathways can further boost axon growth ca-pacity in injured adult retinal ganglion neurons, we performed the optic nerve regeneration experiments using double LSL-kaBraf: Ptenf/f mice. Both the numbers and length of regener-ating axons were increased in the optic nerve of the LSL-kaBraf: Ptenf/f mice compared with those in single LSL-kaBraf or in Ptenf/f mice (Fig. 9 A). The synergistic effect of activating both B-RAF and PI3-kinase signaling is most apparent at the longest lengths of axon regeneration (Fig. 9 B). Note that the mice in these experiments were from a Bax WT background to en-able direct comparison with the original PTEN deletion data (Park et al., 2008); however, this means that possible survival-promoting effects of the mutant alleles cannot be excluded. 810 B-RAF drives axon growth and regeneration | O’Donovan et al.
- 43. JEM Vol. 211,No. 5 Ar t icle The extent of regeneration achieved by direct genetic ac-tivation of specific intracellular signaling pathways, including B-RAF, DLK, PI3-kinase–mTOR, KLF, and JAK–STAT path-ways (Smith et al., 2009; Yan et al., 2009; Park et al., 2010; Blackmore et al., 2012; Shin et al., 2012; Lang et al., 2013), com-pares favorably with what has been reported for application of growth factors such as NGF, BDNF, GDNF, and CNTF (Lykissas et al., 2007; Zhang et al., 2009; Allen et al., 2013; Leibinger et al., 2013). Growth factors as promoters of regeneration are hobbled by two major issues. First, the signaling machinery that enables growth factors to drive axon growth in the developing nervous system is not expressed at sufficient levels in the adult nervous system (Hollis et al., 2009). Indeed, growth inhibitory signaling molecules such as the SOCS family or phosphatases are up-regulated upon maturation (Lu et al., 2002; Smith et al., 2009; Park et al., 2010; Gatto et al., 2013). Therefore, the most promising studies using growth factors have combined them with genetic intervention to up-regulate growth factor receptors or down-regulate their intrinsic inhibitors (Hollis et al., 2009; Sun et al., 2011). The second issue is that of undesirable side ef-fects, especially that of neuropathic pain caused by neurotrophin administration (Obata et al., 2006; Jankowski and Koerber, 2009). Development of “painless” neurotrophins (Capsoni et al., 2011) may improve the usefulness of this family of growth factors in the context of regeneration. Future combined activation of several growth signaling pathways with blockade of growth inhibitory pathways may lead to realistic treatment options for patients with loss of vision, sensation, or locomotion. MATERIALS AND METHODS Mouse models. Mouse breeding and genotyping were performed as de-scribed previously (Mercer et al., 2005; Zhong et al., 2007). The animal proto-col was approved by the Institutional Animal Care and Use Committee at Weill Cornell Medical College, and experiments were conducted in accordance with the National Institutes of Health Guide for the Use and Care of Laboratory Animals. The LSL-kaBraf mouse line was provided by C.A. Pritchard (Univer-sity of Leicester, Leicester, England, UK; Mercer et al., 2005). The TrkAtaulacZ mouse was provided by L. Reichardt (University of California, San Francisco, San Francisco, CA; Moqrich et al., 2004). Nestin-Cre deleter and Bax-null mice were generated in R. Klein’s laboratory (Max Planck Institute of Neuro-biology, Martinsried, Germany; Tronche et al., 1999) and S.J. Korsmeyer’s labo-ratory (Dana-Farber Cancer Institute, Boston, MA; Knudson et al., 1995), respectively. The brn3a-CreERT2 deleter mouse line was generated by J. Zhong in W.D. Snider’s laboratory (University of North Carolina at Chapel Hill, Chapel Hill, NC). All mice were on a mixed 129Sv and C57BL/6 background. We used littermates as controls throughout. Generation of the brn3a-CreERT2 deleter mouse line. Brn3a is a POU domain transcription factor that is selectively expressed in DRG neurons. Using a brn3a promoter construct (Eng et al., 2001), we generated a brn3a-Cre- ERT2 deleter mouse line using the pronuclear injection technique (Fig. 6 A). Western blotting and immunohistochemical staining. Western blot-ting and immunohistochemical staining were performed as described previ-ously (Zhong et al., 2007). An equal amount of total protein was loaded in each lane. The antibodies used were as follows: TrkA, Brn3a, and PlexinA1 antibodies were provided by L. Reichardt, E. Turner (University of Califor-nia, San Diego, La Jolla, CA), and T. Jessell (Columbia University, New York, NY), respectively. Antibodies against MEK1/2 (9122), pMEK1/2 (9121), ERK1/2 (9102), pERK1/2 (9101), pAKT (9271 and 9275), phospho-p70S6K 811 The developmental phenotypes we observed in the B-RAF gain-of-function embryos were generally comple-mentary to those previously observed in B-RAF/C-RAF loss-of-function mice (Zhong et al., 2007). In contrast to nociceptors’ peripheral projections, the development of their central projections does not depend on NGF/TrkA signaling (Patel et al., 2000; Harrison et al., 2004; Zhong et al., 2007). Notably, we found that activation of B-RAF resulted in over-growth of both proprioceptive and nociceptive axons in the spinal cord, whereas the expression of two known repulsive signaling molecules in the dorsal cord and DRG, Sema6 and PlexinA1, remained unaltered. Thus, kaB-RAF appears to acti-vate a normally quiescent axon growth signaling pathway in the central sensory axons that seems to be unaffected by repulsive guidance cues. The importance of this effect, the lack of response to re-pulsive or inhibitory cues, becomes clear in the context of re-generation of central sensory branches after dorsal root crush injury. Sensory axons expressing kaB-RAF robustly regener-ated into the DREZ and spinal cord. The regeneration failure of DRG axons after dorsal root avulsion injuries has been variously attributed to the lack of intrinsic growth capacity, to extrinsic growth barriers such as glia-associated growth inhibitors at the DREZ, and to prema-ture synaptic differentiation (Han et al., 2012; Smith et al., 2012). Application of neurotrophic factors acting via tyrosine kinase receptors has shown substantially enhanced regeneration (Ramer et al., 2000; Wang et al., 2008; Harvey et al., 2010), even func-tional recovery with the systemic administration of artemin (Wang et al., 2008), although these results await independent replication. Future studies will test whether a combination of RAF activation with trophic growth factors can further en-hance axon regeneration and reinnervation of presumptive tar-gets in the spinal cord. Compared with spinal cord lesions, the optic nerve’s sim-ple structure allows for clear evaluation of both lesion and re-generation. In recent years, the optic nerve model has revealed several intracellular signaling pathways that can drive CNS axon regeneration, most prominently the PI3-kinase–mTOR and the JAK–STAT pathways, engaged by growth factor tyro-sine kinase receptors and cytokines (Park et al., 2008; Smith et al., 2009; Buchser et al., 2012; Leibinger et al., 2013; Pernet et al., 2013). Combined deletion of endogenous inhibitors of these two pathways enhanced regeneration above the level reported for deletion of either gene alone (Sun et al., 2011). Activation of PI3-kinase–mTOR via PTEN deletion also en-hanced regenerative growth in an SCI model (Liu et al., 2010), suggesting that results obtained in the optic nerve crush model may generally translate to SCI models. Here, we show that the classic growth factor signaling module RAF–MEK enables axon regeneration in the optic nerve at least as powerfully as any previously reported single molecule manipulation and that the combination of kaB-RAF with activation of PI3-kinase– mTOR via PTEN deletion enhances optic nerve axon regen-eration even more strongly than would be expected for a simple additive effect.
- 44. 3R01EY022409-01S1 from theNational Eye Institute (NEI), and grant ZB1-1102-1 from the Christopher Dana Reeve Foundation to J. Zhong. Z. He is supported by grants 5R01EY21526 and EY021342 from NEI. Y.-J. Son is supported by grant 1R01NS079631 from the National Institute of Neurological Disorders and Stroke and grants from Shriners Hospitals for Children and the Muscular Dystrophy Association. H. Zou is supported by grants from the National Institutes of Health (1R01NS073596) and the Irma T. Hirschl/Monique Weill-Caulier Foundation. K.J. O’Donovan is a Goldsmith fellow. The authors declare no competing financial interests. Submitted: 24 August 2013 Accepted: 18 March 2014 REFERENCES Agthong, S., J. Koonam, A. Kaewsema, and V. Chentanez. 2009. Inhibition of MAPK ERK impairs axonal regeneration without an effect on neuro-nal loss after nerve injury. Neurol. Res. 31:1068–1074. http://dx.doi.org/ 10.1179/174313209X380883 Allen, S.J., J.J. Watson, D.K. Shoemark, N.U. Barua, and N.K. 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Retinal ganglion cells do not extend axons by default: promotion by neurotrophic signaling and electrical activity. Neuron. 33:689–702. http://dx.doi.org/10.1016/S0896-6273(02)00602-5 (9206), phospho-mTOR Ab (2971), and pGSK3 (9336) were obtained from Cell Signaling Technology. III-Tubulin (AA10) was purchased from Invitrogen; Sema6D (S-16) was purchased from Santa Cruz Biotechnol-ogy, Inc.; C-RAF antibody (610151) was purchased from BD. Parvalbumin antibody (PV28) was obtained from Swant. CGRP antibodies (AB5920 and AB5705) were obtained from EMD Millipore. All Western blot and immu-nohistochemical experiments were repeated with tissue from at least three embryos for each genotype, and these embryos were obtained from a differ-ent litter for each experiment. Littermate controls were used throughout. LacZ staining. E16.5 embryos were fixed in 4% paraformaldehyde and stained with X-gal using EMD Millipore’s Tissue Base staining solution ac-cording to the manufacturer’s protocol. After imaging of axon skin innervation, embryos were dehydrated using a methanol in PBS dilution series (25–50%, 75–95%, and 100%), followed by incubation in 50% methanol: 50% benzoyl alcohol/benzoyl benzoate (BABB), and subsequently cleared in 100% BABB (Sigma-Aldrich). Specimens were imaged with a M205A stereomicroscope equipped with a DFC310FX color digital camera system (Leica). DRG culture. For CGRP expression assay, E12.5 DRGs derived from LSL-kaBraf:Bax/:nes-Cre embryos were cultured with 100 ng/ml NGF (Alomone Labs) and skin-conditioned medium for 8 d. DRG neurons cultured in N2-supplied MEM (without NGF) were used as control. Both cultures where treated with FdU (5-fluoro-2-deoxyuridine; Sigma-Aldrich). Culture media were changed every 12 h. The cells were fixed 8 d thereafter, and the cells were stained for CGRP and TrkA. For axon growth and neuron survival assays, DRGs were dissected from E12.5 embryos with the desired genotypes as described previously (Markus et al., 2002; Zhong et al., 2007). The cells were dissociated and plated on laminin-coated coverslips. Cells were then cultured in serum-free media supplemented with 1× N2 (Invitrogen) and 1% BSA. FdU, NGF, or AAV2-Cre (Vector Laboratories) were added as described previously (Markus et al., 2002; Zhong et al., 2007). Dorsal root crush. Surgeons and all other personnel performing experi-ments and analyses were blinded as to genotypes. 10–14-wk-old mice were injected s.c. with tamoxifen (5 μg/10 g body weight) for a consecutive 5 d. After another 2 d, i.e., 1 wk from the first tamoxifen injection, crush injury of the C5, C6, C7, and C8 dorsal roots was performed using a fine forceps (Dumont #5) as described previously (Di Maio et al., 2011). AAV2-GFP (1010 GC, 7004; Vector Laboratories) was then injected to C6 and C7 DRGs. 2 wk after the crush, mice were perfused, and axon regeneration through the C6 and C7 dorsal roots was analyzed in a thin dorsal slice preparation of whole spinal cord or in cryostat sections. Sciatic nerve crush. Mice were injected with tamoxifen (2 μg/10 g body weight) for a consecutive 5 d, followed by 5 d of rest. Unilateral sciatic nerve crush was then performed as described previously (Zhong et al., 1999). Adult DRGs were collected and cultured as described previously (Zou et al., 2009). Images were taken using a Carl Zeiss LSM710NLO confocal microscope. Axon length was quantified as described previously (Zou et al., 2009). Optic nerve crush. Surgeons and all other personnel performing experi-ments and analyses were blinded as to genotypes. The crush-regeneration and axon counting protocol is adapted from Park et al. (2008). Whole-mount optic nerves were treated with FocusClear (CelExplorer Labs) and scanned using an LSM710NLO multiphoton confocal microscope. OD was deter-mined with the Carl Zeiss ZEN2009 software. We would like to thank Louis Reichardt for the TrkAtaulacZ mice and TrkA antibody, Catrin A. Pritchard for the LSL-kaBraf mice, Eric Turner for the Brn3a promoter, and Thomas Jessell for the Plexin1A antibody. Larry Benowitz provided experimental advice on the optic nerve injury model. Rajiv Ratan, David Ginty, William D. Snider, and Annette Markus are acknowledged for insightful discussion and suggestions. This work was supported by startup funds from the Burke Foundation as well as Whitehall Foundation research grant 2010-08-61, grants 1R01EY022409 and 812 B-RAF drives axon growth and regeneration | O’Donovan et al.
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- 47. The Rockefeller UniversityPress $30.00 J. Exp. Med. 2014 Vol. 211 No. 4 595-604 www.jem.org/cgi/doi/10.1084/jem.20131377 Br ief Def ini t ive Repor t 595 Stroke is one of the leading causes of death and disability worldwide. Clinical and preclinical ex-perimental studies highlight the importance of inflammation in both acute and delayed neuro-nal tissue damage after ischemic stroke; however, the mechanisms and cells involved in this neuro-inflammation are not fully understood. There is currently no available treatment targeting the acute immune response that develops in the brain after transient focal ischemia. Therefore, we sought to identify novel T cell–derived cyto-kines that contribute to acute cerebral reperfu-sion using the mouse model of transient middle cerebral artery occlusion (tMCAO). During the reperfusion of infarcted brain tis-sue, leukocytes accumulate in the injured brain where, in addition to clearing cell debris, they promote secondary tissue injury (Yilmaz and Granger, 2010). Within the acute phase of isch-emic reperfusion (I/R) injury there are multiple waves of cell infiltration of macrophages, neutro-phils, and lymphocytes (Gelderblom et al., 2009). Brain-infiltrating T cells have also been widely reported in stroke and animal models of stroke and are thought to have acute detrimental and delayed protective effects (Magnus et al., 2012). Conventionally, the protective role of T cells has been attributed to the accumulation of regula-tory T cells within the CNS in later stages of re-perfusion injury. These T cells produce a variety of cytokines including TGF and IL-10, which are both antiinflammatory and neuroprotective. (Liesz et al., 2009; Stubbe et al., 2013). In addi-tion to having an established role in delayed neuroprotection, Kleinschnitz et al. (2013) have recently shown that CD4+ CD25+ regulatory T cells also promote acute ischemic injury through interaction with the cerebral vasculature. The acute detrimental effects can be further divided into early (24 h) and late (72 h) phases, with IL-17 production by nonconventional T cells (less common T cell subset associated with mu-cosal tissues) possibly accounting for the latter by promoting neutrophil accumulation (Gelderblom et al., 2012). The mechanisms of the early detrimental ef-fects of T cells after cerebral ischemia are least CORRESPONDENCE Zsuzsanna Fabry: zfabry@facstaff.wisc.edu Abbreviations used: BA, basilar artery; ECA, external carotid artery; ICA, internal carotid artery; I/R, ischemia/reperfu-sion; MCA, middle cerebral artery; PCA, posterior cerebral artery; PComA, posterior com-municating artery; RAG, recombination activating gene; rCBF, regional cerebral blood flow; tMCAO, transient MCA occlusion; TTC, 2,3,5- triphenyltetrazolium chloride; VA, vertebral artery. T cell–derived interleukin (IL)-21 promotes brain injury following stroke in mice Benjamin D.S. Clarkson,1,3 Changying Ling,1 Yejie Shi,2 Melissa G. Harris,1,4 Aditya Rayasam,4 Dandan Sun,2,5 M. Shahriar Salamat,1 Vijay Kuchroo,6 John D. Lambris,7 Matyas Sandor,1 and Zsuzsanna Fabry1 1Department of Pathology and Laboratory Medicine, 2Department of Neurological Surgery, 3Department of Cellular and Molecular Pathology, 4Neuroscience Training Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53792 5Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA 15213 6Center for Neurological Diseases, Brigham and Women’s Hospital Harvard Medical School, Boston, MA 02115 7Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 T lymphocytes are key contributors to the acute phase of cerebral ischemia reperfusion injury, but the relevant T cell–derived mediators of tissue injury remain unknown. Using a mouse model of transient focal brain ischemia, we report that IL-21 is highly up-regulated in the injured mouse brain after cerebral ischemia. IL-21–deficient mice have smaller infarcts, improved neurological function, and reduced lymphocyte accumulation in the brain within 24 h of reperfusion. Intracellular cytokine staining and adoptive transfer experiments revealed that brain-infiltrating CD4+ T cells are the predominant IL-21 source. Mice treated with decoy IL-21 receptor Fc fusion protein are protected from reperfusion injury. In postmortem human brain tissue, IL-21 localized to perivascular CD4+ T cells in the area surrounding acute stroke lesions, suggesting that IL-21–mediated brain injury may be relevant to human stroke. © 2014 Clarkson et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial– Share Alike 3.0 Unported license, as described at http://creativecommons.org/ licenses/by-nc-sa/3.0/).
- 48. of several previouslyreported inflammatory genes, we found that IL-21 was one of the most highly expressed inflammatory genes among those measured (Fig. 1 a). Gene expression levels from arrays were normalized to interquartile spot intensity. Arrays did not differ systemically in gene expression levels be-fore or after normalization (not depicted). This increase in IL-21 gene expression was confirmed by real-time (RT) PCR analy-sis, which detected a 24-fold relative increase in IL-21 gene expression in the ipsilateral ischemic brain tissues compared with the contralateral hemisphere at 24 h reperfusion (Fig. 1 b). IL-21 was not detectable by this method in healthy brain tis-sue (Fig. 1 b). IL-21–deficient mice are protected from acute neuronal injury after cerebral I/R injury Whether IL-21 contributes to ischemic tissue injury had not been directly studied. However, in the last few years it has be-come evident that IL-21 expression is associated with acute rejection in mice after kidney, heart, or liver allograft (Baan et al., 2007; Hecker et al., 2009; Xie et al., 2010). Because these models also involve reperfusion of ischemic tissues, these find-ings support the potential role of IL-21 in I/R injury. We evaluated the levels of cerebral I/R injury in IL-21– deficient (IL-21 KO) mice. Infarct volumes in IL-21 KO mice were reduced to 35% of the infarct volumes observed in con-genic C57BL6/J WT mice as early as 24 h after tMCAO, as measured by triphenyltetrazolium chloride (TTC; Fig. 1 c). Similar effects were also seen at 4 d (not depicted) and 7 d after tMCAO (Fig. 1 d), indicating that IL-21 contributes to both immediate and delayed brain injury and suggesting that in the absence of IL-21 tissue repair can occur. Analysis of intracra-nial vascular anatomy revealed no differences between WT and IL-21 KO mice in the patency of the posterior communicating artery that would account for the observed differences in tissue injury (Fig. 1 g). Nor did we observe differences between WT and IL-21 KO mice in heart rate, or blood pressure before or after tMCAO (Fig. 1 h). WT and IL-21 KO CD4+ T cells, CD8+ T cells, and CD11b+ myeloid cells showed no difference in IL-2, IL-17A, IFN-, or TNF production when stimulated in vitro (Fig. 2, g–l). The reduction in infarct volumes corre-sponded with less weight loss (unpublished data), less spleen atrophy (Fig. 2 c), and improved neurological functioning in IL-21 KO mice compared with WT mice as assessed by both grip strength (Fig. 1 e) and Bederson (Fig. 1 f) scoring 1, 4, and 7 d after tMCAO. We measured accumulation of monocytes and lympho-cytes in the brain after tMCAO in IL-21 KO and WT animals (gating strategy in Fig. 2 a). We did not observe significant dif-ferences in the rate of accumulation of monocytes (CD45high CD11b+Ly6chigh), microglia (CD45intCD11b+), B cells (B220+), T cells (TCR+), NK, or NKT cells (NK1.1+) in the ischemic brain of WT and IL-21 KO mice after 1, 4, and 7 d of reperfusion (unpublished data). In contrast, as early as 1 d after tMCAO, IL-21 KO mice showed significantly dimin-ished cerebral accumulation of CD4+ T cells and CD8+ T cells understood. Several laboratories have reported reduced neuro-logical deficit and infarct volumes at 24–48 h reperfusion in T cell–deficient mice after tMCAO (Yilmaz and Granger, 2010). After tMCAO, recombination activating gene 1–deficient (RAG1 KO) mice, which lack T and B lymphocytes, have sig-nificantly smaller brain injury compared with controls; whereas, adoptive transfer of WT CD4+ helper T cells or CD8+ cytotoxic T cells increases stroke infarct volumes within 24 h after ischemia in these mice (Kleinschnitz et al., 2010). Additionally, TCR-transgenic mice and mice lacking co-stimulatory TCR signaling molecules were fully susceptible to acute I/R injury, indicating that T cell involvement at early time points is anti-gen- independent (Kleinschnitz et al., 2010). These data dem-onstrate that conventional CD4+ or CD8+ T cells exacerbate acute injury after cerebral ischemia independently of TCR li-gation, and this effect seems to be concomitant with an early increase in T cell infiltration into the postischemic brain, which many have reported to be between 3 and 48 h (Yilmaz et al., 2006; Gelderblom et al., 2009). Recent findings suggest that, in the postischemic brain, within hours of reperfusion T cells accumulate in postcapillary segments of periinfarct inflamed cerebral microvasculature characterized by high endothelial expression of chemokines and adhesion molecules. These postcapillary venules have been postulated to allow accumulating immune cells to activate each other and promote platelet adhesion in a process termed thrombo-inflammation (Nieswandt et al., 2011). Much research has been devoted to identifying T cell factors that promote thrombo-inflammation (Barone et al., 1997; Hedtjärn et al., 2002; Yilmaz et al., 2006; Shichita et al., 2009; Gelderblom et al., 2012); however, to our knowledge no study has yet identified the T cell–derived factors responsible for the early increase in infarct volumes at 24 h reperfusion. Here, we present data that identify IL-21 as a key CD4+ T cell–derived inflammatory fac-tor that contributes to increased early ischemic tissue injury. RESULTS AND DISCUSSION Robust up-regulation of IL-21 during cerebral I/R injury IL-21 is closely related to IL-2 and IL-15 and signals through the IL-21 receptor, which is comprised of an IL-21–specific subunit and a common subunit shared with IL-2, IL-7, IL-9, and IL-15. IL-21 is known to regulate immune responses by promoting antibody production, T cell–mediated immunity, and NK cell and CD8+ T cell cytotoxicity. Recently, others have shown that stress signals from necrotic tissue can induce rapid IL-21 production from naive T cells (Holm et al., 2009), and co-stimulation with TLR3 ligands during polyclonal T cell activation significantly increases IL-21 secretion that contributes to small intestine localized pediatric celiac disease (van Leeuwen et al., 2013). To test whether IL-21 is up-regulated in brain after ischemic necrosis induced by MCAO and to better understand the cytokines involved in T cell–mediated cerebral I/R injury, we measured changes in inflammatory gene expression in the brain within 24 h after tMCAO in mice using PCR-based gene array analysis. In addition to verifying the up-regulation 596 IL-21 promotes brain injury after stroke | Clarkson et al.
- 49. Br ief Defini t ive Repor t Figure 1. IL-21 is up-regulated early in mouse brain, and IL-21–deficient mice are protected after tMCAO. GeArray S Series Mouse Autoimmune and Inflammatory Re-sponse gene array of transcripts expressed in pooled brain tissues 24 h after tMCAO or sham procedure (n = 3–6 mice per group). (a) Bar graphs show PCR array spot intensity of genes with a greater than sixfold difference in gene expression in tMCAO compared with sham, normalized to the interquartile mean spot intensity. (b) IL-21 mRNA expression level in ipsilateral hemisphere relative to con-tralateral hemisphere 24 h after tMCAO (n = 3 per group). Mann-Whitney rank sum test *, P 0.05. Plots show median, lower, and upper quartile (box) and range (error bars). Infarct volumes of WT and IL-21 KO mice 24 h after tMCAO (c) and 7 d after tMCAO (d). Represen-tative images of TTC-stained 2-mm brain slices shown below (n = 5–7 mice per group). WT and IL-21 KO mice grip strength (e) and Bederson score (f) at 1, 4, and 7 d after 1 h tMCAO (n = 7–8 for each group). (g) Brain vasculature of C57BL/6 and IL-21KO mice perfused transcardially with carbon lampblack (C198-500; Thermo Fisher Scientific) in 20% gelatin ddH2O. Arrows indicate anterior cere-bral (ACA), MCA, posterior cerebral (PCA), basilar (BA), and vertebral arteries (VA), show-ing point of occlusion (white circle). High magnification images demonstrate no differ-ence in patency of the posterior communicat-ing artery (PComA, arrows). (h) Heart rate and blood pressure of WT and IL-21 KO mice be-fore and after tMCAO (n = 3–4 mice per group). Data are representative of 2–3 inde-pendent experiments. **, P 0.01; ***, P 0.001; ****, P 0.0001 by Student’s t test (single comparison) or one-way ANOVA (mul-tiple comparisons). Error bars indicate SD. in either tissue compared with WT experimental animals. Nor did we observe a difference between WT and IL-21 KO mice in the frequency of lymphocytes producing the antiinflam-matory cytokine IL-10 among B cells (B220+), CD8+ T cells, or CD4+ T cells (unpublished data). These data demonstrate that IL-21 deficiency is protective at acute time points after tMCAO and IL-21 levels in the CNS correlate with early in-filtration of T cells without affecting regulatory T or B cell accumulation or IL-10 cytokine production during the acute period (day 1–4). after tMCAO compared with WT mice (Fig. 2 e) and these differences persisted at day 7 (Fig. 2 f ). These differences were not reflected in the spleen before or after tMCAO (Fig. 2 b). IL-21 has also been shown to be produced by and modulate the function of regulatory T cells (Peluso et al., 2007; Battaglia et al., 2013), which begin to accumulate in the brain after tMCAO. Thus, we compared the frequency of regulatory CD4 T cells expressing the marker Foxp3 in the brain and spleen 24 h after tMCAO in WT and IL-21 KO mice. IL-21 KO mice exhibited no difference in regulatory T cell abundance JEM Vol. 211, No. 4 597
- 50. Figure 2. Lymphocyterecruitment to brain is diminished in IL-21 deficient mice. (a) Gating strategy for leukocytes isolated from brain after MCAO. (b) WT and IL-21 KO spleen cells 24 h after tMCAO or sham procedure (n = 3 mice per group). (c) Relative change in spleen weight of WT and IL-21 KO mice after tMCAO (n = 3–7 mice per group). (d) Percentage of blood and spleen CD4+ T cells expressing IL-21 after 5-h ex vivo stimulation with PMA (10 ng/ml) and Ionomycin (1 μg/ml) 4 d after MCAO or control treatment. (e and f) Leukocyte accumulation in the brain of WT mice compared with IL-21 KO mice 1, 4, and 7 d after tMCAO (n = 3–6 mice per group). (g–k) In vitro cytokine expression by WT and IL-21 KO CD4+ and CD8+ T cells after 5-h stimulation under indicated conditions with or without recombinant mouse IL-21 (100 ng/ml). (l) TNF production by CD11b+ myeloid cells stimulated with LPS (500 ng/ml) for 5 h with or without recombinant mouse IL-21 (100 ng/ml). Cells isolated from n = 3 mice per group. Data are representative of two to four independent experiments. *, P 0.05; **, P 0.01; ***, P 0.001; ****, P 0.0001 by Student’s t test (single comparison) or one-way ANOVA (multiple comparisons). Error bars indicate SD (b–d and g–l) and SEM (e, f). 598 IL-21 promotes brain injury after stroke | Clarkson et al.
- 51. Br ief Defini t ive Repor t IL-21 blockade is protective in tMCAO We treated WT mice with IL-21 receptor Fc protein (IL-21R. Fc) using a previously described protocol (Jang et al., 2009; McGuire et al., 2011; Spolski et al., 2012). We administered 500 μg of IL-21R.Fc i.p. 1 h before tMCAO. As measured by TTC staining, treated mice showed significantly reduced in-farct volumes compared with control-treated mice 24 h after tMCAO (Fig. 4 a). We found a similarly protective effect in mice treated with IL-21R.Fc protein (500 μg i.p.) 2 h after ini-tiation of reperfusion (Fig. 4 a). These differences were associ-ated with decreased locomotor function (decreased resistance to lateral push and increased circling behavior) in control-treated mice compared with those treated with IL-21R.Fc (Fig. 4 b and Video 1). Although we cannot exclude the pos-sibility that IL-21R.Fc exhibits its blocking effect in periph-eral immune compartments, using ELISA for human IgG4 we were able to observe that—upon i.p. injection—soluble IL-21R. Fc accumulates in the CNS of mice after tMCAO (Fig. 4 c). IL-21 presence localizes with CD4 staining in human stroke tissue IL-21+ cells were recently detected in human brain tissue during different neuroinflammatory conditions (Tzartos et al., 2011). Thus, we stained groups of postmortem brain tissue from IL-21 is primarily produced by brain-infiltrating CD4+ T cells We measured intracellular IL-21 production by various cell populations. IL-21–producing cells were not detected by flow cytometry among cells isolated from healthy brain, but could be detected among mononuclear cells isolated from isch-emic brain 24 h after tMCAO. Gating on IL-21+ cells revealed that the majority of these cells were CD4+ T cells (Fig. 3 a). IL-21–producing CD4+ T cells were also detected at low lev-els among cells isolated from blood, but not spleen, of healthy WT mice, and these levels were unaffected after transient ce-rebral ischemia (Fig. 2 d), indicating that the increase in IL-21 production was limited to CD4+ T cells recovered from the postischemic brain. Next, we adoptively transferred WT or IL-21 KO CD4+ T cells into lymphocyte-deficient RAG2 KO mice. Purity of transferred CD4+ T cells was confirmed by flow cytometry to be 95% (Fig. 3 b). As shown previ-ously (Kleinschnitz et al., 2010), we observed markedly re-duced infarcts in RAG KO mice compared with WT mice, and infarct volumes could be restored to WT levels in RAG2 KO by adoptively transferring WT CD4+ T cells. Most im-portantly, RAG2 KO mice that received WT CD4+ T cells had significantly larger infarcts than those receiving IL-21 KO CD4+ T cells (Fig. 3 c). Figure 3. IL-21 is primarily produced by brain-infiltrating CD4+ T cells. (a) Intracellular cytokine staining of lymphocytes isolated from n = 5 pooled healthy WT, ischemic IL-21/, or ischemic WT mouse brains 24 h after tMCAO or sham procedure showing IL-21 versus CD8 expression. Histo-grams show CD4, NK1.1, and TCR expression on IL-21+ cells from ischemic WT brain. (b) CD45, CD4, and LFA-1 expression by negative fractions purified from WT and IL-21/ lymph node cells by CD4+ negative selection using magnetic cell separation before transfer into RAG2/ recipients. (c) Infarct volume in WT mice (n = 4), RAG2/ mice (n = 4), RAG2/ mice + WT CD4 T cells (n = 10), and RAG2/ mice + IL-21/ CD4 T cells (n = 10) 24 h after tMCAO. Representative TTC-stained 2-mm mouse brain slices shown on top. Data are representative of two independent experiments. **, P 0.01; ***, P 0.001; ****, P 0.0001 one-way ANOVA. Error bars indicate SD. JEM Vol. 211, No. 4 599
- 52. Figure 4. Blockadeof IL-21 signaling before or after tMCAO reduces infarct size in WT mice. (a) Infarct volumes 24 h after tMCAO in WT mice treated with 500 μg recombinant mIL-21R.Fc or PBS 1 h before (pretreatment) or 2 h after (posttreatment) surgery. Representative TTC-stained brain slices shown on left (n = 3–4 mice per group). (b) Still image from Video 1 depicting behavioral differences between WT mice posttreated with IL-21R.Fc or PBS. (c) IL-21R.Fc protein levels in the indicated organs 20–24 h after tMCAO in WT mice injected with 500 μg IL-21R.Fc 2 h after start of reperfusion (n = 2–4 mice per group). N.D., not detected. Data are representative of two independent experiments. **, P 0.01; ***, P 0.001, by Student’s t test. Error bars indicate SEM. Representative images of postmortem paraffin-embedded human acute stroke lesions stained with control sera (d), or primary anti-bodies against CD4 (e and g [ii-iii]), IL-21 (f and g [iii]), or eosin (g [i]) visualized with Fast Red (d, e, and g) and/or DAB (d, f, and g [iii]) and counterstained with hematoxylin. High magnification images are shown on right. Arrows indicate positive staining. Bars, 50 μm. 600 IL-21 promotes brain injury after stroke | Clarkson et al.
- 53. Br ief Defini t ive Repor t T cells that can secrete IL-21 were detected within the CSF-filled subarachnoid and perivascular spaces during cere-bral infarction in humans. Neuronal cells express IL-21 receptor and up-regulate autophagy genes in response to IL-21 RT-PCR analysis of primary mouse neurons and murine neu-ronal cell lines (Neuro2A) indicated that IL-21R expression was higher on neuronal cells than on other brain cells, includ-ing astrocytes and endothelial cells (Fig. 5, a and c). This is con-sistent with another report where in situ hybridization detected neuron-restricted IL-21R expression in inflamed human brain tissue (Tzartos et al., 2011). Moreover, treating Neuro2a cells with IL-21 after in vitro oxygen glucose deprivation (OGD) significantly increased cell death as measured by XTT cell patients with acute and chronic stroke lesions. In acute infarcts, rare CD4+ T cells were found in the necrotic brain parenchyma (Fig. 4 g, ii, arrows), which was predominantly infiltrated by foamy macrophages (Fig. 4 g, i). In contrast, CD4+ T cells were consistently found within the Virchow Robins space of vessels bordering acute infarcts (not depicted) and in the subarachnoid space adjacent to meningeal vessels (arrows, Fig. 4 e, i). IL-21 staining was limited almost exclusively to these perivascular spaces. Compared with control stained tissue (Fig. 4 d, i), anti– IL-21 staining labeled cells extensively in the subarachnoid space of deep sulci penetrating the infarcted tissue—showing a similar distribution to CD4+ cells in serial sections (arrows, Fig. 4 f ). Additionally, double staining with antibodies for CD4 and IL-21 revealed the presence of CD4+ IL-21+ cells in this perivascular niche (arrow, Fig. 4 g, iii). In summary, CD4+ Figure 5. IL-21 promotes autophagy expres-sion in neuronal cells after hypoxia/ischemia. (a) Il21r mRNA expression relative to GAPDH expression levels is shown in normoxic and hy-poxic primary mouse neurons after OGD or con-trol treatment. (b) Viability of Neuro2A cells treated with the indicated doses of IL-21 after OGD. (c) Il21r mRNA expression relative to GAPDH in neuronal (Neura2A), astrocytic, and endothelial cell lines (MB114) expressed relative to BMDC expression. (d) ATG6 expression in pri-mary neurons treated with PBS, etoposide, or 32–256 ng/ml rIL-21 for 4 h after 1–2 h oxygen glucose deprivation as measured by RT-PCR. Cells treated in triplicate. (e) Number of ATG6+ cells per field in the same regions of WT and IL-21 KO mouse brains after tMCAO as assessed by im-mune staining (n = 3 mice per group). Arrows indicate ATG-6+ cells in periinfarcted brain tissue of WT and IL-21 KO mice. Bars, 100 μm. Data are representative of two independent experi-ments. *, P 0.05; **, P 0.01; ***, P 0.001; ****, P 0.0001 by Student’s t test (single com-parison) or one-way ANOVA (multiple compari-sons). Error bars indicate SEM. JEM Vol. 211, No. 4 601
- 54. of the superiorthyroid artery, the ECA was dissected distally and coagulated along with the terminal lingual and maxillary artery branches. The internal ca-rotid artery (ICA) was isolated, and the extracranial branch of the ICA was then dissected and ligated. A standardized polyamide resin glue-coated 6.0 nylon monofilament (3021910; Doccol Corp) was introduced into the ECA lumen, and then advanced 9–9.5 mm in the ICA lumen to block MCA blood flow. During the entire procedure, mouse body temperature was kept between 37 and 38°C with a heating pad. The suture was withdrawn 60 min after occlu-sion. The incision was closed, and the mice underwent recovery. Infarction size measurement. After 24 h reperfusion, mice were sacrificed and brains were removed and frozen at 80°C for 5 min. 2-mm coronal slices were made with a rodent brain matrix (Ted Pella, Inc.). The sections were stained for 20 min at 37°C with 2% TTC (Sigma-Aldrich). Infarction volume was calculated with the method reported by Swanson et al. (1990) to compensate for brain swelling in the ischemic hemisphere. In brief, the sec-tions were scanned, and the infarction area in each section was calculated by subtracting the noninfarct area of the ipsilateral side from the area of the contralateral side with National Institutes of Health image analysis software, ImageJ. Infarction areas on each section were summed and multiplied by sec-tion thickness to give the total infarction volume. Gene array and RT-PCR. Ipsilateral brain hemispheres were dissected and stored in RNAlater (QIAGEN) at 4°C until further use. Total RNA was ex-tracted and purified with RNeasy Protect Mini kit (QIAGEN) according to the manufacturer’s instructions. Purified RNA samples were analyzed by GeArray S Serious Mouse Autoimmune and Inflammatory Gene array (SuperArray; Bioscience Corporation). Results from GeArray were filtered for genes with spot intensities higher than the mean local background of the bottom 75% of nonbleeding spots. For RT-PCR, 1 μg total RNA from each sample was reverse transcribed using SuperScript II first strand cDNA syn-thesis kit (Invitrogen). RT-PCR was performed on a Smart Cycler (Model SC 100–1; Cepheid) using IL-21 TaqMan gene expression assay (Mm00517640_m1; Applied Biosystems), RT2 qPCR Primer assay for mouse Becn1 (PPM32434A; SABiosciences), or RT2 qPCR Primer Assay for Mouse IL21r (PPM03762A; SABiosciences). The data were normalized to an internal ref-erence gene, GAPDH. Mononuclear cell isolation and flow cytometry. Brains were removed from perfused animals, weighed, minced, transferred to Medicon inserts, and ground in a MediMachine (BD) for 20–30 s. The cell suspension was washed with HBSS, and cells were resuspended in 70% Percoll (Pharmacia) and over-laid with 30% Percoll. The gradient was centrifuged at 2,250 g for 30 min at 4°C without brake. The interface was removed and washed once for further analysis. CD11b-positive and -negative fractions were isolated using Imag anti-CD11b magnetic particles (BD), following the manufacturer’s protocol. A total of 106 cells were incubated for 30 min on ice with saturating concen-trations of labeled antibodies with 40 μg/ml unlabeled 2.4G2 mAb to block binding to Fc receptors, and then washed 3 times with 1% BSA in PBS. Single-cell suspensions from various tissues were cultured at 37°C in 10% FBS in RPMI 1640 media supplemented with GolgiStop (BD) in the pres-ence of either phorbol myristate acetate (50 ng/ml) and ionomycin (1 μg/ml) for 5 h. After surface staining with antibodies against CD4, NK1.1, and TCR, cell suspensions were fixed and permeabilized by Cytofix/Cy-toperm solution (BD), followed by staining with anti–IL-21 antibodies. Fluorochrome-labeled antibodies against CD45, CD11b, Ly6c, B220, CD4, CD8a, NK1.1, IFN-, and appropriate isotype controls were purchased from BD. Fluorochrome-labeled antibody against IL-21 and TCR was pur-chased from eBioscience. Cell staining was acquired on a FACSCalibur or LSRII (BD) and analyzed with FlowJo (Tree Star) software version 5.4.5. Neurofunctional assessment. Neuromuscular coordination was assessed by grip strength test, as previously described (Kleinschnitz et al., 2010). For this test, mice were placed on a horizontal string midway between two supports. Mice were scored from 0 to 5 as follows: 0, falls off within 2 s; 1, hangs on with viability assay (Fig. 5 b). In subsequent studies we found that treatment of primary neurons with IL-21 up-regulated mRNA levels of the autophagy associated gene ATG6 (Fig. 5 d). These data suggest that IL-21 could directly affect neuronal autoph-agy during ischemic injury, which has been implicated in neu-ronal death in infarcted and periinfarcted brain tissue. Thus, we stained WT and IL-21 KO postischemic brain tissues for ATG6. We observed significantly fewer ATG6+ cells in infarcted brain tissue of IL-21 KO mice compared with WT, suggesting that IL-21 may contribute to increased cerebral autophagy after stroke (Fig. 5 e). In conclusion, we implicate IL-21 as a lymphocyte-derived factor with a pronounced effect on brain injury after focal isch-emia in mice. We also present data demonstrating that IL-21– producing CD4+ T cells are present in the brain of patients with acute stroke. These data warrant investigation of the thera-peutic potential of IL-21–modifying treatments in isolation and combination with current anti-thrombotic treatments for ischemic stroke. MATERIALS AND METHODS Ethics statement. C57BL/6 WT mice were obtained from The Jackson Laboratory. IL-21–deficient mice (IL-21tm1Lex) were purchased from the Mu-tant Mouse Regional Resources Center. All mice underwent 1 h tMCAO and 24 h reperfusion. All animal procedures used in this study were con-ducted in strict compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Wisconsin Center for Health Sciences Research Animal Care Committee. All mice (25 g) were anesthetized with 5% halothane for induction and 1.0% halothane for maintenance vaporized in N2O and O2 (3:2), and all ef-forts were made to minimize suffering. Regional cerebral blood flow (rCBF) measurement. Changes in rCBF at the surface of the left cortex were recorded using a blood perfusion monitor (Laserflo BPM2; Vasamedics) with a fiber optic probe (0.7 mm diam). The tip of the probe was fixed with glue on the skull over the core area supplied by the MCA (2 mm posterior and 6 mm lateral from bregma). Changes in rCBF after MCAO were recorded as a percentage of the baseline value. Mice included in these investigations had 80% relative decrease in rCBF during MCAO. Investigation of intracranial vasculature. WT and IL-21 KO mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). After thoracotomy was performed, a cannula was introduced into the ascend-ing aorta through the left ventricle. Transcardial perfusion fixation was per-formed with 2 ml saline and 2 ml of 3.7% formaldehyde. Carbon lampblack (C198-500; Thermo Fisher Scientific) in an equal volume of 20% gelatin in ddH2O (1 ml) was injected through the cannula. The brains were removed and fixed in 4% PFA overnight at 4°C. Posterior communicating arteries (PComA) connect vertebrobasilar arterial system to the Circle of Willis and internal carotid arteries, and its development affects brain sensitivity to isch-emia among different mouse strains (Barone et al., 1993). Development of PComA in both hemispheres was examined and graded on a scale of 0–3, as reported previously (Majid et al., 2000). 0, no connection between anterior and posterior circulation; 1, anastomosis in capillary phase (present but poorly developed); 2, small truncal PComA; 3, truncal PComA. Focal ischemia model. Focal cerebral ischemia in mice was induced by oc-clusion of the left MCA, as described previously (Longa et al., 1989). Operators performing surgeries were masked to experimental groups. In brief, the left common carotid artery was exposed, and the occipital artery branches of the external carotid artery (ECA) were isolated and coagulated. After coagulation 602 IL-21 promotes brain injury after stroke | Clarkson et al.
- 55. Br ief Defini t ive Repor t Statistical analyses and quality standards. All surgeries were performed in a blinded manner by a third party and measurements masked where possi-ble. Infarct volume measurements from TTC stained sections were averaged from two to three independent blinded observers. Based on power calcula-tions, n = 3–10 sex- and age-matched mice were used for each experiment and group assignment was randomized. Among animals receiving MCAO procedure, 86.5% of WT mice, 93.5% of IL-21KO mice, and 100% of RAG2KO mice were included in analysis. Mice were excluded due to prema-ture death (13.5% of WT mice, 3.2% of IL-21 KO mice) or vessel variation (3.2% of IL-21KO mice). Results are given as means ±1 SD. Multiple com-parisons were made using one-way ANOVA. Where appropriate, two-tailed Student’s t test analysis was used for comparing measures made between two groups. For comparison of RT-PCR data, nonparametric Mann-Whitney rank sum analysis was used. P-values 0.05 were considered significant. Online supplemental material. Video 1 shows groups of WT C57BL6 mice treated with 500 μg IL-21R.Fc or PBS control via i.p. injection. On-line supplemental material is available at http://www.jem.org/cgi/content/ full/jem.20131377/DC1. We thank Satoshi Kinoshita for expert histopathology services, Guoqing Song for assisting in the surgical procedures, Dr. Wenda Gao for providing reagents and protocols for the purification of IL-21R.Fc protein, and members of our laboratory for helpful discussions and constructive criticisms of this work. We also thank Khen Macvilay and Sinarack Macvilay for their expertise provided for cytofluorimetry and immunohistochemistry studies and Samuel (Joe) Ollar for assisting in the OGD procedure. This work was supported by awards from the American Heart Association (pre-doctoral fellowship #12PRE12060020 to B.D.S. Clarkson) and the National Institutes of Health (NS037570 and NS076946 to ZF, AI048087 to M.S. Salamat, and AI068730 to J.D. Lambris). The authors have no competing financial interests. Submitted: 1 July 2013 Accepted: 24 February 2014 REFERENCES Baan, C.C., A.H. Balk, I.E. Dijke, S.S. Korevaar, A.M. Peeters, R.P. de Kuiper, M. Klepper, P.E. Zondervan, L.A. Maat, and W. Weimar. 2007. Interleukin-21: an interleukin-2 dependent player in rejection processes. Transplantation. 83:1485–1492. http://dx.doi.org/10.1097/01.tp.0000264998.23349.54 Barone, F.C., D.J. Knudsen, A.H. Nelson, G.Z. Feuerstein, and R.N. Willette. 1993. Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J. Cereb. Blood Flow Metab. 13:683– 692. http://dx.doi.org/10.1038/jcbfm.1993.87 Barone, F.C., B. Arvin, R.F. White, A. Miller, C.L. Webb, R.N. Willette, P.G. Lysko, and G.Z. Feuerstein. 1997. Tumor necrosis factor-alpha. A mediator of focal ischemic brain injury. Stroke. 28:1233–1244. http://dx.doi.org/ 10.1161/01.STR.28.6.1233 Battaglia, A., A. 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Hecker, W. Padberg, and V. Grau. 2009. Expression of interleukin-21, interleukin-21 receptor alpha and related type I cytokines by intravascular graft leukocytes during acute renal allograft forepaw(s); 2, hangs on with forepaws and moves laterally on string; 3, hangs onto string with forepaws and hindpaw(s); 4, hangs onto string with forepaws, hindpaw(s) and tail; 5, escape to supports. Mice were allowed to rest between trials. Scores for each mouse were determined by averaging 5–10 trials (each lasting 15 s). Global neurological deficit was determined by a modified Bed-erson scoring system: 0, no deficit; 1, forelimb flexion; 2, unidirectional circling after being lifted by tail; 3, spontaneous unidirectional circling; 4, longitudinal rolling upon being lifted by tail; 5, spontaneous longitudinal rolling. Generation of IL-21 receptor Fc fusion protein. Chinese hamster ovary cell line (Korn et al., 2007) expressing the extracellular domain (aa 20–236) of mouse IL-21R fused to the fragment crystallizable (Fc) portion of human IgG4 (IL-21R.Fc) were maintained in UltraCHO (BioWhittaker). IL-21R. Ig was purified from the culture supernatant by passage through a protein G–Sepharose column and concentrated by ultrafiltration. Concentration was determined spectrophotometrically. Purity and molecular weight were con-firmed by sodium dodecyl-sulfate PAGE and human-IgG4 ELISA (eBiosci-ence) following the manufacturer’s instructions. The IL-21R.Fc reagent was tested in vitro for its ability to suppress IL-21–induced T cell proliferation. Immunohistochemistry. Paraffin-embedded postmortem brain tissue sec-tions from individuals with acute and chronic stroke lesions were obtained from the Neuropathology Laboratory of the University of Wisconsin De-partment of Pathology. After rehydration and deparaffinization, sections un-derwent heat-induced antigen retrieval in 10 mM sodium citrate, pH 6.0, for surface antigens or Tris-EDTA (10 mM/1 mM) with 0.05% Tween-20 for intracellular antigens. Sections were blocked for 30 min with secondary serum (10% in Tris-buffered saline) and then stained with primary antibodies, 0.5% chicken anti–IL-21 (Lifespan Biosciences) or prediluted mouse anti- CD4 ([1F6]; ab17131; Abcam) for 1–2 h at 37°C or overnight at 4°C. Nor-mal primary sera (5–10%) were used for negative control. After several washes, secondary antibodies (biotin-labeled goat anti–chicken or biotin-labeled goat anti–mouse; Vector Laboratories) were applied to sections and incubated for 2 h at room temperature. Staining was developed using the VECTA-STAIN ABC-HRP kit (Vector Laboratories) with diaminobenzidine sub-strate (BD) or streptavidin-alkaline phosphatase with Fast Red substrate (Laboratory Vision), following the manufacturer’s instructions. Slides were lightly counterstained with hematoxylin, rinsed with running tap water, and mounted. For frozen mouse sections WT and IL-21 KO mice underwent 1-h tMCAO and 1–7-d reperfusion. Brains were perfused with PBS and 3% for-malin, embedded in OCT, and cut into 8-μm frozen sections for immunohistochemistry. Frozen sections were thawed for 10 min at room temperature and blocked with 5% goat serum solution in PBS for 15 min. Sections were stained with rabbit polyclonal antibodies for ATG6 for 1 h at room tempera-ture, followed by phycoerythrin-labeled goat anti–rabbit IgG (Santa Cruz Biotechnology, Inc.). Images were acquired on a BX40 microscope equipped with a Q-Color 3 camera using Q-Capture software (Olympus). Digital images were processed and analyzed using Photoshop CS4 software (Adobe). Color balance, brightness, and contrast settings were manipulated to generate final images. All changes were applied equally to entire image. Neuronal cell oxygen glucose deprivation. Primary neuronal cultures derived from embryonic day 14–18 mouse cortices were grown to 80% con-fluency in neural basal media supplemented with B27 (2%) and penicillin/ streptomycin (1%), as previously described (Kintner et al., 2010). Astrocytic and microglial contamination was excluded based on the absence of GFAP+ and CD11b+ cells when stained by immunocytochemistry. For OGD, media was replaced with neural basal media with or without glucose and placed in a hypoxic chamber or under normoxic conditions for 2 h at 37°C. Afterward, cells were lysed and mRNA isolated using RNeasy mini kit (QIAGEN). For XTT viability assay, Neuro2A underwent OGD and were treated with dose curve of IL-21 immediately after return to normoxic media and incubated for 4 h at 37°C. XTT labeling mixture (50 μl per well; Roche) was added ac-cording to manufacturer’s instructions, and at18 h fluorescence was read on a GENious Microplate reader (Tecan). Cell number was calculated using a standard curve of known untreated cells kept under normoxic conditions. JEM Vol. 211, No. 4 603
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- 57. The Rockefeller UniversityPress $30.00 J. Exp. Med. 2014 Vol. 211 No. 10 1937-1945 www.jem.org/cgi/doi/10.1084/jem.20140214 Br ief Def ini t ive Repor t 1937 Approximately 50% of (frontotemporal demen-tia) FTLD cases are characterized by cellular aggregation and mislocalization of TDP-43 (i.e., FTLD-TDP). TDP-43 normally localizes to the nucleus and regulates transcriptional con-trol, splicing, and RNA processing (Sephton et al., 2011; Polymenidou et al., 2011). In FTLD-TDP, nuclear depletion of TDP-43 occurs, often in neurons containing cytoplasmic TDP-43 ag-gregates (Neumann et al., 2006). The mecha-nisms underlying TDP-43 mislocalization in FTLD have not been characterized, and whether TDP-43 mislocalization plays a causal role in neurodegeneration remains controversial (Lee et al., 2012). FTLD has sporadic and familial forms. Mutations in the GRN gene that cause progranulin haploinsufficiency are a common cause of familial FTLD-TDP (Baker et al., 2006). Aged Grn-KO mice exhibit FTD-like behav-ioral abnormalities but lack TDP-43 mislocal-ization or neurodegeneration in cortical regions (Ahmed et al., 2010; Yin et al., 2010; Martens et al., 2012). Retinal abnormalities are documented in Alzheimer’s disease (AD), progressive supranu-clear palsy (PSP), Parkinson’s disease, and mul-tiple systems atrophy (Hinton et al., 1986; Bayer et al., 2002; Tamura et al., 2006; Paquet et al., 2007; Albrecht et al., 2012; Helmer et al., 2013). CORRESPONDENCE Ari Gree: agreen@ucsf.edu OR Li Gan: lgan@gladstone.ucsf.edu Abbreviations used: AD, Alzheimer’s disease; CDR, clinical disease rating; FTD, frontotemporal dementia; FTLD, frontotemporal lobar degeneration; GCC, ganglion cell complex; GRN, progranulin; INL, inner nuclear layer; OCT, optical coherence tomography; ONL, outer nuclear layer; PD, Parkinson’s disease; PSP, progressive supranuclear palsy; RGC, retinal ganglion cell; RNFL, retinal nerve fiber layer; PhNR, photopic negative response; TDP-43, TAR DNA/ RNA binding protein 43. Early retinal neurodegeneration and impaired Ran-mediated nuclear import of TDP-43 in progranulin-deficient FTLD Michael E. Ward,1,2 Alice Taubes,1 Robert Chen,2 Bruce L. Miller,2 Chantelle F. Sephton,5 Jeffrey M. Gelfand,2 Sakura Minami,1 John Boscardin,3 Lauren Herl Martens,4 William W. Seeley,2 Gang Yu,5 Joachim Herz,5 Anthony J. Filiano,6 Andrew E. Arrant,6 Erik D. Roberson,6 Timothy W. Kraft,7 Robert V. Farese, Jr.,4 Ari Green,2 and Li Gan1,2 1Gladstone Institute of Neurological Diseases, 2Department of Neurology, 3Department of Medicine, 4Gladstone Institute of Cardiovascular Disease, University of California, San Franciso, San Francisco, CA 94158 5Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390 6Departments of Neurology and Neurobiology and 7Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294 Frontotemporal dementia (FTD) is the most common cause of dementia in people under 60 yr of age and is pathologically associated with mislocalization of TAR DNA/RNA binding protein 43 (TDP-43) in approximately half of cases (FLTD-TDP). Mutations in the gene encoding progranulin (GRN), which lead to reduced progranulin levels, are a signifi-cant cause of familial FTLD-TDP. Grn-KO mice were developed as an FTLD model, but lack cortical TDP-43 mislocalization and neurodegeneration. Here, we report retinal thinning as an early disease phenotype in humans with GRN mutations that precedes dementia onset and an age-dependent retinal neurodegenerative phenotype in Grn-KO mice. Retinal neuron loss in Grn-KO mice is preceded by nuclear depletion of TDP-43 and accompanied by reduced expression of the small GTPase Ran, which is a master regulator of nuclear import required for nuclear localization of TDP-43. In addition, TDP-43 regulates Ran expression, likely via binding to its 3-UTR. Augmented expression of Ran in progranulin-deficient neurons restores nuclear TDP-43 levels and improves their survival. Our findings establish retinal neurodegeneration as a new phenotype in progranulin-deficient FTLD, and suggest a pathological loop involving reciprocal loss of Ran and nuclear TDP-43 as an underlying mechanism. © 2014 Ward et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial– Share Alike 3.0 Unported license, as described at http://creativecommons .org/licenses/by-nc-sa/3.0/).
- 58. RESULTS AND DISCUSSION Early retinal abnormalities in humans with GRN mutations and Grn-KO mice Because retinal neuron loss occurs in other neurodegenerative diseases, we suspected that retinal neurons could be a vulnerable neuronal population in humans with progranulin haploinsufficiency secondary to GRN mutations. Using optical coher-ence tomography (OCT), we measured retinal nerve fiber layer (RNFL) thickness and macular volume in living human control subjects and subjects with GRN mutations. The RNFL consti-tutes the axonal compartment of retinal ganglion cells (RGCs) and, as such, is a surrogate measurement of RGC number. Mac-ular volume is a combined measurement of all of the layers of the retina within the macula. We observed significant reductions in RNFL thickness and macular volume in subjects with GRN Due to the clinical accessibility of the retina, new retinal imaging techniques under development hold promise as potential diagnostic and prognostic modalities for neurode-generative diseases (Koronyo-Hamaoui et al., 2011). However, whether retinal abnormalities are an early or late disease phe-nomenon has not been established. Here, we identify retinal neurodegeneration as a novel disease-related phenotype in human subjects with GRN mutations before clinical symp-toms of dementia. In Grn-KO mouse, retinal neuronal loss is preceded by depletion of nuclear TDP-43. We further explore the role of Ran, a central regulator of nuclear traf-ficking, in TDP-43 nuclear depletion and degeneration in Grn-KO neurons. Our findings suggest a novel relationship between TDP-43 and Ran-mediated nuclear trafficking in FTLD pathogenesis. Figure 1. Progranulin deficiency causes retinal neuron loss in humans with GRN mutations and retinal neuron loss/dysfunction in a progranulin-deficient mouse model of familial FTLD. (A and B) RNFL thinning (A) and macular volume loss (B) occur in humans with progranulin haploinsufficiency caused by GRN mutations and precede dementia onset. Each dot represents value of an individual eye, and bars represent median values. Asymptomatic GRN-mutation carriers (CDR = 0) are shown in blue and symptomatic GRN mutation carriers (CDR score ≥ 0.5) are shown in red. Control age- and sex-matched subjects are represented by gray dots. (C–F) Macular ganglion cell loss occurs in GRN mutation carriers and precedes dementia onset. An automated segmentation algorithm (C) was used to determine the volumes of GCC (D), INL (E), and ONL (F) in the maculae of control and GRN mutation carriers (D–F). Results represent a single cohort of n = 24 control subjects and 12 GRN mutation carrier subjects (7 asymptomatic, 5 symptomatic), and p-values were gen-erated via mixed-effects linear regression analyses. (G) Progranulin expression occurs in the GCL and photoreceptor inner and outer segments (IS, OS) of mouse retinas. Immunostaining of progranulin and DAPI staining of nuclei are shown in a representative retinal cross section. (H) RNFL thinning and loss of inner retinal neurons shown in HE-stained retinal cross sections from 18-mo-old Grn KO mice. Representative sections equidistant to the optic nerve are shown. (I) Quantification of RNFL layer thickness. n = 5–7 mice/age/genotype; *, P 0.05, one-way ANOVA with Tukey’s post-hoc analysis, 2 independent experiments. (J) Loss of Neu-N–positive neurons in the GCL of 18-mo Grn KO retinas. Neu-N–positive cells in the GCL were quantified in sections equidistant to the optic nerve head. n = 5–7 mice/age/genotype; **, P 0.01, one-way ANOVA with Tukey’s post-hoc analysis, 2 independent experiments. (K) Impaired light-evoked RGC electrophysiological responses in aged Grn KO mice. Electroretinograms (ERGs) were performed on 12- and 18-mo-old mice, and the ampli-tude of the photopic-negative response (PhNR, a RGC-specific waveform) was quantified. n = 6 mice/age/genotype; ***, P 0.001 at 18 mo of age via re-peated measures two-way ANOVA with Bonferroni’s multiple comparison test; 2 independent experiments. Bars: (C, G, and H) 50 μm. 1938 Retinal thinning and TDP-43 mislocalization in FTLD | Ward et al.
- 59. Br ief Defini t ive Repor t in the brain (Ahmed et al., 2010; Yin et al., 2010), we ob-served progressive, substantial thinning of the RNFL and a loss of ganglion cell layer (GCL) cells in 18-mo-old Grn-KO mice (Fig. 1, H–J). In agreement with the pathological altera-tions, electrophysiological abnormalities in Grn-KO mouse retinas paralleled GCL neuron loss. At 18 mo, but not 12 mo, of age, Grn-KO mice had substantially reduced amplitudes of photopic negative responses (PhNRs), a measure of RGC function (Fig. 1 K). Amplitudes of a- and b-wave responses were also significantly reduced in an age-dependent manner in Grn-KO mice, indicating additional dysfunction of inner nuclear layer neurons and photoreceptors (unpublished data). Nuclear depletion of TDP-43 in Grn-KO retinal neurons precedes neurodegeneration Loss of nuclear TDP-43 is commonly observed in postmor-tem brain tissue from patients with FTLD-TDP (Neumann et al., 2006; Davidson et al., 2007), including FTLD associated with GRN mutations (Fig. 2 A). However, the mechanism of nuclear depletion of TDP-43 in FTLD is unknown, and it remains unclear if loss of nuclear TDP-43 plays a causal role in FTLD pathogenesis. At 18 mo of age, levels of nuclear TDP-43 mutations compared with controls, indicating that retinal neu-ron loss occurred in these subjects (Fig. 1, A and B; and Fig. S1). A substantial number of the GRN mutation carriers enrolled in our trial (7/12) were cognitively asymptomatic. Intrigu-ingly, we found that this subgroup of asymptomatic GRN mutation carriers also had significant reductions in RNFL thickness and macular volume (Fig. 1, A and B). We then ana-lyzed individual neuronal layers of the macula via an auto-mated segmentation algorithm, to determine the volumes of the ganglion cell complex (GCC), inner nuclear layer (INL), and outer nuclear layer (ONL) in the maculae of control and GRN-mutation carriers (Fig. 1 C). Significant thinning of the GCC and INL, but not ONL, was observed in GRN muta-tion carriers (Fig. 1, D–F). The volume of GCC was signifi-cantly reduced even in asymptomatic carriers, further implicating RGC loss as an early neurodegenerative phenotype in sub-jects with progranulin deficiency (Fig. 1 D). We then determined if a similar phenotype occurred in Grn-KO mice. Total retinal progranulin expression levels were similar to those in the brain, based on ELISA (unpublished data), with prominent expression in RGCs and photorecep-tors (Fig. 1 G). Despite a lack of significant neurodegeneration Figure 2. Nuclear depletion of TDP-43 occurs in retinal neurons before neurodegeneration. (A) Post-mortem brain tissue from a human GRN mutation car-rier was co-stained with the nuclear marker DAPI (blue) and an anti–TDP-43 antibody (green). Normal TDP-43 distribution is still observed in some neurons (arrow-head), but other neurons exhibit nuclear depletion of TDP43 (arrow) with or without apparent cytoplasmic inclusions. Bar, 10 μm. (B) Immunofluorescence confo-cal microscopy of TDP-43 in 18-mo-old retinal GCL neurons. Brightness of the TDP-43 channel was in-creased in insets to highlight loss of nuclear TDP-43 in Grn KO neurons. Bar, 1 μm. (C) Scatter plot of nuclear and cytoplasmic intensities of TDP-43 in GCL neurons of 18-mo-old Grn KO mice. n = 121–174 cells from 6 mice/genotype; ***, P 0.001, mixed-effects multivari-ate linear regression mode; 2 independent experiments. Bars represent median values. (D) Significant reduction in the nuclear/cytoplasmic TDP-43 ratio was observed in 12-mo-old Grn-KO GCL neurons (**, P 0.01) and 18-mo-old Grn-KO GCL neurons (**, P 0.01). n = 217–357 cells from 5–7 mice/age/genotype, mixed-effects multivariable linear regression model; 2 independent experiments. Mean ± SEM is shown. JEM Vol. 211, No. 10 1939
- 60. TDP-43 levels correlatedwith those of nuclear Ran (Fig. 3 C). Moreover, in the inferior frontal gyrus of three patients with FTLD-TDP due to GRN mutations, we found a significant correlation between nuclear depletion of TDP-43 and Ran (Fig. 3, D and E). To understand the mechanisms underlying the intimate correlation between nuclear TDP-43 and Ran, we next as-sessed whether Ran mRNA is altered in the brains of FTLD-TDP- 43 patients, in which TDP-43 is mislocalized. By mining an existing mRNA-expression database comparing healthy control versus GRN-mutation-carrying FTD sub-jects (Chen-Plotkin et al., 2008), we found that cortical Ran expression was reduced by 60% in human subjects carrying a GRN mutation (P = 0.04). As TDP-43 regulates the expression of thousands of genes, in many cases by binding directly to mRNAs and altering their stability (Polymenidou et al., 2011), we explored the possibility that Ran mRNA is a substrate of TDP-43. Indeed, analyses of a published unbiased screen of the TDP-43–RNA interactions (Sephton et al., 2011) revealed that TDP-43 binds to the 3 UTR of Ran mRNA (Fig. 4 A). Moreover, inhibiting TDP-43 expression by shRNA-mediated knockdown significantly reduced levels of Ran mRNA (Fig. 4 B) and protein (Fig. 4, C–D) in N2A cells. Ran mRNA levels were also reduced in retinas of aged Grn KO mouse (Fig. 4 E), consistent with our observations of nuclear depletion were strikingly reduced in Grn-KO retinal GCL neurons, whereas levels of cytoplasmic TDP-43 were unchanged (Fig. 2, B and C). Depletion of nuclear TDP-43 also occurred in 12-mo-old Grn-KO mice, before significant GCL neuron loss (Fig. 2 D). Interestingly, neither nuclear nor cytoplasmic TDP-43 inclusions were found in the 100 Grn-KO GCL neurons we examined (Fig. 2 B). Thus, in progranulin-deficient FTLD-TDP, nuclear depletion of TDP-43 and neurodegeneration can occur independent of cytoplasmic TDP-43 accumulation/aggregation. These results are consistent with observations that TDP-43, especially nuclear TDP-43, is re-quired for neuron survival (Wegorzewska and Baloh, 2011; Igaz et al., 2011; Arnold et al., 2013). TDP-43 regulates Ran mRNA levels and requires Ran for nuclear localization We then explored how nuclear clearing of TDP-43 occurs in FTLD. The small GTPase Ran is a master regulator of nuclear transport (Melchior et al., 1995), and Ran accessory proteins are necessary for nuclear TDP-43 localization (Nishimura et al., 2010). We hypothesized that Ran expression might be altered in our retinal FTLD model and contribute to nuclear TDP-43 depletion. Indeed, nuclear Ran was significantly de-pleted in Grn-KO GCL neurons (Fig. 3, A and B), and nuclear Figure 3. Nuclear clearing of TDP-43 and Ran are pathologically associated in FTLD-TDP. (A) 18-mo-old GCL neurons from WT and Grn-KO retinas were co-stained for TDP-43 and Ran. Nuclei were labeled with DAPI. (B) Nuclear Ran levels in 18-mo-old GCL neurons. n = 165–278 cells from 6 mice/gen-otype; *, P = 0.019, linear regression model; 2 independent experiments. Scatter plot of individual cell intensities with medians shown. (C) Nuclear Ran and TDP-43 intensities are correlated in Grn-KO GCL neurons. Each dot represents a single cell. n = 165 cells from 6 Grn-KO mice; r = 0.8963; P 0.001, Spear-man’s rho; 2 independent experiments. (D) Immunofluorescence co-staining of GRN mutant human cortex shows depletion of Ran and TDP-43 in the same neuron (noted with an arrow; compare to neurons with high lev-els of TDP-43 and Ran [arrowhead]). (E) TDP-43 and Ran levels correlate in cortical neurons from human GRN-mutation carriers. Shown are the correlation analyses of nuclear Ran and TDP-43 intensities of individual neurons from post-mortem brain. n = 111–141 cells from each of 3 subjects;, r = 0.56; P 0.001. The serum progranulin levels were 19.3–21.2 ng/ml for R493X carrier (control patients: 41.3 ± 15.5 ng/ml). Spearman’s rho. Bars: 2 μm (A), 10 μm (D). 1940 Retinal thinning and TDP-43 mislocalization in FTLD | Ward et al.
- 61. Br ief Defini t ive Repor t Figure 4. Maintenance of functional TDP-43 and Ran by an interdependent feedback loop and improved survival of Grn-KO neurons by exogenous Ran expression. (A) Snapshot of unique reads from the TDP-43 RIP library mapped to the Ran gene shown. Reads mapped to the 3-UTR of Ran indicate TDP-43 binding. No reads mapped to the Ran gene from the Ctrl RIP library. (B) TDP-43 knockdown in N2A cells results in a reduction in steady-state Ran mRNA levels, as measured by Q-PCR. n = 12 wells/group; ***, P 0.001, Student’s t test; 3 independent experiments. (C) Representative Western blot showing reduced Ran protein levels after TDP-43 knockdown. (D) Quantification of (C). n = 9 wells/group, ***, P 0.001, Student’s t test; 2 independent experiments). (E) Ran mRNA expression is reduced in aged Grn KO retinas. Q-PCR results from homogenized whole retinas from 10–12-mo-old mice shown. n = 15–16 mice/genotype; *, P = 0.014; Stu-dent’s t test; 2 independent experiments. Bars, 10 μm. (F–G) Ran is necessary for nuclear localization of TDP-43. (F) Representative images showing subcellular localization of TDP-43 in cortical neurons cotransfected with TDP-43-GFP and either empty vector, mCherry-Ran, mCherry-RanQ69L, or mCherry-RanT24N. TDP- 43-GFP is present in the nuclei of neurons transfected with mCherry and mCherry-Ran, but is significantly reduced in nuclei of neurons transfected with either of the Ran mutants (arrowheads, dashed lines). (G) Quantification of the ratios of nuclear/cytoplasmic TDP-43-GFP. n = 21 cells/transfection; ***, P 0.001, one-way ANOVA with Tukey’s post-hoc analysis; 2 independent experiments. (H–I) N2A cells were transfected with siRNA against Grn. Levels of Ran (H) and TDP-43 (I) were quantified via Western blot 7 d after transfection. n = 6 wells/group; **, P = 0.002 (TDP-43); **, P = 0.006 (Ran); 2 independent experiments. (J) Living wild-type or Grn KO primary neurons transfected with GFP + empty vector (control) or GFP + Ran were imaged longitudinally by automated microscopy at 24–48-h intervals for 7–9 d. Kaplan-Meir survival analysis was used to create cumulative risk of death functions for each population of transfected neurons. ***, P 0.001 (log-rank test); n = 423 neurons (WT Ctrl), 518 neurons (KO Ctrl), 427 neurons (WT Ran), and 463 neurons (KO Ran); 3 independent experiments pooled. (K) Primary cortical neurons from wild-type or Grn KO mice were transduced with AAV-GFP (control) or AAV-GFP-P2A-Ran. 1 wk later, neurons were fixed and processed for TDP-43 immunostaining. Nuclear TDP-43 levels were quantified via Volocity. n = 101–478 cells imaged from 6–12 wells of a 96-well dish; *, P 0.05 (mixed-effects multivariate linear regression model); 3 independent experiments. Means ± SEM shown (B, D, E, G–I, and K). of TDP-43 in the retinas of Grn KO mice (Fig. 3 A). These find-ings suggest that nuclear depletion of TDP-43 in progranulin-deficient neurons could down-regulate Ran. Ran is required for nuclear transport of the majority of pro-teins that shuttle between the nucleus and cytoplasm (Stewart, 2007). To determine if inactivation of Ran is sufficient to cause JEM Vol. 211, No. 10 1941
- 62. depletion of nuclearTDP-43 and Ran as a potential mecha-nism of neurodegeneration in FTLD-TDP. In this model, loss of function of TDP-43 via nuclear depletion contributes to neurodegeneration and can occur without cytoplasmic TDP-43 aggregation. Loss of Ran expression, potentially in combination with other associated nuclear transport factors, impairs transport of TDP-43 to the nucleus (Nishimura et al., 2010). In turn, loss of nuclear TDP-43 lowers Ran levels, which could further deplete nuclear TDP-43. These data may point toward novel therapeutic strategies aimed at re-storing nucleocytoplasmic transport as a means to improve neuronal survival in neurodegenerative diseases. MATERIALS AND METHODS Human subjects. Subjects enrolled through the UCSF Memory and Aging Center in whom GRN mutations were identified and age- and sex-matched control subjects without a history of neurological disease were invited to par-ticipate in our study. A standardized clinical evaluation was performed on all GRN mutation carriers at the UCSF Memory and Aging Center by board-certified neurologists who had additional training in behavioral neurology. For GRN mutation carriers, based on the results of this clinical evaluation, subjects were then subgrouped into asymptomatic GRN mutation carriers (CDR = 0, n = 7) and symptomatic GRN mutation carriers (CDR ≥ 0.5, n = 5). One GRN mutation carrier had a prior diagnosis of age-related macular degenera-tion. This subject was included in the analysis (exclusion of this subject from analysis did not meaningfully affect statistical significance of RNFL thinning or macular volume loss). No other control subjects or GRN mutation carriers had a history of ophthalmological disease or ocular surgery. Written informed consent was obtained from all participants with ca-pacity. Written informed consent was obtained from a designated surrogate decision maker in subjects deemed unable to provide informed consent due to diminished capacity, but we only enrolled subjects who were able to as-sent. The UCSF Committee on Human Research (CHR) approved this pro-tocol, and the study was performed in accordance with the Declaration of Helsinki. Retinal imaging. We performed spectral domain optical coherence to-mography (OCT) at the UCSF Neurodiagnostics Center using a Heidelberg Spectralis instrument (Heidelberg Engineering, Heidelberg, Germany). A trained technician blinded to patient diagnosis and to genotype (when rele-vant) performed all scans and repeated each measurement at least three times. Mean RNFL thickness was determined using a peripapillary B-scan 3.4-mm from the center of the papilla. Images were evaluated by a blinded technician to meet prespecified image quality criteria, including signal intensity and beam uniformity. For this analysis, we analyzed and averaged the RNFL thickness and macular volume of all interpretable scans. RNFL thickness and macular volume was measured using automated software provided by Hei-delberg. Segmentation analysis of macular scans was then performed to de-termine the volume of individual neuronal layers via a proprietary, validated computerized algorithm (Heidelburg Engineering, Heidelburg, Germany). Layers analyzed included the ganglion cell complex (GCC; comprising ganglion cell neuronal cell bodies, their dendrites, and axons projecting from underlying inner nuclear layer neurons), the inner nuclear layer (INL), and the outer nuclear layer (ONL). Statistical analysis for human subjects. RNFL thickness, macular vol-umes, and segmented macular volume were analyzed in human subjects. We used multiple linear regression analysis to compare differences between GRN mutation carriers and unaffected controls. Adjustment for age and sex did not meaningfully change the results, so we elected to report unadjusted values. To account for inter-eye correlations, when two eyes from the same individual were analyzed, the standard error was adjusted using the clustered nuclear depletion of TDP-43, we expressed dominant–negative RanQ69L (which cannot hydrolyze GTP) or RanT24N (which is nucleotide-free or GDP-bound) in cortical neurons. Both of these Ran mutants caused TDP-43 mislocalization (Fig. 4, F and G), indicating that TDP-43 requires functional Ran for import into the nucleus. Enhancing Ran expression improves the survival of progranulin-deficient neurons The results of the prior experiments suggested a model of a reciprocal depletion of nuclear TDP-43 and Ran: loss of nu-clear TDP-43 down-regulates Ran mRNA, and Ran dys-function depletes nuclear TDP-43. Indeed, acute knockdown of progranulin levels in N2A cells via siRNA reduced both TDP-43 and Ran protein expression (Fig. 4, H and I). To directly test this model, we hypothesized that augmenting Ran expression would increase nuclear TDP-43 and improve the survival of Grn-KO neurons. An automated microscopy approach was used to quantify the effect of Ran expression on neuron survival (Arrasate and Finkbeiner, 2005). In this assay, individual GFP-transfected neurons are repeatedly im-aged over multiple days, thus generating longitudinal survival curves for cohorts of neurons from different genetic back-grounds and/or those expressing different plasmids. Previous studies have established that progranulin-deficient cortical neurons exhibit enhanced vulnerability in culture (Guo et al., 2010; De Muynck et al., 2013). Indeed, primary Grn-KO cortical neurons had shorter lives than wild-type neurons (Fig. 4 J). Expression of exogenous Ran enhanced the sur-vival of Grn-KO neurons, but not wild-type neurons (Fig. 4 J). Consistent with our findings in the retina of Grn-KO mice, primary Grn-KO cortical neurons had decreased nuclear TDP-43 expression, which was increased by expression of exogenous Ran (Fig. 4 K). In conclusion, our findings provide strong evidence of early retinal abnormalities in GRN mutation carriers. Reti-nal thinning occurred in cognitively asymptomatic GRN mutation carriers (who are at high risk for future dementia), indicating that retinal neuron loss is an early phenomenon that can precede clinical symptoms in FTD. Because small changes in retinal thickness can be reproducibly measured longitudinally via OCT, retinal imaging could be a useful modality for assessing response to therapeutics in early dis-ease stages of progranulin-deficient FTD. Further study will be needed to establish the rate of retinal degeneration in GRN mutation carriers. The reasons why RGCs are particu-larly susceptible to death in GRN mutation carriers are un-clear. RGCs have long axons and due to their central role in vision they have a high metabolic demand; both of these char-acteristics may contribute selective vulnerability in the set-ting of progranulin deficiency. We also observed a retinal neurodegenerative phenotype in Grn-KO mice that parallels our observations in humans, establishing the retina as a new model system in which to study mechanisms of neurodegeneration in FTLD-TDP. Our findings support a pathogenic loop involving reciprocal 1942 Retinal thinning and TDP-43 mislocalization in FTLD | Ward et al.
- 63. Br ief Defini t ive Repor t around the DAPI-stained nuclei at the z-position center of the nuclei, and used to quantify mean intranuclear TDP-43 intensity. Cytoplasmic intensity of TDP-43 was determined by drawing a perinuclear region of interest in the cytoplasm. This mode of cytoplasmic intensity quantification was found to be more accurate than tracing around the entire cell soma, given the relatively high density of ganglion cells. Nuclear/cytoplasmic intensity ratios represent the mean nuclear intensity/mean cytoplasmic intensity per cell. For Ran quan-tification, intranuclear intensity was determined by drawing a ROI slightly inside of the nuclear envelope. Data were transformed into log10 intensity, and a mixed-model regression of the intensity variable versus genotype and/or age that controlled for clustering by mouse was applied to assess sta-tistical significance in STATA. Transfection of cortical neurons with Ran mutants. Postnatal day 0 rat cortical neurons were isolated and cultured in Neurobasal-A with B27. 3–5 d after isolation, they were transfected with human TDP-43-GFP + mCherry, mCherry-huRan, mCherry-huRanT24N, or mCherry-huRanQ69L. 1 d after transfection, neurons were fixed and imaged on a spinning disk confocal microscope. Images of mCherry-positive neurons were taken with the same acquisition settings across transfection groups, and the TDP-43-GFP signal in the nucleus and cytoplasm was quantified. Differences in nuclear/cytoplas-mic ratio across groups was assessed with one-way ANOVA with Tukey’s post-hoc analysis. Immunostaining of postmortem brains from human subjects with GRN mutations. De-identified post-mortem brain tissue (left inferior frontal gyrus) from FTLD subjects with previously documented GRN muta-tions, deemed nonhuman subject material as per UCSF CHR guidelines, was obtained from the UCSF Neurodegenerative Disease Brain Bank. Tis-sue was embedded in paraffin and 10-μm sections were made. Antigen re-trieval was performed using IHC World’s antigen retrieval solution as per manufacturer’s guidelines, followed by Sudan Black treatment and primary antibody incubation overnight at 4°C (anti–TDP-43, 1:3,000; Protein Tech; anti-Ran, 1:1,000; BD), followed by secondary antibody and DAPI-staining. For quantification of staining, confocal images of DAPI-stained nuclei, TDP- 43, and Ran were taken at equal intensities across multiple fields of view in the cortex. Nuclear TDP-43 and Ran levels were quantified and analyzed as was done in mouse RGCs. TDP-43 RIP. The TDP-43-RNA immunoprecipitation dataset was gen-erated from pull-down experiments conducted as part of a previous study using the methods described therein (Sephton et al., 2011). TDP-43 knockdown, progranulin knockdown, Q-RT-PCR, and Western blot analysis. N2A cells were grown in DMEM (low glucose) + 10% FCS and transfected with Mission TDP-43 shRNA construct #752 or con-trol shRNA construct (Sigma-Aldrich) via Lipofectamine 2000. 6–8 d after transfection, N2A cells were harvested for RNA or protein analysis. RNA was prepared via RNeasy columns (QIAGEN), transcribed into cDNA, and Ran or cyclophilin (control) RNA levels were analyzed via Q-RTPCR. Equal amounts of total protein from knockdown and control cells were im-munoblotted against tubulin (loading control), TDP-43, or Ran, and then quantified using a Licor imaging system, with relative levels of Ran/tubulin quantified for each sample. For progranulin knockdown experiments, N2A cells were transfected with control siRNA (Thermo Fisher Scientific) or Grn#1 siRNA. Greater than 95% knockdown was observed by Western blot and progranulin ELISA by 5 d after transfection. Samples were processed for Ran and TDP-43 quantification, as above. For Ran mRNA analysis of mouse retinas, whole retinas were isolated from freshly perfused mice and nonneu-roretinal tissue was dissected away. RNA preparation and Q-RTPCR was performed as above. Longitudinal neuronal survival analysis. Longitudinal survival analysis of individual GFP-transfected neurons was essentially performed as described previously (Barmada et al., 2010), with the following modifications: cortical sandwich estimator. P 0.05 was considered significant. Analyses were per-formed using STATA 12.0. Mice. Wild-type and Grn-KO mice were obtained from R.V. Farese’s labo-ratory (University of California, San Francisco, CA; Martens et al., 2012). Mice used in experiments shown in Figs. 1–3 were of a mixed background consisting of 62.5% C57BL/6J, 12.5% 129Sv/Jae, and 25% FVB. Mice used in experiments shown in Fig. 4 were fully backcrossed into C57BL/6J. Age-and sex-matched mice from the same genetic background were used as con-trols for Grn KO mice. RNFL and GCL neuron quantification. 16-μm transverse sections of WT and Grn-KO mouse eyes were made and stained with HE/Neu-N, and the center sections (as determined by the center of the optic nerve head) were imaged via light/fluorescent microscopy, respectively. The area of the RNFL was measured in ImageJ and divided by the length of the RNFL across the field of view to determine thickness (equidistant from the optic nerve head across mice). For GCL neuron quantification, sections were stained with anti-NeuN antibody and subsequently imaged via fluorescence microscopy. The number of Neu-N positive cells in the GCL in individual fields of view were counted and divided by the length of the GCL using sec-tions equidistant from the optic nerve head across mice. Statistical analysis was conducted with a one-way ANOVA followed by Tukey multiple com-parison test. Electroretinography. After overnight dark adaptation, mice were anesthe-tized under dim red illumination with 0.1 mg/kg ketamine and 10 mg/kg xylazine. Under anesthesia, both eyes were treated with 0.5% proparacaine followed by a mixture of 2.5% phenylephrine and 1% tropicamide for pupil dilation. The mice were kept warm using a 37°C heating pad (Deltaphase Isothermal Pads; Braintree Scientific). A gold reference electrode was electri-cally connected to the cornea of one eye and a platinum wire, mounted on a fiber-optic cable, was connected to the cornea of the other eye. Electrical continuity was made using hydroxypropyl methylcellulose (Goniosol). Light stimuli were delivered directly into the eye through the tip of the fiber optic. Stimulus intensity was controlled by calibrated neutral density filters, and stimulus wavelength was 500 nm (±5 nm; narrow band filter) or 505 nm (±17 nm; broad band filter). Responses were recorded from threshold up to light 1,000,000 fold brighter in darkness, and the photopic responses were recorded in the presence of rod-saturating background lights. Electrical re-sponses were amplified (Astro-med CP122W; DC-300Hz) and digitized at 2 KHz (Real-Time PXI Computer; National Instruments). Antibodies used. Rabbit anti-TDP-43 (Protein Tech), 1:1,000; rabbit C-terminal anti–TDP-43 (produced by G. Yu; UT Southwestern, Dallas, TX), 1:1,000 each; goat anti-Ran (Santa Cruz Biotechnology, Inc.), 1:200; and mouse anti-Ran (BD), 1:250–1:1,000. Immunostaining. 16-μm transverse sections of WT and Grn-KO mouse eyes were made from paraffin embedded tissue and mounted on silanized slides. After de-waxing and rehydration, sections were blocked for 1 h at room temperature in PBS/0.5% Triton/10% donkey serum. Primary anti-bodies were incubated with sections overnight at 4°C, and then slides were washed and stained with secondary Alexa Fluor–conjugated antibodies (1:300; Invitrogen) for 2 h at room temperature. After washing, samples were mounted with #1.5 coverslips using Prolong-Gold antifade reagent with DAPI (Invitrogen). All fluorescent imaging was performed on an inverted confocal Ti microscope (Eclipse; Nikon) with a Nipkow spinning disk at-tachment and EM camera (Hamamatsu). Quantification of TDP-43 and Ran. Sections from equal retinal eccen-tricity were imaged with a spinning disk confocal microscope, and fields of view of a similar distance from the center of the retinal sections were imaged. Acquisition settings were identical between samples, and all samples used for quantification were stained on the same day. Regions of interest were drawn JEM Vol. 211, No. 10 1943
- 64. This project wasfunded by the Consortium for Frontotemporal Dementia Research (L.G., G.Y., E.D.R. J.H., B.V.F), The Bluefield Project to Cure FTD (M.W., fellowship), R01 AG036884 (L.G), the UCSF Resource Allocation Program (A.G.), the UCSF Alzheimer’s Disease Research Center (M.W.), #P50 AG023501 (B.F), R01NS079796 (G.Y.), R01NS075487 (E.D.R), T32HD071866 (A.E.A), K08EY023610, (M.W) the Chartrand Foundation and Clinical Science Translational Institute (M.W., A.G.), the Howard Hughes Medical Institute (A.G.), the Alzheimer’s Association (G.Y.), the Welch Foundation (G.Y.), R37HL63762 (J.H), the Brightfocus Foundation (J.H), the Alzheimer’s Drug Discovery Foundation (B.M). The authors report no competing financial interests. Submitted: 31 January 2014 Accepted: 5 August 2014 REFERENCES Ahmed, Z., H. Sheng, Y.-F. Xu, W.-L. Lin, A.E. Innes, J. Gass, X. Yu, C.A. Wuertzer, H. Hou, S. Chiba, et al. 2010. 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Progranulin deficiency leads to enhanced cell vulnerability and TDP-43 transloca-tion in primary neuronal cultures. Brain Res. 1366:1–8. http://dx.doi .org/10.1016/j.brainres.2010.09.099 Helmer, C., F. Malet, M.-B. Rougier, C. Schweitzer, J. Colin, M.-N. Delyfer, J.-F. Korobelnik, P. Barberger-Gateau, J.-F. Dartigues, and C. Delcourt. 2013. Is there a link between open-angle glaucoma and neurons from postnatal day 0 wild-type or Grn KO mice were dissociated and plated on PDL-coated 96 well-dishes (microclear bottom dishes; Greiner) at a density of 90,000 cells/well in Neurobasal-A + B27 supple-ment. 3–5 d after plating, neurons were transfected via Lipofectamine 2000 with GFP + empty vector or GFP + mouse Ran cDNA at a ratio of 1:3 to ensure that all fluorescent cells co-expressed the second plasmid. The day after transfection, wells containing GFP-expressing neurons were imaged at 5× magnification with an automated microscope (Array Scanner XTI; Thermo Fisher Scientific). The same fields of view were reimaged every 24–48 h for a total of 7–9 d after transfection. Adjacent fields of view from individual wells were stitched together into montages via ImageJ, and a time series across days for each well was then generated using a custom-made macro in ImageJ. The survival of individual GFP-transfected neu-rons over time was then assessed by loss of GFP fluorescence. Kaplan-Meier and cumulative risk of death curves were made with R software, and sta-tistical significance of differences in survival between cohorts of neurons was determined with the log-rank test. Tukey multiple comparison test was used for comparisons involving more than two groups. Rescue of neuronal TDP-43 expression. Primary cortical neurons from postnatal day 0 wild-type or Grn KO pups were isolated and plated as de-scribed above. Neurons were transduced with AAV2-GFP (Virovek) or AAV2-GFP-P2A-human Ran (in which bicistronic GFP and untagged Ran co-expression is driven by a single chicken--actin-CMV promoter, with a P2A sequence separating GFP and Ran sequences). 7 d after transduction, neurons were fixed and immunostained with anti–TDP-43 antibody. Indi-vidual wells were imaged, and regions of interest of nuclei from GFP-positive cells were generated via Volocity from background-subtracted images. Using these regions of interest, integrated density measurements of total TDP-43 levels in each nuclei were then calculated. A mixed-model regression of the intensity variable versus genotype and AAV-vector that controlled for clus-tering by well was applied to assess statistical significance in STATA. Statistics. Design and execution of statistical tests were done in collabora-tion with a professional biostatistician (J. Boscardin, University of California, San Francisco, CA). For human retinal imaging data and for nuclear and cy-toplasmic TDP-43 and Ran immunostaining in retinal neurons and in cul-tured cortical neurons, significance was determined via mixed-effects linear regression analyses, accounting for interindividual, within-eye correlations in the human studies and intramouse clustering in rodent models. In experi-ments involving two comparison groups, unpaired two-tailed Student’s t tests were used to assess for differences. In experiments involving more than two comparison groups, ANOVA test with post-hoc tests (Tukey or Bon-ferroni, as specified in the text) were used. For longitudinal neuronal survival analysis, cumulative risk of death curves were generated with R software, and statistical significance was determined with the log-rank test. P 0.05 was considered significant. Unless otherwise noted, statistical testing was performed using Prism and Stata 12.0 software. Study approval. For retinal imaging, written informed consent was ob-tained from all participants with capacity; in subjects deemed unable to pro-vide informed consent due to diminished capacity, written consent was obtained from a designated surrogate decision-maker and subjects provided assent. The study protocol was approved by the UCSF Committee on Human Research (IRB # 11–05333). For studies involving mice, all procedures were approved by the Institutional Animal Care and Use Committee at UCSF (#AN087501-02A) and UAB (#101109282, #130309617). Online supplemental material. Fig. S1 shows clinical characteristics of human subjects who underwent retinal imaging. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20140214/DC1. We would like to thank Dr. Sami Barmada for his thoughtful input on the project and assistance with analysis of neuronal survival experiments, Yungui Zhou and Marcel Alavi for technical assistance, Dr. Anna Karydas for her assistance with genetic analysis of human subjects, and Dr. Laura Mitic for her feedback. 1944 Retinal thinning and TDP-43 mislocalization in FTLD | Ward et al.
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- 67. Ar t icle The Rockefeller University Press $30.00 J. Exp. Med. 2014 Vol. 211 No. 6 1049-1062 www.jem.org/cgi/doi/10.1084/jem.20131751 1049 Accumulating evidence suggests that cerebro-vascular risk factors play an important role in Alzheimer’s disease (AD) pathophysiology. Many AD patients suffer from altered cerebral blood flow, damaged cerebral vasculature, and increased cerebral microinfarcts (de la Torre, 2004; Brundel et al., 2012), and a majority of patients with de-mentia present with both AD and vascular pa-thologies (MRC CFAS, 2001; Viswanathan et al., 2009). Furthermore, cerebral amyloid angiopathy (CAA), which is the deposition of the -amyloid (A) peptide within cerebral blood vessels, re-sults in degenerative vascular changes (Thal et al., 2008; Smith and Greenberg, 2009). Patients with both CAA and neurological pathology including neurofibrillary tangles and neuritic plaques have more severe cognitive impairment than patients with only AD pathology or CAA alone (Pfeifer et al., 2002), and reduction of CAA levels in AD transgenic mice leads to memory improvement (Park et al., 2013). Interestingly, the Nun Study showed that one-third of the participants who had neurological AD pathol-ogy were actually not demented at the time of death, but when AD pathology was concomitant with brain infarcts, there was a high prevalence of dementia found in participants (Snowdon et al., 1997; Mortimer, 2012). Thus, the identifi-cation of a molecular association between these vascular and neurological pathologies could aid in more efficient diagnoses and effective treat-ments for AD. Recent studies have suggested that fibrin-ogen, a primary protein component of blood clots, serves as a molecular link between the vascular and neurological abnormalities ob-served in AD patients. Normally, fibrinogen is found in the blood and is excluded from the brain via the blood–brain barrier (BBB). How-ever, it has been shown that: 1) fibrinogen is often localized to CAA in the brain’s blood ves-sels and brain parenchyma in AD patients and in mouse models of AD (Paul et al., 2007; Ryu and McLarnon, 2009; Cortes-Canteli et al., 2010; Klohs et al., 2012); 2) fibrin deposition in the vasculature increases BBB dysfunction and neu-rovascular damage in AD mice (Paul et al., 2007; CORRESPONDENCE Sidney Strickland: strickland@rockefeller.edu Abbreviations used: AD, Alzheimer’s disease; BBB, blood– brain barrier; CAA, cerebral amyloid angiopathy; FP, fluorescence polarization; HTS, high-throughput screen; SPR, surface plasmon resonance; TAMRA, 5-carboxy-tetrameth-ylrhodamine; tPA, tissue plas-minogen activator. A novel A-fibrinogen interaction inhibitor rescues altered thrombosis and cognitive decline in Alzheimer’s disease mice Hyung Jin Ahn,1 J. Fraser Glickman,2 Ka Lai Poon,1 Daria Zamolodchikov,1 Odella C. Jno-Charles,1 Erin H. Norris,1 and Sidney Strickland1 1Laboratory of Neurobiology and Genetics and 2High Throughput Screening Resource Center, The Rockefeller University, New York, NY 10065 Many Alzheimer’s disease (AD) patients suffer from cerebrovascular abnormalities such as altered cerebral blood flow and cerebral microinfarcts. Recently, fibrinogen has been iden-tified as a strong cerebrovascular risk factor in AD, as it specifically binds to -amyloid (A), thereby altering fibrin clot structure and delaying clot degradation. To determine if the A–fibrinogen interaction could be targeted as a potential new treatment for AD, we designed a high-throughput screen and identified RU-505 as an effective inhibitor of the A–fibrinogen interaction. RU-505 restored A-induced altered fibrin clot formation and degradation in vitro and inhibited vessel occlusion in AD transgenic mice. Furthermore, long-term treatment of RU-505 significantly reduced vascular amyloid deposition and microgliosis in the cortex and improved cognitive impairment in mouse models of AD. Our studies suggest that inhibitors targeting the A–fibrinogen interaction show promise as therapy for treating AD. © 2014 Ahn et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial– Share Alike 3.0 Unported license, as described at http://creativecommons.org/ licenses/by-nc-sa/3.0/).
- 68. the background AlphaLISAsignal. Therefore, the actual in-hibitory efficacy of these compounds could be higher than the results from dose–response experiments of AlphaLISA. In addition, avidity effects may cause higher IC50 values in AlphaLISA than FP. There could be multiple A–fibrinogen interactions between acceptor bead and donor bead in Alph-aLISA (Fig. 1 B), and therefore blocking one interaction may not reduce the signal. Because both AlphaLISA and the FP assay are based on optical measurements, colored compounds could significantly modify the measurement through inner filter effects. Thus, we confirmed the potency of our candidates using a pull-down assay. All five compounds showed inhibitory effects, whereas RU-505 had significant inhibitory efficacy (Fig. 2 A). These combined experiments show that the compounds identified are inhibitors of the A–fibrinogen interaction. Soluble oligomeric A has been hypothesized to be the primary toxic species in AD (Cleary et al., 2005). Therefore, we tested which form of A, monomer or oligomer (prepared as in Stine et al. [2011]), interacts with fibrinogen and whether RU-505 can selectively inhibit the interaction of one or the other. Using the AlphaLISA assay, we found that both A42 monomer and oligomer interact with fibrinogen, but the affin-ity of oligomer for fibrinogen binding is 4 times higher than that of the monomer (Fig. 2 B). RU-505 inhibits the interaction of both monomer and oligomer with fibrinogen, but has higher inhibitory efficacy against the monomer–fibrinogen interaction than the oligomer (Fig. 2 C). Validation of hit compounds using in vitro clotting assay Because the interaction between A42 and fibrinogen in-duces a structurally abnormal fibrin clot and delays fibrin clot degradation during fibrinolysis (Ahn et al., 2010; Cortes- Canteli et al., 2010; Zamolodchikov and Strickland, 2012), one of the main objectives of our study was to identify com-pounds that restore A-induced delayed fibrinolysis. When fibrinogen associates into a fibrin meshwork after cleavage by thrombin, the fine structure of this fibrin clot scatters light and the solution increases in turbidity. Thus, the kinetics of turbidity can be used as a read-out to analyze fibrin network formation and degradation. We tested whether our hits re-stored A-induced altered thrombosis and fibrinolysis in vitro. Each hit compound (20 μM) or vehicle (0.4% DMSO) was incubated for 10 min with purified human fibrinogen and plasminogen in the presence or absence of A42. Fibrin clot formation and degradation were analyzed by measuring tur-bidity immediately after adding thrombin and tissue plasmin-ogen activator (tPA) to the mixture. In the presence of A42, the maximum turbidity of the fibrin clot was decreased be-cause A altered fibrin clot structure and the dissolution of the fibrin clot was delayed (Fig. 2 D; red). RU-505 restored the A-induced decrease in turbidity during fibrin clot for-mation (Fig. 2 D; green) and significantly reduced the delay in fibrin degradation in the presence of A (Fig. 2 E). We also tested other hit compounds, including RU-965, using the turbidity assay, but none had significant effects (Fig. 2 F and Cortes-Canteli et al., 2010); 3) A binds specifically to fibrino-gen; and 4) fibrin clots formed in the presence of A have an abnormal structure, making them resistant to degradation by fibrinolytic enzymes (Ahn et al., 2010; Cortes-Canteli et al., 2010). Overall, these results indicate that in the presence of A, any fibrin clots formed might be more persistent and may ex-acerbate neurovascular damage and cognitive impairment. Therefore, molecules that block this interaction without affect-ing clotting in general could restore altered thrombosis and fi-brinolysis and protect against vascular damage in AD patients, and could be used as therapeutic agents. RESULTS Hit identification and optimization using high-throughput screening To investigate this idea, we designed a high-throughput screen (HTS) to identify small molecules that inhibit the interaction between A and fibrinogen. Low molecular weight compounds were screened using fluorescence polarization (FP) and Alpha- LISA assays in a complementary fashion to cross check the activity of the hit compounds and to ensure the removal of false-positive artifacts. Primarily, 93,000 compounds were screened using FP, which measured the changes in the anisotro-phy induced by binding of a 5-carboxy-tetramethylrhodamine (TAMRA)–labeled A peptide to fibrinogen (Fig. 1 A). Then, hits from FP were screened using AlphaLISA to independently confirm the activity of the inhibitors identified in the FP assay (Fig. 1 B). After both steps, we selected only drug-like com-pounds using Lipinski’s Rule of Five, which allowed us to de-termine which chemical compounds have pharmacological properties that would make them likely orally active drugs in humans (Lipinski et al., 2001). We also filtered out artifactual compounds using a quenching assay, which identifies insoluble compounds, singlet oxygen quenchers, and biotin mimetics in-terfering with the AlphaLISA signal. We identified several candi-date compounds with half-maximal inhibitions (IC50) between 10 and 50 μM from the dose-response assays using both FP and AlphaLISA assays (Table 1). To expand and improve our candidate compounds, we purchased a focused analogues compound library, based on combinatorial variations of scaffolds from the primary hit compounds. These analogues were screened at three different concentrations (5, 10, and 20 μM) using AlphaLISA. Next, we selected only drug-like compounds using Lipinski’s Rule of Five and also included the quenching assay. If inhibition by quenching was 30%, the compounds were removed from fur-ther analyses because these compounds were more likely to be false positives. Finally, we screened the active nonquenching compounds in concentration-response experiments with freshly dissolved powders using both FP and AlphaLISA assays. We identified five drug-like compounds with IC50 3 μM by FP and IC50 10 μM by AlphaLISA (Fig. 1 C and Table 2). In some cases, the maximum inhibition of several com-pounds in AlphaLISA was lower than that of FP. There are several possible reasons for these differences. First, some hit compounds showed negative quenching values, which increases 1050 A-fibrin interaction inhibitor as AD treatment | Ahn et al.
- 69. JEM Vol. 211,No. 6 Ar t icle to the sensor chip surface, and RU-505 was injected for 2 min at 30 μl/min. Sulindac sulfide was used as positive control, and sulindac was used as negative control (Richter et al., 2010). To analyze the correlation between HTS and SPR, we used an analogue of RU-505, RU-4180 (Fig. 2 H), which did not in-hibit the A–fibrinogen interaction in AlphaLISA assay. Al-though RU-4180 weakly binds to A42 (green; Fig. 2 G), RU-505 showed strong binding to A42 (blue; Fig. 2 G). Fur-thermore, because it is known that sulindac sulfide binds A, we tested whether it could inhibit the A–fibrinogen interac-tion by AlphaLISA and found that it had no effect. These results suggest that RU-505 inhibits the A–fibrinogen interaction 1051 not depicted). Moreover, RU-505 did not have any effect on fibrin clot formation and degradation in the absence of A (Fig. 2 D, purple). This result suggests that RU-505 could effectively restore A-induced altered fibrin clot structure and delayed degradation without affecting normal clot for-mation and fibrinolysis. The interaction between A and RU-505 To elucidate how RU-505 inhibits the A–fibrinogen inter-action, surface plasmon resonance (SPR) was used to analyze the binding characteristics of RU-505 (Fig. 2 G). Hexaflu-oroisopropanol- treated monomerized A42 was immobilized Figure 1. The chemical structure and dose–response curve of A–fibrinogen interaction inhibitors. (A) TAMRA–labeled A peptide was bound to fibrinogen and the test compound, and the anisotropy of TAMRA–A–fibrinogen binding was deter-mined by FP. (B) Biotin-labeled A42, which binds a streptavidin donor, was incubated with fibrinogen, which binds a protein A ac-ceptor bead coated with antifibrinogen anti-body. A42 and fibrinogen interactions bring the beads in close proximity, resulting in the excitation of the donor beads and release of singlet oxygen molecules that triggers light emission in acceptor beads (AlphaLISA [AL]). (C) The half-maximal inhibitory concentration (IC50) values of the indicated compounds were determined by dose–response FP and AL ex-periments and are indicated inside the panel (red, FP; blue, AL). A quenching test was also performed to calculate how much each hit compound interfered with the AL signal at 10 μM concentration. Quenching values are indicated below the dose–response curve. n = 3–4 repeats per assay and all error bars indicate SEM. Data are representative of at least three independent experiments.
- 70. Number of compounds% of picked compounds Inhibition cut-off (%) Total library compound - 93,716 - - Primary assay hits using FP 20 μM 3,010 3.21 75 Secondary assay hits using AlphaLISA (AL) 12.5 μM 167 0.18 50 Filtering using quenching (AL) 12.5 μM 97 0.1 30 Lipinski’s rule 87 0.09 ≤1 violation Validation/dose–response Hits (FP and AL) 0.31–40 μM 26 0.028 IC50 50 μM Dose–response using fresh compound (FP 0.07–40 μM 10 0.011 IC50 50 μM and AL) and the length of occluded vessels was measured 5 min after the addition of each concentration of FeCl3 for both Tg6799 and WT. There was no significant difference in the percentage of occluded vessels before FeCl3 treatment or after 5 and 10% FeCl3 treatment among groups (Fig. 3 B). However, there was a significant difference between the percentage of occluded vessels after 15% FeCl3 treatment in vehicle-treated WT and Tg6799 mice (Fig. 3, B and C). Approximately half (52.7 ± 12.1%) of the vessels were occluded in vehicle-treated WT mice, but 95.6 ± 3.5% of vessels were occluded in vehicle-treated Tg6799 mice. RU-505 treatment significantly lowered the vessel occlusion in Tg6799 mice to 60.7 ± 8.7%, but did not change vessel occlusion in WT mice (54.2 ± 11.8%). These results suggest that our lead compound significantly restored altered thrombosis and fibrinolysis in AD mice without affect-ing normal thrombosis and fibrinolysis in WT littermates. Treatment with RU-505 reduced vascular A deposition CAA has been implicated in vascular degeneration of AD (Chen et al., 2006; Okamoto et al., 2010). Our previous stud-ies showed that the A–fibrinogen interaction increases A fibrillization (Ahn et al., 2010). Thus, we investigated whether treatment of Tg6799 mice with RU-505 for 4 mo could de-crease A deposition in blood vessels. A deposits were stained using Congo red, and blood vessels were labeled using lam-inin (green; Fig. 4 A). We quantified CAA area in the cortex Table 1. Workflow of high-throughput primary screening Step Test compound concentration through A binding, but RU-4180 does not inhibit the inter-action because its affinity for A is too weak. Moreover, from the case of sulindac sulfide, A binding itself is not enough to inhibit the interaction between A and fibrinogen. To inhibit the A–fibrinogen interaction, a compound requires at least two features: 1) an A-specific binding moiety and 2) a moi-ety responsible for inhibiting fibrinogen’s binding to A. RU-505 restored altered thrombosis in AD mice To assess whether our lead compound could restore A- induced altered thrombosis and fibrinolysis in vivo, we exam-ined cerebral blood flow and thrombosis in a transgenic mouse model of AD, Tg6799 mice (Oakley et al., 2006), with or without long-term treatment of RU-505. Blood flow and thrombosis were analyzed by a FeCl3-induced thrombosis model combined with intravital microscopy (Cortes-Canteli et al., 2010). We administered RU-505 or vehicle (35 mg/kg dose, every other day) to 4-mo-old Tg6799 and WT litter-mates for 4 mo (analyzed at 8 mo of age). Brains of 8-mo-old Tg6799 or WT mice were exposed by craniotomy, and blood flow was observed using injected fluorescence-conjugated dex-tran (Fig. 3 A). Three concentrations of FeCl3 (5, 10, and 15%) were incrementally administered to the brain surface to in-duce thrombosis. Clot formation was revealed by the appear-ance of an enlarging shadow superimposed on normal blood flow (Fig. 3 A and Videos 1–4). The length of all visible vessels with 20 μm diam was measured before FeCl3 treatment, Table 2. Workflow of high-throughput screening using a focused library Step Test compound concentration Number of compounds % of picked compounds Inhibition cut-off Library compound - 2,092 - - AlphaLISA (AL) assay hits 5, 10, and 20 μM 327 15.6 Inhibition 35% at 5 μM and 50% at 10 μM Filtering using quenching (AL) 10 μM 58 2.77 Quenching 27% at 10 μM and inhibition 55% at 10 μM Lipinski’s rule 50 2.39 ≤1 violation Validation/dose response hits (FP and AL) 0.01–20 μM 5 0.24 IC50 3 μM (FP) and 10 μM (AL) Selection criteria and number of compounds selected during each step of screening. 1052 A-fibrin interaction inhibitor as AD treatment | Ahn et al.
- 71. Figure 2. RU-505inhibited the A–fibrinogen interaction and restored A-induced altered fibrin clot formation and degradation. (A) Can-didate compounds (10 μM) were incubated with biotinylated A42 and fibrinogen, and pull-down assays were performed using streptavidin–Sepharose. All samples were analyzed by Western blot. Dot blots were performed to control for amounts of A pulled down. Control (Ctrl) lane contains only A and fibrinogen without any compound (one-way ANOVA and Bonferroni post-hoc test; *, P 0.05; n = 3–4 independent experiments). (B) The binding affinity between fibrinogen and monomeric or oligomeric biotinylated A42 was measured using the AL assay. (n = 3–4 experiments, data are representative of three independent experiments). (C) The inhibitory efficacy of RU-505 on the interaction between fibrinogen and monomeric or oligomeric biotin-LC-A 42 was accessed in dose–response experiments using the AL assay. (n = 3–4 experiments, data are representative of three independent experiments). (D) RU-505 or DMSO was incubated with fibrinogen in the presence or absence of A42, followed by plasminogen, thrombin, tPA, and CaCl2. Fibrin clot formation was assessed by measuring turbidity (n = 3 experiments, data are representative of three independent experiments). (E and F) The time to fibrin clot degradation was analyzed by measuring time to half lysis. Control clot half lysis time was set to 100% for each experiment and all other values were calculated relative to controls. (***, P 0.001; n = 3 experiments, data are representative of three independent experiments). (G and H) A42 was immobi-lized on the SPR sensor chip surface, and the interaction of the indicated compounds with A42 was analyzed using Biacore 3000. Sulindac sulfide (known to bind A42) was a positive control, and sulindac was negative control. (H) Chemical structure of RU-4180. Data are representative of three to four independent experiments. All values are means and SEM. JEM Vol. 211, No. 6 Ar t icle This result indicates that inhibition of the A–fibrinogen in-teraction by RU-505 reduced A deposits in blood vessels of 1053 AD mice. Treatment with RU-505 improved cognitive impairment of AD mice Because RU-505 restored A-induced altered thrombosis and impaired fibrinolysis in vitro and in vivo, we explored whether long-term RU-505 treatment could have behavioral by measuring Congo red deposits inside blood vessels, and A plaque deposition was quantified by measuring Congo red outside blood vessels. The CAA area of RU-505–treated Tg6799 mice (0.025 ± 0.006%, cortex) was significantly de-creased from that of vehicle-treated Tg6799 mice (0.046 ± 0.004%, cortex; Fig. 4 C). However, there was no significant difference in A plaque area in the cortex between RU-505– and vehicle-treated Tg6799 mice (Fig. 4, B and D). WT mice did not exhibit any CAA-specific pattern of Congo red staining.
- 72. Figure 3. RU-505prevented altered thrombosis and fibrinolysis in AD transgenic mice. (A) After craniotomy, three concentrations of FeCl3 (5, 10, and 15%) were incrementally administered to the surface of the brains of vehicle- or RU-505–treated WT and Tg6799 mice (Videos 1–4), and clotting of cerebral blood vessels (20 μM) was imaged (bars, 200 μm). Representative intravital images shows the surface of the brains of vehicle- or RU-505– treated WT and Tg6799 mice before FeCl3 treatments or 5 min after 15% FeCl3 treatments. (B and C) Frequency of clotted vessels was calculated at in-creasing concentrations of FeCl3 (B) and was plotted for 15% FeCl3 treatment (C; ***, P 0.001; n = 5 mice per group). All values are means and SEM. longer latency to reach the closed target hole (Fig. 5 D) and significantly fewer visits to the target hole compared with ve-hicle- treated WT and RU-505–treated Tg6799 mice (Fig. 5 E). These results suggested that vehicle-treated Tg6799 mice have impaired spatial learning and memory, and RU-505 treat-ment restored the cognitive impairment of Tg6799 mice. When we measured total distance traveled during probe trials, Tg6799 mice moved significantly less than WT mice. How-ever, there was no significant difference in distance traveled between RU-505–treated and untreated Tg6799 mice (Fig. 5 F). This result suggests that the better performance of RU-505– treated Tg6799 mice compared with untreated Tg6799 mice is likely caused by memory improvement and not effects on locomotion. To further address any possible issue of hypoactivity in the Tg6799 mice and to test whether RU-505 treatment had a similar effect on a different strain of AD transgenic mice, we administered RU-505 to 4-mo-old TgCRND8 mice (Chishti et al., 2001) for 3 mo (analyzed at 7 mo-of-age) as a pilot ex-periment. During training, RU-505 treatment did not lead to Results are from two independent experiments. effects on AD mice. 7-mo-old Tg6799 mice treated for 3 mo with RU-505 were tested using contextual fear conditioning to assess possible cognitive changes. RU-505 treatment had no effect on baseline freezing behavior in WT and Tg6799 mice (Fig. 5 A). When we evaluated contextual memory of Tg6799 and WT mice 24 h after training, vehicle-treated Tg6799 mice showed a severe memory deficit compared with vehicle-treated WT mice (Fig. 5 B). RU-505–treated Tg6799 mice exhibited significantly improved memory compared with their vehicle-treated AD counterparts, whereas long-term treatment of RU-505 in WT mice did not impact basal freezing behavior or contextual memory. We also explored cognitive performance of RU-505– treated Tg6799 mice with the Barnes maze, a behavioral test that assesses spatial learning and memory in rodents (Walker et al., 2011). Vehicle-treated Tg6799 mice took a signifi-cantly longer amount of time to find the target hole com-pared with vehicle-treated WT and RU-505–treated Tg6799 mice (Fig. 5 C). During the memory retention test in the probe trials, vehicle-treated Tg6799 mice also had significantly 1054 A-fibrin interaction inhibitor as AD treatment | Ahn et al.
- 73. JEM Vol. 211,No. 6 Ar t icle Long-term treatment with RU-505 decreased the level of infiltrated fibrinogen and microgliosis in AD mice To understand the mechanisms underlying improvement in cognitive function of RU-505–treated AD mice, we analyzed cortical fibrinogen infiltration and microgliosis in Tg6799 mice after four months of RU-505 treatment. BBB permeability is increased in mouse models of AD (Paul et al., 2007) and infiltrated fibrinogen might bind to A and become resistant to degradation in the paren-chyma. If RU-505 can inhibit the interaction between in-filtrated fibrinogen and A in the parenchyma, the level of infiltrated fibrinogen could be decreased in the brain of AD mice. In addition, activation of microglia is highly increased 1055 improvement in spatial learning in TgCRND8 mice (Fig. 6 A); however, this treatment significantly reduced the latency to reach the target hole during the probe trial compared with vehicle-treated TgCRND8 mice (Fig. 6 B). Furthermore, the number of visits to the target hole during the probe trial was significantly higher in RU-505–treated TgCRND8 mice compared with vehicle-treated TgCRND8 mice (Fig. 6 C). In addition, vehicle-treated TgCRND8 mice showed similar locomotor activity during probe trials (Fig. 6 D), indicating that the impaired per-formance of vehicle-treated TgCRND8 mice in Barnes maze test is more likely caused by deficits in spatial memory. These re-sults suggest that treatment of RU-505 substantially improved the deficits in spatial memory of TgCRND8 mice. Figure 4. CAA pathology in AD trans-genic mice was reduced after long-term treatment with RU-505. (A) A deposits within cortical blood vessels of vehicle- or RU-505–treated Tg6799 mice were visualized using Congo red and laminin (green) labeling (bars, 100 μm). (B) Representative pictures showing parenchymal A deposition in un-treated and treated mice (bars, 100 μm). (C and D) CAA and A plaques in (A) and (B) were quantified from 7–10 sections per mouse (n = 5 mice per group; *, P 0.05). All values are means and SEM. Results are from two independent experiments.
- 74. Figure 5. RU-505restored cognitive function in Tg6799 mice. (A) Freezing behavior was measured before electric foot shock during the training day to assess the basal freezing tendency of each group of mice. (n = 8–10 mice per group). (B) Contextual memory was assessed by measuring freezing behavior upon reexposure to the training chamber 24 h after fear conditioning training. (*, P 0.05; **, P 0.01; n = 8–10 mice per group). Results are from two independent experiments. (C–E) Spatial learning and memory retention of WT and Tg6799 mice was assessed using the Barnes maze after 3 mo of treatment with RU-505 or vehicle. One target hole was connected to a hidden escape chamber. (C) During training trials, latency to poke the target hole was measured. Significance was assessed using two-way ANOVA analysis with repeated measure (WT/vehicle vs. Tg6799/vehicle: F[1,120] = 40.47; P 0.001; Tg6799/vehicle vs. Tg6799/RU-505: F[1,108] = 11.97; P 0.01; n = 10–14 mice per group). Differences in latency were assessed by Bonferroni post hoc analysis. (D–F) During the Barnes maze probe trial, latency to reach the closed target hole (D), number of visits to the target hole (E), and total traveled distance (F) were measured ([E] *, P 0.05; **, P 0.01; ***, P 0.001; n = 10–14 mice per group; [F] ***, P 0.001; n = 10–14 mice per group). All results of the Barnes maze are from three independent experiments. deposition (green; Fig. 7 A) outside the endothelial cells of blood vessels that were labeled using CD31 (red; Fig. 7 A), and the area of activated microglia that were labeled using CD11b (red; Fig. 7 B). The levels of infiltrated fibrinogen and micro-gliosis were highly increased in the cortex of Tg6799 com-pared with WT mice (Fig. 7, C and D), and these increases were significantly decreased by RU-505 (Fig. 7, C and D). in AD patients and mouse models of AD, and an increase of inflammation in the brain is correlated with memory im-pairment (Bayer et al., 1999; Dhawan and Combs, 2012; Vom Berg et al., 2012). Therefore, we measured the level of infiltrated fibrinogen and microgliosis in the cortex of Tg6799 or WT littermate mice after RU-505 treatment. We quantified fibrinogen Figure 6. RU-505 restored spatial retention memory in TgCRND8 mice without affecting motor behavior. (A) The spatial memory of vehicle- or RU-505–treated WT and TgCRND8 mice was assessed using Barnes maze. (B and C) Spatial memory of RU-505–treated WT and TgCRND8 mice was tested using the Barnes maze probe trials. Time to reach the target hole (B), the number of visits to the closed target hole (C), and total distance traveled (D) were assessed (n = 7–11 mice per group). The results cor-roborate those in Fig. 5 and are from one experiment. All values are means and SEM. 1056 A-fibrin interaction inhibitor as AD treatment | Ahn et al.
- 75. JEM Vol. 211,No. 6 Ar t icle RU-505. In addition, the increased infiltrated fibrinogen in AD mice may interact with A in the tunica media of arteri-oles, which could be another target region for inhibition of A-induced exacerbated thrombosis, as well as for prevention of CAA formation. One question that arises from our results is why RU-505 treatment reduced vascular amyloid deposits, but not paren-chymal plaque. Amyloid accumulates in the tunica media of arterioles in CAA, and the tunica media is much closer to the intravascular region than the parenchyma. Therefore, the fibrinogen levels in the tunica media should be much higher than in the parenchyma. For this reason, the A–fibrinogen interaction could be a major factor of A fibrillization in CAA, but have only minor effects on A fibrillization on paren-chymal 1057 plaques. We primarily investigated the interaction between A42 and fibrinogen in this study, but the ratio of A40 to A42 is higher in CAA. Another question is how RU-505, an inhibitor of the A42–fibrinogen interaction, could reduce CAA, which is primarily composed of A40. One possibility is that A40 also interacts with fibrinogen even though its DISCUSSION The present study shows that the novel compound, RU-505, restored A-induced altered thrombosis and delayed fibrinolysis in vitro and in vivo by inhibiting the A–fibrinogen interaction. We also demonstrate that long-term RU-505 treatment can reduce vascular amyloid deposits, infiltrated fibrinogen, and microgliosis in the cortex of a transgenic mouse model of AD. Finally, this novel A–fibrinogen interaction inhibitor improved the cognitive decline of two different strains of AD transgenic mice. Using pharmacokinetics, we found that RU-505 is highly permeable to the BBB because RU-505 levels in the brain were equal to or greater to that in the blood over a 24-h period after single subcutaneous injection. The half-life of RU-505 was 3.7 h in the blood and 12.4 h in the brain. Therefore, the intravascular and the tunica media of arterioles are the most likely regions for RU-505 action. Several studies have shown that the amount of soluble A significantly in-creases in the vicinity of amyloid deposits in blood vessels (Shinkai et al., 1995; Suzuki et al., 1994), and the intravascu-lar area near CAA might be a major target of inhibition by Figure 7. Long-term treatment with RU-505 reduced the level of infiltrated fibrinogen and microgliosis in the cortex of Tg6799 mice. (A) Fibrinogen localized outside of endothelial cells of blood vessels was labeled with FITC-conjugated antifibrinogen antibody (green), and endothelial cells were labeled using anti-CD31 antibody (red; bars, 50 μm). (B) Activated microglia were visualized by staining for CD11b (red). DAPI staining (blue) was used to show integrity of tissue (bars, 100 μm). (C and D) Total fibrinogen area (C) and microgliosis (D) were quantified from 3 sections per mouse (n = 3–4 mice per group; *, P 0.05; ***, P 0.001). All values are means and SEM. Results are from two independent experiments.
- 76. over 3 mo,which minimized this issue. Our future direction would modify RU-505, and find less toxic analogues with sim-ilar or better efficacy. For more than a decade, A has been the major target for developing AD therapies. Most of these efforts focused on using antibodies to lower A levels, preventing A aggregation, or re-ducing A production. However, none of these methods were successful as the treatments did not show clinical efficacy or caused serious adverse side effects such as aseptic meningoen-cephalitis (Gilman et al., 2005; Mangialasche et al., 2010). How-ever, numerous studies still support the hypothesis that A plays an important role in the pathogenesis of AD (Tanzi and Bertram, 2005; Jonsson et al., 2012). Therefore, new strategies for anti-A therapy are necessary for developing novel treatments for AD. In-hibiting the interaction between A and its binding proteins could be an alternative therapeutic approach, and our study shows that a small molecule, bioavailable inhibitor of the A– fibrinogen interaction, RU-505, significantly restored altered thrombosis and improved cognitive deficits observed in AD transgenic mouse models. Therefore, treatment of the neurovas-cular pathology observed in AD using an inhibitor of the A–fibrinogen interaction may be a valuable strategy for devel-oping novel AD therapeutics. MATERIALS AND METHODS Animals Tg6799 mice (The Jackson Laboratory) are double transgenic mice for APP/ Presenilin 1 that coexpress five early onset familial AD mutations on a mixed background C57BL/6 x SJL (Oakley et al., 2006). TgCRND8 mice (pro-vided by A. Chishti and D. Westaway, University of Toronto, Canada) have three APP mutations (K670N, M671L, and V717F) driven by the human prion protein promoter on a mixed background C57 x C3H/C57 (Chishti et al., 2001). RU-505 was prepared in 2.5% EtOH, 4.5% Cremophor RH40 (Sigma-Aldrich), and 14% D5W (5% dextrose in water) in saline. We admin-istered 35 mg/kg dose of RU-505 or vehicle to Tg6799 mice and 25 mg/kg dose or vehicle to TgCRND8 mice subcutaneously every other day. Non-transgenic (WT) littermates were used in all experiments. The assigned geno-type of all the mice used in the experiments throughout the paper was double-checked by taking tail tissue the day of sacrifice. Only male mice were used in experiments, and all animals were maintained in The Rocke-feller University Comparative Biosciences Center and treated in accordance with protocols approved by The Rockefeller University Institutional Animal Care and Use Committee. Primary compound screening Approximately 93,000 compounds were screened using HTS. Compound screening libraries that include known off-patent drugs, natural products, and combinatorially elaborated active pharmacophores were purchased from several vendors listed in Table 3. The primary assay used FP to measure the changes in the anisotropy induced by binding of TAMRA-labeled A42 (Anaspec) to fibrinogen. TAMRA-A42 (2 nM) was mixed with 300 nM fibrinogen (EMD Millipore) and 20 μM compounds (dissolved in 1% DMSO [final]) in 50 mM PBS, pH 7.4, 0.001% Tween 20, and 0.001% BSA as 50 μl final volume in black 384-well plates (Greiner) at RT. After binding reached equilibrium, polarization measurements were recorded with a Perkin-Elmer EnVision plate reader with excitation at 490 nm and emission at 535 nm. The FP response was monitored and plotted as milli-Polarization (mP) units. Compounds that showed 75% inhibition of the A–fibrinogen inter-action in the FP assay were selected for screening by AlphaLISA as a second-ary assay. Compounds (12.5 μM) were plated in white 384-well plates affinity is 10 times less than A42, and RU-505 has a strong inhibitory efficacy on A40–fibrinogen interaction (unpub-lished data). Therefore, RU-505 could reduce CAA through inhibition of both A42- and A40–fibrinogen interaction. Second, despite the higher levels of A40 in vascular amy-loid, A42 is also essential for vascular amyloid deposition in transgenic mice overexpressing human APP (Van Dorpe et al., 2000; McGowan et al., 2005) and AD human patients (Roher et al., 1993; Shinkai et al., 1995). A42 could act as a nucleation seed of amyloid deposit in the vessel walls and accelerate deposition of A40 (Van Dorpe et al., 2000; Yoshiike et al., 2003; McGowan et al., 2005). Therefore, even though the ratio of A40 to A42 is higher in vascular amyloid, the A42–fibrinogen interaction could be critically involved in CAA formation. Fibrinogen is a proinflammatory mediator in several dis-eases and induces the activation of microglia in the nervous system (Adams et al., 2007; Davalos and Akassoglou, 2012). Our study showed that RU-505 treatment reduced the level of infiltrated fibrinogen and activated microglia in the brain of AD transgenic mice. One possible mechanism for this re-duction is that RU-505 binds A, inhibits the A–fibrinogen interaction, and facilitates fibrinogen degradation. The decreased level of infiltrated fibrinogen could result in the decrease of microgliosis. The other possible mechanism is that long-term RU-505 treatment reduced vascular amyloid deposits and prevented BBB leakage. This recovery of a healthy BBB could reduce fibrinogen infiltration and inflammation in the paren-chyma of Tg6799 mice. Increased levels of plasma fibrinogen are associated with cognitive deficits (Xu et al., 2008), AD risk (van Oijen et al., 2005), and brain atrophy (Thambisetty et al., 2011), and in-creased levels of fibrinogen have also been found in the CSF of AD patients (Craig-Schapiro et al., 2011; Vafadar-Isfahani et al., 2012). Moreover, several studies have shown that anti-coagulant treatment improves cognition in mouse models of AD and dementia patients (Ratner et al., 1972; Walsh et al., 1978; Cortes-Canteli et al., 2010; Timmer et al., 2010). How-ever, anticoagulant therapy can cause severe problems in elderly patients who have a more fragile vasculature because it may increase the incidence of major systemic bleeding. There-fore, drugs should specifically block the A–fibrinogen inter-action so that A-induced altered blood clot formation and degradation can be restored without affecting general hemo-stasis. RU-505 successfully targeted only A-induced altered blood clot formation and did not affect general clot forma-tion and degradation (Fig. 2 D and Fig. 3). The maximum tolerated dose of RU-505 after single in-travenous dose in mice was between 100 and 200 mg/kg. When we treated Tg6799 mice and WT littermates with two doses (100 and 50 mg/kg) of RU-505 every other day for 3 mo, we found that 100 mg/kg for long-term treatment was toxic to the AD mice, but 50 mg/kg showed no clinical signs of toxicity except local chronic inflammation at the injection site. To address the issue of local inflammation, we lowered the dose to 35 mg/kg for Tg6799 or 25 mg/kg for TgCRND8 1058 A-fibrin interaction inhibitor as AD treatment | Ahn et al.
- 77. JEM Vol. 211,No. 6 Ar t icle biotinylated A42 (Anaspec) and 5 nM fibrinogen (EMD Millipore) for 1 h at room temperature in 500 μl of binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 0.1% BSA, and protease inhibitor mix-ture). The samples were gently rotated for 1 h at room temperature with 30 μl streptavidin–Sepharose high performance beads (GE Healthcare). After incubation, the beads were washed five times with binding buffer, and non-reducing sample buffer was added to the beads for elution. Western blots were performed using antifibrinogen antibody (Dako). Dot blots were per-formed using anti-A antibody 4G8 (Covance) to show comparable amounts 1059 of A were also being pulled down. The binding assay between fibrinogen and monomeric or oligomeric A42 Biotin-A42 monomers and oligomers were prepared as in (Stine et al., 2011). In brief, biotin-LC-A42 (Anaspec) was monomerized by treatment with hexafluoroisopropanol, dissolved to 5 mM with dimethyl sulfoxide, then diluted to 100 μM with cold PBS, and sonicated. Monomeric biotin- LC-A42 was incubated at 4°C for 24 h for oligomeric preparation. 1 nM fibrinogen was mixed with increasing concentrations of monomeric or oligomeric biotin-LC-A42 (0.5–20 nM) for 30 min at room temperature and the binding affinity was measured using AlphaLISA assay. The inhibitory efficacy of RU-505 on the interaction between fibrinogen and monomeric or oligomeric biotin-LC-A42 was accessed in dose–response experiments using AlphaLISA assay. In vitro thrombosis and fibrinolysis assay To test whether hit compound have an effect on fibrin clot formation and lysis, 20 μM of each compound (dissolved in 0.4% DMSO [final]) or DMSO control was incubated with fibrinogen (1.5 μM) in the presence or absence of A42 (3 μM) for 10 min and then mixed with plasminogen (0.25 μM) in 20 mM Hepes buffer (pH 7.4) with 137 mM NaCl. Fibrin clot formation and degradation was analyzed measuring turbidity right after adding throm-bin (0.5 U/ml), tPA (0.15 nM), and CaCl2 (5 mM) in a final volume of 150 μl. Assays were performed at RT in High Binding 96-well plates (Thermo Fisher Scientific) in triplicate and were measured at 450 nm using a Spectramax Plus384 reader (Molecular Devices). SPR SPR experiments were performed to test whether our lead compounds bind to A42 as described previously (Richter et al., 2010). Biacore 3000 instru-ment and CM5 sensor chips (GE Healthcare) were used for this assay. Hexa-fluoroisopropanol- treated monomerized A42 was immobilized to the sensor chip surface by amine coupling. Compounds were diluted to 40 μM from DMSO stock solutions in PBS as running buffer (final 2% DMSO) and injected for 2 min at a flow rate of 30 μl/min using the KINJECT command. After the dissociation phase the chip was rinsed with 20 mM HCl. Corre-sponding DMSO dilutions were used as a buffer blank, and a solvent correc-tion assay was performed to correct the difference of DMSO response between empty reference surface and protein-immobilized surface. Sulindac sulfide and sulindac were used as positive control and negative controls, re-spectively (Richter et al., 2010). In vivo toxicity study Maximum tolerated dose studies were performed to determine the toxicity of RU-505 (AMRI) and to identify the optimal dose for in vivo assays. Sin-gle injection toxicity was performed at Absorption Systems LP (Exton, PA), and four different doses (200, 100, 50, and 20 mg/kg mouse) of RU-505, along with saline and vehicle, were injected into male and female CD-1 mice intravenously. Mortality and overt clinical signs of toxicity were moni-tored for 2 d. All animals dosed with 200 mg/kg were found dead after single intravenous injection, and no clinical signs of toxicity were observed after single dose of 20, 50, or 100 mg/kg for 2 d after injection. Therefore, the maximum tolerated dose of RU-505 after single intravenous dose in mice was established as 100 mg/kg. (Greiner) and were incubated with 10 nM biotinylated A42 (Anaspec) and 1 nM fibrinogen for 30 min at RT in final volume of 10 μl assay buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 0.1% BSA). The mixture was incubated with anti–fibrinogen antibody (Dako), 20 μg/ml streptavidin-conjugated donor, and protein A–conjugated accep-tor beads (PerkinElmer) for 90 min at RT. Samples were read by a PerkinElmer EnVision plate reader. Hit compounds from the secondary assay were evaluated using Lipinski’s Rule of Five to determine whether each chemical compound has properties that make it a potential usable drug. If compounds violated more than one of Lipinski’s Rule of Five, those compounds were removed from our list. The AlphaScreen TruHits kit (PerkinElmer) was used to detect those compounds that react with singlet oxygen and thus unspecifically quench the assay. The AlphaScreen TruHits kit also allows for the identification of color quenchers, light scatterers (insoluble compounds), and biotin mimetics interfering with the AlphaLISA signal. If inhibition by quenching was more than 30% at 10 μM compound, those compounds were removed from our list. After completing the quenching test, we screened hit compounds in a dose–response experiment with various compound concentrations (0.01–20 μM) using FP and Alpha- LISA. The data were fitted to sigmoidal dose–response equation (Y = Bottom + (Top – Bottom)/1 + 10(logIC50 X) × Hill coefficient)) using GraphPad Prism 4 to calculate half-maximal inhibition (IC50) of each compound. Compounds with IC50 50 μM in both FP and AlphaLISA were purchased as powder and were retested in dose–response experiments using both assays. Analogue compound screening To improve our candidate compounds, we had access to the ChemNavigator database, which has 50 million commercially available compounds and software for Tanimoto-based similarity searching. We purchased 2,000 analogue com-pounds through ChemNavigator or directly from Albany Molecular Research Inc. These analogues were tested using AlphaLISA at 5, 10, and 20 μM. We se-lected compounds which have 50% inhibition at 10 μM and a proportional in-hibitory effect at 5 or 20 μM. Drug-like compounds were evaluated using Lipinski’s Rule of Five, and false-positive compounds were filtered out using the AlphaScreen TruHits kit (PerkinElmer) as described above. Compounds with IC50 10 μM in both FP and AlphaLISA were selected using dose–response ex-periments. Selected compounds were purchased as powder and were retested in dose–response experiments using both assays. Finally, we identified hit com-pounds of with IC50 3 μM in FP and IC50 10 μM AlphaLISA assay. Pull-down assay Hit compounds were tested using a pull-down assay as described previously (Ahn et al., 2010). In brief, compounds at 10 μM were incubated with 100 nM Table 3. Vendor list for primary screening library Provider No. of compound from each provider ChemDiv 21,986 Prestwick 1,110 Cerep 4,000 ChemBridge 5,000 Microsource 2,000 AMRI 50,000 Biofocus 7,750 GreenPharma 240 Sigma LOPAC 1,280 Prof. Derek Tan (Memorial Sloan- Kettering Cancer Center, New York, NY) 350 Total 93,716
- 78. Immunohistochemistry for infiltrated fibrinogen and microgliosis Mice were saline/heparin-perfused, and 20 μm coronal brain cryostat sections were fixed with 50% methanol and 50% acetone. For fibrinogen and endothe-lial cell staining, brain sections were incubated with FITC-conjugated an-tifibrinogen antibody (Dako) and anti-CD31 antibody (BD) overnight. For activated microglia staining, brain sections were incubated with anti-CD11b antibody (DSHB) overnight. After immunohistochemistry, brain sections were analyzed with a confocal microscope (Inverted DMI 6000; Leica) equipped with HyD detectors and HCX PL APO CS (10× NA 0.4 and 20× NA 0.7) objective lenses at room temperature. The imaging medium was air for both the objective lenses used and Leica Application Suite Advanced Fluorescence was used for image collection as software. Each set of stained sections was processed under identical gain and laser power setting and under identical brightness and contrast settings. Images of brain section were acquired and thresholded using ImageJ. The total area of infiltrated fibrinogen or activated microglia was analyzed as percentage of total cortex area with the analyzer blinded to treatment of mice. The average of 3 different sections from each mouse was determined (n = 3–4 mice per group). Behavioral analysis All behavioral experiments were performed and analyzed with a researcher blinded to genotype and treatment. We administered 35 mg/kg of RU-505 or vehicle to 4-mo-old Tg6799 mice and WT littermates and 25 mg/kg or vehicle to 4-mo-old TgCRND8 mice and WT littermates subcutaneously every other day for three months (analyzed at 7 mo of age). Mice were han-dled and allowed to acclimate to the testing room for 10 min per day for at least 5 d. Contextual fear conditioning During training, Tg6799 mice and WT littermates (n = 8–10 per group) were allowed to explore the training chamber (Med Associates, Inc.) for the first 2 min, and then received three mild footshocks (2 s, 0.7 mA) spaced 1 min apart. Mice were removed from the training chamber 30 s after the last foot shock. Contextual learning was assessed 24 h after training by reexpos-ing mice to the same training chamber for 3 min. Mouse behavior during training and testing was recorded, and freezing behavior was measured by observing mice every 5 s. Barnes maze The Barnes maze apparatus (TAP Plastics) consisted of a white circular plat-form (92 cm diam) with 20 equally spaced holes (5 cm in diameter; 7.5 cm between holes). Among these holes, one hole (target hole) was connected to a hidden black escape chamber. Bright lights (600 lx) were used to motivate the mice to find the target hole and enter into the escape chamber. Visual clues surrounded the maze. To remove any lingering scent on the maze from the previous animal, the platform and escape box were cleaned using 50% ethanol between mice. The entire experiment was recorded and analyzed using the Ethovision video tracking system (Noldus). Tg6799 mice. Training consisted of two training trials per day over a pe-riod of 7 d (n = 10–14 per group). During each trial, mice were placed in the center of the maze in a black starting box for 30 s. After 30 s, the box was re-moved, and mice were allowed to freely explore and find the target hole within 2 min. Latency to poke the target hole was recorded. If mice did not enter into the escape chamber within 2 min, they were gently guided into the escape chamber and placed in the chamber for 30 s. To assess memory retention, a probe trial was conducted 24 h and 3 d after the last training. The target hole was closed like the other 19 holes, and the escape chamber was removed. Holes were kept in the same position as during the training. Mice were placed in the center of the maze in a black starting box for 30 s. After 30 s, the box was removed, and mice were allowed to freely explore for 90 s. The number of visits into each hole and the latency to reach the target hole were recorded. For analysis, scores of each mouse from both probe trials were combined and averaged. Because AD treatment would be long-term and toxicity of long-term treatment can be different from toxicity after a single injection, we treated Tg6799 mice and WT littermates with two doses (100 and 50 mg/kg) of RU-505 every other day for 3 mo, and overt clinical signs of toxicity were monitored. After 3 mo, mice were sent to the Laboratory of Comparative Pathology at Memorial Sloan-Kettering Cancer Center for complete nec-ropsy and hematology reports to determine the effects of our lead compound after long-term treatment. Pharmacokinetics The pharmacokinetics of and BBB permeability to RU-505 were deter-mined by assessing the drug’s decay in blood plasma and brain homogenates over a 24-h period after subcutaneous injection (35 mg/kg) into WT mice of the same genetic background as Tg6799. Blood was collected in heparinized tubes after cardiac puncture 0.5, 1, 2, 4, 6, and 24 h after RU-505 administra-tion. After perfusion, brains were collected and homogenized with PBS. Plasma and brain homogenates were sent to Apredica and were analyzed by LC/MS/MS using an Agilent 6410 mass spectrometer coupled with an Agi-lent 1200 HPLC. This analysis revealed that RU-505 penetrates the blood brain barrier, and RU-505 levels in the brain were equal to or greater than in the blood (3 μM). In vivo thrombosis assay To observe blood circulation and to induce thrombosis, a cranial window was prepared as described previously (Cortes-Canteli et al., 2010). In brief, a cra-nial window was prepared over the parietal cortex of 7-mo-old Tg6799 mice and WT littermates which were treated with RU-505 (35 mg/kg) or vehicle for three months (n = 5 per group). Mice were anesthetized by i.p. injection of 500 mg/kg tribromethanol and 0.04 mg/kg atropine and placed in a cus-tom built restraint system. A 2.5-mm circular craniotomy was prepared using 5–10 circular brush strokes with a fine dental drill bit, and a 4-mm plastic ring surrounding the window was attached with dental acrylic and cyanoacrylate adhesive. Sterile saline was applied periodically to protect the brain surface and prevent drying. For imaging of blood flow, 100 μl of 5 mg/ml 2 MDa FITC-conjugated dextran (Sigma-Aldrich) dissolved in PBS was ad-ministered by retroorbital injection. During the entire imaging session, the body temperature of mice was kept at 37.5°C using a TC-1000 Mouse com-plete temperature control system (CWE Inc.). Increasing concentrations of FeCl3 (5, 10, and 15%) were added directly to the brain surface with an interval of 5 min, and thrombosis was recorded using Olympus IX71 microscope equipped with Hamamatsu Orca ER B/W digital camera. MetaMorph acquisition software and 5× objective lens (NA = 0.25) were used for image collection. The total length of vessels with a 20 μm diam before FeCl3 treatment and the total length of occluded vessels at 5 min after each FeCl3 treatment (5, 10, and 15%) were measured. All analysis was performed using National Institutes of Health ImageJ software with the analyzer blinded to genotype and treatment of mice. Immunohistochemistry for CAA and A plaques Mice were saline/heparin-perfused, and 20-μm coronal brain cryostat sec-tions were fixed with 4% paraformaldehyde. Brain sections were incubated with rabbit anti–laminin antibody (Sigma-Aldrich) overnight and stained for 30 min with 0.2% Congo Red (Sigma) in 70% isopropanol. After immuno-histochemistry, brain sections were analyzed with a microscope (Axiovert 200; Carl Zeiss) equipped with Plan-Neofluar (10× NA 0.3, and 20× NA 0.5) objective lenses at room temperature. The imaging medium was air for both the objective lenses used. The AxioCam color camera (Carl Zeiss) and AxioVision software (Carl Zeiss) were used for image collection. Each set of stained sections was processed under identical gain and laser power set-ting and under identical brightness and contrast settings. Images of all the areas with CAA and A plaques were acquired and thresholded using Image J. The total area of CAA and A plaques was analyzed as percentage of total cortex area with the analyzer blinded to treatment of mice. The av-erage of 7–10 different sections from each mouse was determined (n = 5 mice per group). 1060 A-fibrin interaction inhibitor as AD treatment | Ahn et al.
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During training, mice were placed in the center of the maze in a black starting box for 30 s. After 30 s, the box was removed, and mice were allowed to freely explore and find the target hole for 5 min. To assess memory retention, probe trials were conducted 24 h and 6 d after the last training. Mice were allowed to freely explore for 2 min during probe trials. The number of visits into each hole and the latency to reach the target hole were recorded. For analysis, scores of each mouse from both probe trials were combined and averaged. Statistical analysis All numerical values presented in graphs are mean ± SEM. Statistical signifi-cance of most experiments was determined using two-tailed t test analysis comparing control and experimental groups. The pull-down assay (Fig. 2 A) was analyzed using one-way ANOVA and Bonferroni post hoc test. Com-parison of training curves from the Barnes maze (Fig. 5 C and Fig. 6 A) was analyzed using two-way ANOVA with repeated measure and Bonferroni post hoc test. Online supplemental material Video S1–S4 show intravital visualization of blood flow and blood vessel occlusion in Tg6799 mice or WT littermates that were treated with RU-505 or vehicle as increasing concentrations of FeCl3 (5%, 10%, and 15%) were added directly to the brain surface. Online supplemental material are avail-able at http://www.jem.org/cgi/content/full/jem.20131751/DC1. The authors thank The Rockefeller University Bio-Imaging Resource Center for technical assistance, as well as Dr. Marta Cortes-Canteli, Dr. Zu-Lin Chen, and members of the Strickland Laboratory for discussion. This work was supported by the Thome Memorial Medical Foundation, Alzheimer’s Drug Discovery Foundation, National Institutes of Health (NS50537), Woodbourne Foundation, Mellam Family Foundation, May and Samuel Rudin Family Foundation, and the Blanchette Hooker Rockefeller Fund. The authors have no conflicting financial interests. Submitted: 20 August 2013 Accepted: 27 March 2014 REFERENCES Adams, R.A., J. Bauer, M.J. Flick, S.L. Sikorski, T. Nuriel, H. Lassmann, J.L. Degen, and K. Akassoglou. 2007. The fibrin-derived gamma377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease. J. Exp. Med. 204:571–582. http://dx.doi.org/10.1084/jem.20061931 Ahn, H.J., D. Zamolodchikov, M. Cortes-Canteli, E.H. Norris, J.F. Glickman, and S. Strickland. 2010. Alzheimer’s disease peptide beta-amyloid inter-acts with fibrinogen and induces its oligomerization. Proc. Natl. Acad. Sci. 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- 81. Ar t icle Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer’s disease Jong Kil Lee,1,2,3 Hee Kyung Jin,1,4 Min Hee Park,1,2,3 Bo-ra Kim,1,2,3 Phil Hyu Lee,5 Hiromitsu Nakauchi,6 Janet E. Carter,7 Xingxuan He,8 Edward H. Schuchman,8 and Jae-sung Bae1,2,3 1Stem Cell Neuroplasticity Research Group, 2Department of Physiology, Cell and Matrix Research Institute, School of Medicine, 3Department of Biomedical Science, BK21 Plus KNU Biomedical Convergence Program, 4Department of Laboratory Animal Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Korea 5Department of Neurology and Brain Research Institute, Yonsei University College of Medicine, Seoul 120-752, Korea 6Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan 7Mental Health Sciences Unit, Faculty of Brain Sciences, University College London, London WC1E 6DE, England, UK 8Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029 In Alzheimer’s disease (AD), abnormal sphingolipid metabolism has been reported, although the pathogenic consequences of these changes have not been fully characterized. We show that acid sphingomyelinase (ASM) is increased in fibroblasts, brain, and/or plasma from patients with AD and in AD mice, leading to defective autophagic degradation due to lysosomal depletion. Partial genetic inhibition of ASM (ASM+/) in a mouse model of famil-ial pathological findings, including reduction of amyloid- (A) deposition and improvement of memory impairment. Similar effects were noted after pharmacologic restoration of ASM to the normal range in APP/PS1 mice. Autophagic dysfunction in neurons derived from FAD patient induced pluripotent stem cells (iPSCs) was restored by partial ASM inhibition. Overall, these results reveal a novel mechanism of ASM pathogenesis in AD that leads to defective autophagy due to impaired lysosomal biogenesis and suggests that partial ASM inhibition is a potential new therapeutic intervention for the disease. The Rockefeller University Press $30.00 J. Exp. Med. 2014 Vol. 211 No. 8 1551-1570 www.jem.org/cgi/doi/10.1084/jem.20132451 AD (FAD; amyloid precursor protein [APP]/presenilin 1 [PS1]) ameliorated the autopha-gocytic 1551 defect by restoring lysosomal biogenesis, resulting in improved AD clinical and Alzheimer’s disease (AD) is the most common form of dementia. It is characterized clinically by progressive loss of memory, and pathologi-cally by the presence of neuritic plaques and neurofibrillary tangles (Selkoe, 2001). There are profound biochemical alterations in multiple pathways in the AD brain, including changes in amyloid- (A) metabolism, tau phosphoryla-tion, and lipid regulation, although to date the underlying mechanisms leading to these complex abnormalities, as well as the downstream conse-quences, remain largely unknown (Yankner et al., 2008; He et al., 2010; Mielke et al., 2012). Sphingolipid metabolism is an important pro-cess for tissue homeostasis that regulates the formation of several bioactive lipids and second messengers that are critical in cellular signaling (Lahiri and Futerman, 2007; Wymann and Schneiter, 2008). In the brain, the proper bal-ance of sphingolipid metabolites is essential for normal neuronal function, and subtle changes in sphingolipid homeostasis may be intimately in-volved in neurodegenerative diseases including AD (Cutler et al., 2004; Grimm et al., 2005; Hartmann et al., 2007; Grösgen et al., 2010; Haughey et al., 2010; Mielke and Lyketsos, 2010; Di Paolo and Kim, 2011; Tamboli et al., 2011). Recently, our studies and those of others (Katsel et al., 2007; He et al., 2010) have shown that the activity of several sphingolipid metabolizing enzymes, including acid sphingomyelinase CORRESPONDENCE Jae-sung Bae: jsbae@knu.ac.kr Abbreviations used: A, amyloid-; AC, acid ceramidase; AD, Alzheimer’s disease; ALP, autophagy–lysosome pathway; AMI, amitriptyline-hydrochloride; AP, alkaline phosphatase; ApoE4, apolipoprotein E4; APP, amyloid precursor protein; ASM, acid sphingomyelinase; AV, autophagic vacuole; CM, conditioned medium; EM, electron microscope; FAD, familial AD; i.c., intracerebral; iPSC, induced pluripotent stem cell; Lamp1, lysosomal-associated membrane protein 1; LBPA, lysobisphosphatidic acid; LC3, microtubule-associated protein 1 light chain 3; M6P, mannose-6- phosphate; NPD, Niemann- Pick disease; PD, Parkinson’s disease; PS1, presenilin 1; SA--gal, senescence-associated- -galactosidase; TFEB, tran-scription factor EB. J.K. Lee and H.K. Jin contributed equally to this paper. © 2014 Lee et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial– Share Alike 3.0 Unported license, as described at http://creativecommons.org/ licenses/by-nc-sa/3.0/).
- 82. Figure 1. ASMis increased in AD and complete ASM gene deficiency exacerbates pathology of APP/PS1 mice. (A and B) ASM was estimated in the blood plasma (A; control, n = 30; AD, n = 40; and PD, n = 20) and fibroblast (B; control, n = 24; PS1-FAD, n = 24; ApoE4, n = 24; and PD, n = 12) with AD, PD, or normal controls. (C) ASM activity did not show passage differences between AD and normal fibroblasts (n = 8 per passage group). (D) Detection of sphingomyelin, ceramide, and AC in plasma (control, n = 20–22; and AD, n = 33–35) and fibroblast (control, n = 12; PS1-FAD, n = 18; and ApoE4, n = 18). (E) Crossing scheme to generate WT, APP/PS1, ASM/, and APP/PS1/ASM/ mice. PCR-based genotyping to detect WT, APP/PS1, ASM/, and APP/PS1/ ASM/ mice. (F) Survival curves of WT (n = 26), APP/PS1 (n = 30), ASM/ (n = 30), and APP/PS1/ASM/ (n = 25) mice. (G) Body weights of WT, 1552 Role of ASM in the pathogenesis of AD | Lee et al.
- 83. JEM Vol. 211,No. 8 Ar t icle 1553 (ASM), are abnormal in the brains of AD patients. ASM is ex-pressed by almost all cell types and has an important house-keeping role in sphingolipid metabolism and membrane turnover. It is mainly located within the endosomal/lysosomal compartment but is associated with the cellular stress response and may become preferentially transported to the outer leaf-let of the cell membrane under conditions of cell stress ( Jenkins et al., 2009). Mutations in the ASM gene (SMPD1) lead to the type A and B forms of the lysosomal storage disorder Niemann-Pick disease (NPD). In addition to its role in NPD, the importance of ASM in numerous signaling processes, including cell death, inflammation, and autophagy, has been extensively documented in several pathological conditions (Santana et al., 1996; Górska et al., 2003; Petrache et al., 2005; Lang et al., 2007; Smith and Schuchman, 2008; Teichgräber et al., 2008; Sentelle et al., 2012). However, the role of ASM in AD and the cellular mechanisms that link ASM and AD have not been fully characterized. This lack of understanding between the correlation of altered ASM levels and AD patho-physiology led us to explore the mechanisms underlying ASM’s role in AD pathogenesis. Here, we show for the first time that increased ASM activity in AD causes a defect of au-tophagic degradation due to disruption of lysosomal biogen-esis and integrity, and that partial inhibition of ASM activity leads to restoration of autophagy and improvement of patho-logical and clinical findings in AD mice. RESULTS ASM activity is increased in AD patients We first sought to confirm whether sphingolipid metabolism is altered in AD patient samples. We examined ASM and acid ceramidase (AC) activities, and the levels of several sphingo-lipids, including sphingomyelin and ceramide, in samples from normal individuals and AD patients. Consistent with previous results (He et al., 2010), ASM was significantly increased in plasma and fibroblasts from individuals with AD compared with normal aged individuals (Fig. 1, A and B). To assess whether increased ASM activity was an AD-specific signature, we analyzed ASM activity in samples from individuals with Parkinson’s disease (PD). The activity of ASM was not ele-vated in PD-derived samples compared with normal (Fig. 1, A and B). ASM activity also did not show passage differences between AD and normal fibroblasts (Fig. 1 C). Sphingomyelin levels were decreased in the AD plasma compared with nor-mal (Fig. 1 D). No significant differences in the ceramide and AC levels were found between the two groups (Fig. 1 D). These results confirmed that elevation of ASM, an important sphingolipid-modulating factor, is AD specific and may influ-ence disease progression and/or pathogenesis. Partial ASM inhibition in AD mice reduces pathology To investigate the influence of ASM on AD pathology, we first generated amyloid precursor protein (APP)/presenilin 1 (PS1) double mutant and APP/PS1/ASM/ triple mutant mice (Fig. 1 E). AD-related pathologies in APP/PS1 mice normally begin at 6–7 mo of age; however, our APP/PS1/ ASM/ mice died young (Fig. 1 F). We presume that the early death of the APP/PS1/ASM/ animals was due to their ASM/ phenotype because these animals (originally developed as a model of the neurodegenerative type A NPD) usually die by 6–8 mo of age. The APP/PS1/ASM/ mice showed significantly decreased body weight compared with APP/PS1 mice (Fig. 1 G), and indicators of brain injury, such as cell death and inflammation, were significantly increased (Fig. 1, H–J). These data demonstrated that complete deletion of ASM in APP/PS1 mice exacerbated brain pathology, and that APP/PS1/ASM/ mice were not suitable to examine the correlation of ASM and AD pathology. To overcome these obstacles, we generated APP/PS1/ ASM+/ triple mutant mice (with partial genetic deletion of the ASM gene; Fig. 2, A and B). Similar to AD patients, ASM activity was elevated in plasma, brain, and fibroblasts of 9-mo-old APP/PS1 mice (Fig. 2 C), likely due to the stress response re-lated to the progression of AD-like disease in these animals. Next, to further investigate the cell contribution of increased ASM activity in AD mouse brain, we isolated neurons and microglia from the brain. Although ASM activity was slightly increased in APP/PS1 microglia compared with WT microg-lia, the degree of ASM increase was greater in neurons than microglia (Fig. 2 D), indicating that neurons were the main contributor of elevated ASM activity in AD mouse brain. Importantly, ASM activity in age-matched APP/PS1/ ASM+/ mice was significantly decreased compared with the APP/PS1 mice to levels within the normal range or lower (Fig. 2 C). Other sphingolipid factors were unaltered in the APP/PS1/ASM+/ mice except sphingomyelin, which was modestly reduced in APP/PS1 mice and elevated in the triple mutant animals (Fig. 2 E). To determine whether the reduced ASM activity in the APP/PS1/ASM+/ mice affected AD pathology, we first determined the A profile. Thioflavin S staining, immunofluorescence, and ELISA results of A40 and A42 showed significantly lower A levels in the 9-mo-old APP/PS1/ ASM+/ mice compared with age-matched APP/PS1 mice (Fig. 2, F–I). In APP/PS1/ASM+/ mice, cerebral amyloid an-giopathy and C-terminal fragment of APP were also reduced (Fig. 2 J; and see Fig. 4, D and F). There were no significant differences of tau hyperphosphorylation between the two groups (Fig. 2 K). APP/PS1, ASM/, and APP/PS1/ASM/ mice were determined at the indicated ages (n = 6–7 per group). (H–J) Brain sections from 7-mo-old mice were immunostained with anti–active caspase3 (H; n = 4 per group; bars, 50 μm), anti-GFAP (I; n = 4 per group; bars, 100 μm), and anti–Iba-1 (J; n = 4 per group; bars, 100 μm). Data are representative of three independent experiments. A–D and G, Student’s t test. H–J, one-way ANOVA, Tukey’s post hoc test. *, P 0.05; **, P 0.01. All error bars indicate SEM.
- 84. Figure 2. Partialgenetic inhibition of ASM leads to decreased AD pathology in the APP/PS1 mice. (A) Generation of the APP/PS1/ASM+/ mice. (B) Body weights of WT, APP/PS1, ASM+/, and APP/PS1/ASM+/ mice were determined at 9 mo of age (n = 14 per group). (C) ASM activity in blood plasma (n = 14–15 per group), brain (n = 13–14 per group), and fibroblast (n = 8 per group) derived from WT, APP/PS1, ASM+/, and APP/PS1/ASM+/ mice. (D) ASM activity was assessed in neuron and microglia isolated from mouse brain (WT, n = 8; APP/PS1, n = 6; and APP/PS1/ASM+/, n = 6). (E) Detection of sphingomyelin, ceramide, and AC in 1554 Role of ASM in the pathogenesis of AD | Lee et al.
- 85. Figure 3. Partialgenetic inhibition of ASM prevents memory impairments in APP/PS1 mice. (A) Learning and memory was assessed by Morris water maze test in the WT (n = 13), APP/PS1 (n = 12), ASM+/ (n = 12), and APP/PS1/ASM+/ (n = 12) mice (B–F) Probe trial day 11. (B) Time spent in target platform and other quadrants was measured. (C and D) Path length (C) and swim speed (D) were analyzed. (E) The number of times each animal entered the small target zone during the 60-s probe trial. (F) Representative swimming paths at day 10 of training. (G) The freezing response during the training session. Bars show exposure to the tone and arrows the application of the footshock. (H) The results of contextual and tone tasks (WT, n = 14; APP/PS1, n = 14; ASM+/, n = 13; and APP/PS1/ASM+/, n = 13). Data are representative of three independent experiments. A, C, D, E, and H, one-way ANOVA, Tukey’s post hoc test. B, Student’s t test. *, P 0.05; **, P 0.01. All error bars indicate SEM. JEM Vol. 211, No. 8 Ar t icle 1555 Next, to assess the potential effect of partial genetic ASM inhibition on learning and memory in APP/PS1 mice, we per-formed the Morris water maze task and fear conditioning. Aged APP/PS1 mice showed severe deficits in memory formation and APP/PS1/ASM+/ mice were largely protected from this defect (Fig. 3). Collectively, these results suggested that re-stored ASM activities to the normal range in APP/PS1 mice decreased A load and improved learning and memory. Partial ASM inhibition reverses defective autophagy in AD mice A reduction in APP/PS1/ASM+/ mouse cerebral amyloido-sis could be due to a decreased inflammatory response, atten-uated APP expression, or activation of proteases involved in A degradation. We first assessed the apoptotic and inflam-matory responses in brain samples derived from APP/PS1 and APP/PS1/ASM+/ mice but did not detect differences between plasma (n = 8–10 per group), brain (n = 7–9 per group), and tail (n = 5–6 per group) fibroblast. (F) Mice brain sections were stained with thioflavin S in APP/PS1 and APP/PS1/ASM+/ mice. The relative area occupied by A plaques were determined (n = 6–7 per group; bars, 100 μm). (G–I) Analysis of A40 and A42 depositions from the mice brain samples using immunofluorescence staining (G and H; n = 6–7 per group; bars, 200 μm) and ELISA kits (I; n = 8 per group). (J and K) Confocal laser microscope images and quantification of cerebral amyloid angiopathy (J; n = 6 per group; bars, 50 μm) and tau hyperphosphorylation (K; n = 6 per group; bars, 20 μm) in APP/PS1 and APP/PS1/ASM+/ mice. Data are representative of two (D and K), three (B, C, and E), or four (F–J) independent experiments. B–E, one-way ANOVA, Tukey’s post hoc test. F–K, Student’s t test. *, P 0.05; **, P 0.01; ***, P 0.005. All error bars indicate SEM.
- 86. Figure 4. Geneticinhibition of ASM does not affect inflammatory pathway and processing of APP. Brain sections of APP/PS1 and APP/PS1/ASM+/ mice were stained with active caspase3 (A; n = 5 per group; bars, 50 μm; arrows indicate active caspase3-positive cells) and GFAP antibody (B; n = 6 per group; bars, 100 μm). (C) mRNA levels of proinflammatory cytokines or antiinflammatory cytokines (n = 4–5 per group). (D) Mouse brain lysates were tested for APP and -CTF levels using Western blot analysis. (E and F) Quantification of APP (E) and -CTF (F) levels (n = 6 per group). (G) Western blot analysis for Bace-1 levels (n = 6 per 1556 Role of ASM in the pathogenesis of AD | Lee et al.
- 87. JEM Vol. 211,No. 8 Ar t icle To examine how genetic inhibition of ASM affected the autophagic pathway in AD, we also analyzed fibroblasts and brain samples derived from 9-mo-old WT, APP/PS1, APP/ PS1/ASM+/, and ASM+/ mice. Compared with WT, APP/ PS1 mice showed increased LC3-II, similar to human AD fibroblasts. This enhanced LC3-II level was reduced in APP/ PS1/ASM+/ mice. Beclin-1 expression did not vary between the groups (Fig. 5, F, G, K, and L). Metabolic analysis of pro-tein turnover (Cuervo et al., 2004) was assessed using fibro-blasts from WT, APP/PS1, and APP/PS1/ASM+/ mice. Under culture conditions that induced autophagy (absence of serum), degradation of long-lived proteins was significantly lower in cells from APP/PS1 mice compared with WT mice but was increased in cells derived from APP/PS1/ASM+/ mice (Fig. 5 I). The differences of cell senescence levels in cultured mice fibroblasts were not found between the groups, indicating that these changes were not related to the cell se-nescence (Fig. 5 J). The levels of cathepsin D, a lysosomal hy-drolase, were elevated in APP/PS1 mice compared with WT. Enhanced cathepsin D level was ameliorated in APP/PS1/ ASM+/ mice (Fig. 5, F, G, K and L). However, the activity of cathepsin D was not changed between the groups (Fig. 5, H and M). This result indicates that the elevated levels of ca-thepsin D in APP/PS1 mice did not ultimately translate into a significant increase of enzyme activity. We also analyzed the expression of p62, indicator of autophagic turnover. Increased p62 levels in APP/PS1 mice were reduced in APP/PS1/ ASM+/ mice (Fig. 5, F, G, K and L). To corroborate the immunoblotting results, we performed electron microscope (EM) analysis using mouse brain samples. As previously reported (Yu et al., 2005), APP/PS1 mouse brain regions showed an increased number of autophagic vacuoles (AVs), whereas brains of APP/PS1/ASM+/ mice showed a reduced number of these vesicles, albeit still higher than WT mice (Fig. 5 N). The endocytic pathway is also considered a major con-tributor to A deposition in AD (Ginsberg et al., 2010; Li et al., 2012). To determine whether the endocytic pathway was affected by partial ASM inhibition, we examined Rab5 and Rab7 expression in our animals. The expression pattern of these proteins showed no difference between the groups (Fig. 5 O). Although additional studies of endocytic pathway are required to identify the exact mechanism, our results showed that en-docytic pathway was not a main mechanism by ASM inhibi-tion. Collectively, these results revealed dysfunctional changes in the turnover of AVs in the APP/PS1 mice, and that partial genetic ASM inhibition could reverse this abnormality and improve autophagic degradation of proteins. 1557 the two strains (Fig. 4, A–C). To determine whether reduc-tion of ASM activity affected APP expression, we compared the levels of APP in the two strains. We found that partial ge-netic inhibition of ASM did not influence the overall expres-sion levels of APP (Fig. 4, D and E). We also examined the A generating enzyme Bace-1 using brain homogenates. Bace-1 was slightly decreased in APP/PS1/ASM+/ mice compared with APP/PS1 mice, but the reduction did not reach statisti-cal significance (Fig. 4 G). To address A clearance by mi-croglia, we analyzed microglia activation and A degrading enzyme release by microglia but again did not detect differ-ences (Fig. 4, H and I). Similarly, these changes did not show any differences between APP/PS1 and APP/PS1/ASM+/ mice in 5-mo-old young mice (Fig. 4, J–M). There were also no significant differences of brain pathology, such as apopto-sis, inflammation, and A deposition, between the WT and ASM+/ mice (Fig. 4, A–I). Overall, these data suggested that the partial inhibition of ASM in APP/PS1/ASM+/ did not alter major inflammatory pathways or expression of APP. Dysfunction of the normal proteolytic degradation system also could affect AD pathogenesis and lead to enhanced A deposition (Lee et al., 2010b). Autophagy, a major degradative pathway of the lysosomal system, is known to be markedly impaired in AD (Boland et al., 2008). We found that the microtubule-associated protein 1 light chain 3 (LC3)–II levels were significantly increased in human AD-derived fibroblasts compared with control fibroblasts (Fig. 5, A–C). Increased LC3-II levels could stem from overinduction of autophagy or may be a product of reduced autophagic turnover and defects in the latter stages of autophagic degradation. We therefore measured the level of beclin-1 expression in the AD cells, which is part of a kinase complex responsible for autophagy induction (Zeng et al., 2006), and found that it did not vary between the groups (Fig. 5, A and B). To examine autophagic turnover of protein, we then ana-lyzed proteolysis of long-lived proteins (Lee et al., 2010b) in control and AD fibroblasts. When autophagic/lysosomal degra-dation was induced through serum withdrawal, proteolysis was increased in control fibroblasts but not significantly changed in PS1 and apolipoprotein E4 (ApoE4)–derived AD patient fibro-blasts (Fig. 5 D). Lysosome stability relating to autophagy also could be affected by lysobisphosphatidic acid (LBPA) binding with ASM (Kirkegaard et al., 2010). We therefore measured LBPA immunofluorescence intensity in control and AD fibroblasts, and found that it did not vary between the groups (Fig. 5 E). Collectively, these data indicated that the autophagosome ac-cumulation in AD is due to dysregulation of autophagic pro-tein degradation, similar to previous results (Lee et al., 2010b). group). (H) Immunofluorescence images of Iba-1 in the APP/PS1 and APP/PS1/ASM+/ mouse brain (bars, 100 μm). The relative area occupied by Iba-1–positive cells was quantified (n = 6 per group). (I) The expression of NEP, IDE, and MMP9 was measured in the brain with quantitative real-time RT-PCR (n = 4–5 per group). (J) Immunofluorescence images of GFAP-positive cells in the 5-mo-old WT, APP/PS1, and APP/PS1/ASM+/ mouse brain (bars, 100 μm). The relative area occupied by GFAP-positive cells was quantified (n = 6–7 per group). (K) Western blot analysis and quantification for APP and -CTF levels in the 5-mo-old mice (n = 6 per group). (L) Western blot analysis for Bace-1 levels in the 5-mo-old mice (n = 6 per group). (M) Immunofluorescence images of Iba-1 in the in the 5-mo-old mouse brain (bars, 100 μm). The relative area occupied by Iba-1–positive cells was quantified (n = 6 per group). Data are representative of three (A–H) or two (J–M) inde-pendent experiments. A–C, G–J, L, and M, one-way ANOVA, Tukey’s post hoc test. E, F, and K, Student’s t test. *, P 0.05. All error bars indicate SEM.
- 88. ASM elevation causesdefective autophagic degradation by lysosomal depletion To gain more direct insights into the relationship of ASM and autophagic dysfunction, we treated human fibroblasts and neu-rons with recombinant 1–10 μM ASM and determined the LC3-II and p62 levels. ASM strongly accelerated LC3-II and Figure 5. Partial genetic inhibition of ASM reverses defective autophagy in APP/PS1 mice. (A) Western blot analysis of LC-3 and beclin-1 levels in controls, PS1-FAD, and ApoE4 fibroblasts. (B) LC3-II and beclin-1 levels were quantified (n = 4 per group). (C) Immunocytochemistry for LC3 in controls, PS1-FAD, and ApoE4 fibroblast (n = 4–5 per group; bars, 20 μm). (D) Degradation of long-lived proteins was measured in controls, PS1- FAD, and ApoE4 fibroblasts (n = 6 per group). (E) Representative images and quantification of LBPA in control, PS1-FAD, and ApoE4 fibroblast (n = 4 per group; bars, 50 μm). (F) Western blot analyses for LC3, beclin-1, p62, and cathepsin D in tail fibroblast derived from WT, APP/PS1, ASM+/, and APP/PS1/ ASM+/ mice. (G) Densitometric analysis of LC-3-II, beclin-1, p62, and cathepsin D (n = 7–8 per group). (H) Cathepsin D activity in mice tail fibroblast (n = 4 per group). (I) Rates of proteolysis of long-lived proteins in fibro-blasts (n = 6 per group). (J) Representative images and quantification data of SA--gal staining in the mice tail fibroblasts (n = 5 per group; bars, 50 μm). (K) Western blot analy-ses for LC3, beclin-1, p62, and cathepsin D in the brains of 9-mo-old WT, APP/PS1, ASM+/, and APP/PS1/ASM+/ mice. (L) Densitometric quantification of LC-3-II, beclin-1, p62, and cathepsin D (n = 6–8 per group). (M) Cathep-sin D activity in brain extracts of WT, APP/ PS1, ASM+/, and APP/PS1/ASM+/ mice (n = 4 per group). (N) EM images and quantifica-tion data of cortical region. Higher magnifi-cation of boxed area shows detail of AVs (arrow; n = 5 per group; bars: [low magnifi-cation] 2 μm, [high magnification] 1 μm). (O) Western blot analysis of Rab5 and Rab7 levels in the brain lysates (n = 5 per group). Data are representative of two (A–E and N) or three (F–M and O) independent experiments. B–O, one-way ANOVA, Tukey’s post hoc test. *, P 0.05. All error bars indicate SEM. p62 levels in human fibroblasts and neurons in a concentration-dependent manner (Fig. 6, A–C). The level of beclin-1 ex-pression was not affected by ASM (Fig. 6, A and C), indicating that the accumulation of autophagosomes was not due to the biogenesis pathway. ASM is found in a secretory and a lysosomal form ( Jenkins et al., 2009), and the mannose-6-phosphate (M6P) 1558 Role of ASM in the pathogenesis of AD | Lee et al.
- 89. Figure 6. Autophagicprocesses are affected by lysosomal ASM. (A and C) Western blot analysis of LC3, Beclin-1, and p62 in human fibroblast (A; n = 7 per group) and human neuron (C; n = 6 per group). (B) Immunocytochemistry and quantification for LC3 after ASM treatment (n = 7–8 per group; bars, 20 μm). (D) ASM activity was assessed in the ASM-treated fibroblast with or without M6P (n = 6 per group). (E) Confocal microscopic analysis of Lamp1- and ASM-positive vesicles (bars, 20 μm). (F and G) The effect of lysosomal ASM on LC3-II expression. (F) 10 μM ASM was added to fibroblast for 24 h with or without 10 mM M6P. LC3-II expression was determined by Western blot analysis (n = 5–6 per group). (G) LC3-II levels were examined in 10 μM ASM-treated fibroblast with or without M6P receptor suppression using siRNA (n = 5–6 per group). (H) The effect of 10 μM ASM on cell viability was estimated by MTT assay (n = 5–6 per group). (I and J) Representative images and quantification data of LC3 (I; bars, 20 μm) and SA--gal staining (J; bars, 100 μm) in P5, P10, and P20 human fibroblasts. NH4Cl and H2O2 were used for positive control (n = 5 per group). Data are representative of three (B, E, I, and J) or four (A, C, D, and F–H) independent experiments. A, C, F, G, I, and J, one-way ANOVA, Tukey’s post hoc test. B, D, and H, Student’s t test. *, P 0.05; **, P 0.01; ***, P 0.005. All error bars indicate SEM. JEM Vol. 211, No. 8 Ar t icle ASM-treated cells compared with nontreated cells. This en-hanced ASM activity was reduced in the presence of M6P (Fig. 6 D). To confirm whether the ASM treatment reached the lysosomes, we also examined the colocalization of ASM and ly-sosomes using immunocytochemistry. Double immunostaining 1559 receptor system is involved in trafficking of ASM to the lyso-some (Dhami and Schuchman, 2004). To elucidate which ASM form affected lysosomal/autophagic dysfunction, cells were incubated with ASM alone, or in the presence of M6P. As ex-pected, the activity of ASM was significantly increased in
- 90. Figure 7. ASMcauses abnormal autophagic protein degradation by altering ALP. (A) Autophagic flux assay. Human fibroblasts were cultured in: (1) complete medium with or without 10 μM ASM in the presence or absence of NH4Cl (left), (2) complete medium or starvation condition in the presence or absence of NH4Cl (middle), or (3) complete medium or starvation condition with or without 10 μM ASM (right). The LC3-II levels were examined by West-ern blotting (n = 6–7 per group). (B) The accumulation of p62 was assessed in the human fibroblast cultured with 10 μM ASM, 20 mM NH4Cl, or starvation 1560 Role of ASM in the pathogenesis of AD | Lee et al.
- 91. JEM Vol. 211,No. 8 Ar t icle To further investigate the relationship of elevated ASM and defective lysosomal/autophagic degradation, we evalu-ated alteration in lysosomal pH using the acidotropic dye LysoTracker red. H2O2- and NH4Cl-treated cells were used as positive and negative controls, respectively. Flow cytometry and fluorescent microscopic analysis did not show any differ-ences of lysosomal pH between ASM- and vehicle-treated fibroblast (Fig. 7 E). Recently, the transcription factor EB (TFEB) was identified as a master regulator of the autophagy– lysosome pathway (ALP) and lysosome biogenesis (Settembre et al., 2011). Enhancement of TFEB function is able to stim-ulate ALP function and promote protein clearance. To ex-amine whether ASM could affect the ALP and lysosome biogenesis, we tested endogenous levels of TFEB. ASM-treated fibroblasts and neurons showed significantly decreased TFEB levels (Fig. 7, F and G). Also, the levels of the lysosomal structural protein Lamp1 were decreased in ASM-treated cells (Fig. 7, F–H). To further validate our observation, we inves-tigated TFEB subcellular localization after ASM treatment. Interestingly, ASM-treated cells showed a reduced TFEB expression in the nuclear compartment (Fig. 7 I). Similarly, the expression levels of TFEB target genes related to lyso-some were significantly decreased in ASM-treated fibroblasts (Fig. 7 J). Conversely, to determine whether autophagic deg-radation affected ASM, we evaluated ASM activity in fibro-blasts after NH4Cl treatment. Blocking of autophagic degradation via NH4Cl did not show any significant changes of ASM ac-tivity (Fig. 7 K). These results further suggested that lyso-somal ASM acts not as an inducer but rather as an inhibitor of autophagic protein degradation by reducing ALP function and lysosome biogenesis. To examine the in vivo effect of ASM activation on au-tophagic dysfunction, we introduced conditioned medium (CM) from cultured ASM-overexpressing cells into C57BL/6 mice via intracerebral (i.c.) and intravenous (i.v.) injections. ASM-CM–treated mice showed elevated ASM activity in the brain and plasma (Fig. 8, A and B), as well as increased LC3-II without changes of beclin-1 expression in the brains (Fig. 8, C and D). Cathepsin D level was increased in the ASM-CM (i.c.)–treated mice compared with control mice, but actual activity was not changed (Fig. 8, C and E). p62 also was in-creased in the ASM-CM (i.c.)–treated mice compared with control mice (Fig. 8 C). ASM-CM (i.c.)–treated mice further exhibited abnormal ALP function, indicative of decreased TFEB and Lamp1 levels (Fig. 8 F). Although ASM-CM (i.v.)– treated mice showed slightly increased cathepsin D and p62 1561 of ASM and lysosomal-associated membrane protein 1 (Lamp1) showed that most ASM-positive vesicles were colocalized with Lamp1-positive vesicles, indicating that treated ASM was located in lysosome (Fig. 6 E). The ASM-induced autophago-some accumulation was significantly decreased by inhibition of lysosomal ASM uptake using M6P or M6P receptor siRNA (Fig. 6, F and G). These results suggested that elevation of ly-sosomal ASM may lead to autophagic dysfunction in AD. ASM treatment did not affect the cell survival (Fig. 6 H). The levels of LC3 and cell senescence in the fibroblast also did not show the differences with passage number, indicating that ASM caused the abnormal autophagy (Fig. 6, I and J). As discussed above, the accumulation of AVs in cells can result from either autophagy induction or the blockade of au-tophagic degradation. To further distinguish between these pos-sibilities, we performed an autophagic flux assay (Rubinsztein et al., 2009) in the presence or absence of NH4Cl that blocks autophagic degradation but does not affect autophagosome formation. It was hypothesized that if ASM treatment en-hanced autophagy induction, in the presence of NH4Cl (which inhibits degradation) a considerable increase in LC3- II would be expected due to the combined effects of blocking degradation and enhancing induction. However, compared with NH4Cl treatment alone, dual treatment of fibroblasts with ASM and NH4Cl did not show any significant changes of LC3-II (Fig. 7 A, left). In contrast, the addition of NH4Cl (Fig. 7 A, middle) or ASM (Fig. 7 A, right) in serum starvation culture resulted in a significant but similar increase of LC3-II levels. Furthermore, we measured the autophagic flux by de-tecting the abundance of p62. The levels of p62 were mark-edly increased in the cells treated with ASM or NH4Cl (Fig. 7 B). We also performed LC3 flux assay in human AD fibroblasts and 9-mo-old WT, APP/PS1, and APP/PS1/ ASM+/ mice fibroblasts. Autophagic flux was measured by assessing the changes of LC3-II in the presence and absence of NH4Cl–mediated lysosomal inhibition. Under basal con-dition, human AD and APP/PS1 fibroblasts showed signifi-cantly increased LC3-II levels compared with normal cells. NH4Cl–induced lysosome inhibition led to marked increase of LC3-II levels in the normal fibroblasts, but this increase was significantly less in the AD cells (Fig. 7, C and D). APP/ PS1/ASM+/ fibroblast showed similar pattern in LC3-II increase compared with normal cell (Fig. 7 D). Collectively, these results indicated that enhanced lysosomal ASM in AD caused a defect of autophagic degradation but not induction. condition (n = 4 per group). (C) Western blot analysis of LC3-II levels in controls, PS1-FAD, and ApoE4 fibroblasts in the presence or absence of NH4Cl (n = 6 per group). (D) Western blot analysis for LC3-II levels in fibroblasts derived from WT, APP/PS1, and APP/PS1/ASM+/ mice in the presence or absence of NH4Cl (n = 6 per group). (E) Effect of ASM on lysosomal pH. FACS and histological analysis of fibroblasts stained with LysoTracker red (n = 5 per group; bars, 20 μm). H2O2- and NH4Cl-treated cells were used as positive and negative controls, respectively. (F and G) Western blot analyses for TFEB and Lamp1 in human fibroblasts (F; n = 6 per group) and neurons (G; n = 6 per group) after treatment with ASM. (H) Immunocytochemistry of Lamp1 in control and ASM-treated fibroblast (n = 5 per group; bars, 20 μm). (I) Western blot analysis for nuclear localization of TFEB in ASM-treated cells (n = 5 per group). (J) Quantitative real-time PCR analysis of TFEB-target gene expression in normal (n = 6) and ASM-treated (n = 10) fibroblasts. (K) ASM activity was estimated in the fibroblast with or without NH4Cl (n = 5 per group). Data are representative of two (E, H, and I) or three (A–D, F, G, J, and K) independent experiments. A, B, and E–G, one-way ANOVA, Tukey’s post hoc test. C, D, and H–K, Student’s t test. *, P 0.05; **, P 0.01. All error bars indicate SEM.
- 92. Figure 8. ASMcauses autophagic dysfunction in vivo by sequestrating ALP function. (A and B) ASM was estimated in the brain and blood plasma of C57BL/6 mice after ASM-CM treatment into the hippocampus (A; i.c., n = 6 per group) or tail vein (B; i.v., n = 6 per group). (C and D) Western blot analyses for LC3, beclin-1, p62, and cathepsin D in the brains of C57BL/6 mice after ASM-CM treatment into the hippocampus (C; n = 5–6 per group) or tail vein (D; n = 4–5 per group). (E) Cathepsin D activity in the brain extracts of C57BL/6 mice after ASM-CM treatment (n = 4 per group). (F and G) Protein expression of TFEB and Lamp1 in the brains after ASM-CM treatment into the hippocampus (F; n = 5–6 per group) or tail vein (G; n = 5 per group). (H) Protein expression of TFEB and Lamp1 in the brains of 9-mo-old WT, APP/PS1, ASM+/, and APP/PS1/ASM+/ mice (n = 6–7 per group). Data are repre-sentative of three independent experiments. A–G, Student’s t test. H, one-way ANOVA, Tukey’s post hoc test. *, P 0.05. All error bars indicate SEM. levels and decreased ALP function proteins, this did not reach statistical significance (Fig. 8, D and G). The activity of cathep-sin D was also not changed (Fig. 8 E). These relatively modest effects of ASM-CM (i.v.) treatment on autophagy dysfunction might be due to presence of the blood–brain barrier because only a slight increase of ASM activity in the brain was achieved by these treatments, and the activity of ASM did not reach those of APP/PS1 mice. 1562 Role of ASM in the pathogenesis of AD | Lee et al.
- 93. Figure 9. Pharmacologicalrestoration of ASM to the normal range improves pathology in AD mice. (A) Protocol of AMI treatment in APP/PS1 mice. (B) ASM was estimated in the blood plasma (n = 12–14 per group) and brain (n = 9–10 per group) of APP/PS1 mice after AMI treatment. (C) Sphin-gomyelin, ceramide, and AC were determined using UPLC based methods in the plasma (n = 9 per group) and brain (n = 8 per group). (D) Mice brain sec-tions were stained with thioflavin S to detect A (bars, 200 μm). The relative area occupied by A plaques were determined (n = 6 per group). (E–G) A40 JEM Vol. 211, No. 8 Ar t icle 1563
- 94. To examine whetherpartial genetic inhibition of ASM affected the ALP in APP/PS1 mice, we also analyzed TFEB and Lamp1 levels in the brain samples derived from WT, APP/PS1, ASM+/, and APP/PS1/ASM+/ mice. Compared with WT, APP/PS1 mice showed significantly decreased TFEB and Lamp1 expression, which were increased in APP/PS1/ ASM+/ mice (Fig. 8 H). Together, these findings show for the first time a direct correlation of lysosomal ASM and the function of the ALP, and suggest that abnormal autophagic degradation in AD may be due to the effects of elevated ASM expression on this pathway. Pharmacological restoration of ASM to the normal range improves pathology in AD mice The ASM-mediated lysosomal/autophagic dysfunction in AD prompted us to examine possible therapeutic implications of this pathway. To decrease ASM in APP/PS1 mice, we undertook pharmacological inhibition using amitriptyline-hydrochloride (AMI) for 4 mo (Fig. 9 A). AMI is a known inhibitor of ASM that can cross the blood–brain barrier. At 9 mo of age, AMI-treated APP/PS1 mice exhibited decreased ASM activity compared with vehicle-treated mice (Fig. 9 B). Other sphingo-lipid metabolites were not changed (Fig. 9 C). A levels were decreased in the AMI-treated APP/PS1 mice compared with the nontreated littermates (Fig. 9, D–G). The levels of LC3-II, p62, and cathepsin D were decreased in the AMI-treated APP/ PS1 mice (Fig. 9, H and I). Actual activity of cathepsin D was not changed by AMI treatment (Fig. 9 J). AMI treatment sig-nificantly increased TFEB and Lamp1 protein levels in APP/ PS1 mice (Fig. 9, H and I). Similarly, APP/PS1 mice treated with AMI showed recovery of memory function (Fig. 9, K–P). Overall, these positive but relatively moderate results (e.g., A levels) in AMI-treated APP/PS1 mice might be due to under dosing of the animals. We speculate that this may be improved in the future by adjusting the dose or using modified, more potent drugs of a similar class. Restoration of ASM ameliorates autophagic dysfunction in the AD patient–specific cells To further validate our observation made by partial ASM in-hibition in AD mice, we studied possible changes in autophagy dysfunction in human AD fibroblast after ASM inhibition. Elevated ASM levels in human AD fibroblasts (PS1–familial AD [FAD] and ApoE4) were restored to normal range by ASM siRNA treatment (Fig. 10 A). ASM siRNA-treated human AD fibroblasts (PS1-FAD and ApoE4) showed decreased LC3-II and p62 accumulation compared with control siRNA-treated cells (Fig. 10 B). Also, ASM siRNA was able to increase lysosome levels (as judged by Lamp1 expression) by activating TFEB in the human AD fibroblasts (Fig. 10 C). Many insights into the pathogenesis in neurodegenerative disease have come from investigating postmortem brain tis-sues due to the difficulty of invasive access to living human CNS. The recent developments in induced pluripotent stem cells (iPSCs) and induced neurons have allowed investigation of pathogenesis of neurological diseases in vitro (Kondo et al., 2013). To explore whether the observed effects of ASM in previous results are paralleled by similar alterations in AD human neurons, we first established iPSCs with PS1 mutation (PS1 iPSC-2, -4, and -21) by transduction of human fibroblast with retroviruses encoding OCT4, SOX2, KLF4, and c-Myc. The PS1-iPSC cell line was shown to be fully reprogrammed to pluripotency, as judged by colony morphology, alkaline phosphatase (AP) staining, expression of pluripotency-associated transcription factors and surface markers, karyotype stability, and generation of teratomas (Fig. 10, D–G). To establish whether the PS1 mutation may affect neuronal differentia-tion, PS1 iPSC and control iPSC lines were induced to dif-ferentiate into neurons for 10 d. Consistent with previous results (Kondo et al., 2013), no obvious differences in the abil-ity to generate neurons were observed between control and PS1-iPSCs (Fig. 10 H). A42 secretion level was increased in PS1 iPSC-derived neurons compared with control iPSC-derived neuron (Fig. 10 I). Next, we investigated whether elevated ASM in fibroblasts was also evident increased in PS1 iPSC and iPSC-derived neurons. The ASM activity in PS1 iPSC was not changed ex-cept for PS1-4 iPSC in comparison to those in control iPSC, but the activity of ASM was significantly higher in PS1 iPSC-derived neurons compared with control iPSC-derived neu-ron (Fig. 10 J). Elevated ASM levels in PS1 iPSC-derived neurons were restored to normal range by ASM siRNA treat-ment (Fig. 10 J). Neurons from PS1-4 iPSCs also had signifi-cantly higher abnormal autophagic markers than neurons from control iPSC (Fig. 10 K). ASM siRNA treatment significantly decreased the protein level of abnormal autophagic markers in PS1 iPSC-derived neurons (Fig. 10 K). To corrob-orate the immunoblotting results, we performed EM analysis using control and PS1 iPSC-derived neurons. As expected, PS1 iPSC-derived neurons exhibited increased AV accumula-tion, whereas ASM siRNA-treated PS1 iPSC-derived neu-rons showed a reduced number of these vesicles (Fig. 10 L). and A42 in the brains of AMI treated or nontreated APP/PS1 mice were assessed using immunofluorescence staining (E and F; n = 8 per group; bars, 200 μm) and ELISA kits (G; n = 6 per group). (H and I) Western blot analyses and quantification for LC3, Beclin-1, p62, cathepsin D, TFEB, and Lamp1 in the brains of APP/PS1 mice treated with AMI or control (n = 6–8 per group). (J) Cathepsin D activity in the brain extracts of AMI-treated or nontreated APP/PS1 mice (n = 4 per group). (K) Escape latencies of APP/PS1 mice treated with AMI or control over 10 d (WT, n = 14; nontreated APP/PS1, n = 10; and AMI-treated APP/PS1, n = 12). (L–O) Probe trial day 11. (L and M) Path length (L) and swim speed (M) were recorded and analyzed. (N) Time spent in target platform and other quadrants was measured. (O) The number of times each animal entered the small target zone during the 60-s probe trial. (P) Repre-sentative swimming paths at day 10 of training. Data are representative three independent experiments. B–J and N, Student’s t test; K–M and O, one-way ANOVA, Tukey’s post hoc test. *, P 0.05; **, P 0.01. All error bars indicate SEM. 1564 Role of ASM in the pathogenesis of AD | Lee et al.
- 95. Figure 10. Restorationof ASM to the normal level reverses impaired autophagy in the AD patient-specific cells. (A) SMPD1 gene suppression by ASM-siRNA in human fibroblasts. ASM activity was assessed after ASM siRNA treatment in the control and AD fibroblast (n = 6 per group). (B) LC3-II and p62 levels were examined in human AD fibroblast with or without ASM inhibition. siRNA-mediated suppression of ASM reduced LC3-II and p62 levels in PS1-FAD (left; n = 7 per group) and ApoE4 fibroblast (right; n = 6 per group). (C) Protein expression of TFEB and Lamp1 in the PS1-FAD and ApoE4 JEM Vol. 211, No. 8 Ar t icle 1565
- 96. 2007). Moreover, ithas been shown that the abnormal au-tophagic flux in AD may be due to dysfunction at the late au-tophagy stage associated with the lysosome (Lai and McLaurin, 2012; Zhou et al., 2012). Similar to previous results (Lee et al., 2010b; Lai and McLaurin, 2012), we found that the impaired autophagic flux in AD was associated with reduced autopha-gic degradation due to decreased ALP function. In addition, we show for the first time that this is directly linked to ele-vated ASM activity. Suppression of ASM expression or inhibi-tion of its uptake and delivery to lysosomes using M6P reversed abnormal autophagic degradation. These findings indicate that increased lysosomal ASM plays a negative role in AD by causing autophagic dysfunction, suggesting that therapeutic strategies to restoring ASM activity to the normal range may be beneficial for AD pathology. This was further studied in the AD mice by finding that partial genetic or systemic inhibition of ASM activities in these animals largely reversed autophagic pathology by restoring ALP function, as well as reducing the accumulation of incom-pletely digested substrates within the autophagic-lysosomal compartments (e.g., LC3-II and p62). A accumulation also was reduced in response to ASM inhibition, as was cathepsin D expression. There are several challenges associated with inter-pretation of cathepsin D levels in AD. Although some reports have shown that cathepsin D activities were decreased in AD (Lee et al., 2010b), many studies indicate that cathepsin D is elevated in AD and contributes to the pathogenesis, such as A formation (Cataldo et al., 2004; Lai and McLaurin, 2012; Zhou et al., 2012). A recent paper suggested that COP9 signalosome deficiency increased cathepsin D levels but reduced the au-tophagic degradation. They suggested that these results were associated with a failure of lysosomal assembly of cathepsin D because only a lysosomal cathepsin D could affect autophagic degradation (Su et al., 2011). In this study, we have found that maturation of cathepsin D was increased in AD mice, but the actual enzyme activity was not changed between the groups. This result indicated that the elevated levels of cathepsin D did not ultimately translate into a significant increase of enzyme activity. Based on these papers and our data, a plausible inter-pretation of increased cathepsin D in our AD mice is that AD microenvironment attempts to increase cathepsin D synthesis, but this does not have a direct impact on lysosomal function because the activity of the enzyme is unchanged. Therefore, Decreased TFEB target genes in PS1 iPSC-derived neurons were also significantly increased by ASM siRNA treatment (Fig. 10 M). These results confirm that abnormal autophagy observed in AD mice and human fibroblasts by ASM also occur in AD patient neurons, and restoration of ASM back to normal levels is able to ameliorate autophagic dysfunction by restoring lysosomal biogenesis in AD patient cells. DISCUSSION Although the exact causes of AD are unknown, the complex interactions of genetic and environmental factors are likely play important roles in the pathogenesis. ASM activity is known to be increased by environmental stress and in various diseases, and is elevated in AD patients (He and Schuchman, 2012). One downstream consequence of increased ASM is elevated ceramide, contributing to cell death, inflammation, and other common disease findings. Although elevated ASM is known to occur in AD, the cellular mechanisms that link ASM and AD have not been fully characterized. The data presented here suggest a previously unknown role of ASM in the down-regulation of lysosomal biogenesis and inhibition of lysosome-dependent autophagic proteolysis. The findings also establish proof of concept for ASM inhibitor therapy in AD. Our previous study showed that sphingolipid metabolism was severely impaired in the human AD brain, and that ASM activity was positively correlated with the A levels (He et al., 2010). Consistent with our previous study, we found that ASM was significantly increased in fibroblasts, brain, and/or plasma from patients with AD and in AD mice, although other sphin-golipid factors were unaltered. There are some differences be-tween the previous and this study. For example, the previous results showed increased ceramide level in AD, but we could not found significant changes of sphingolipid factors includ-ing ceramide in AD compared with normal samples. These differences might be related to the fact that once formed, ce-ramide can rapidly enter several metabolic pathways. It may be used for either the biosynthesis of complex lipids or bro-ken down into sphingosine, which itself is rapidly converted to sphingosine 1 phosphate. Accumulation of abnormal AVs has been observed in AD (Lee et al., 2010b; Nixon and Yang, 2011), and the autophagy pathway is increasingly regarded as an important contributor to A-mediated pathogenesis in AD (Yu et al., 2005; Nixon, fibroblast after ASM inhibition (n = 5–6 per group). (D–G) Generation of PS1 iPSC lines from patient fibroblast. (D) Established iPSCs showed embryonic stem cell–like morphology (Phase; bar, 1 mm), AP activity (bar, 200 μm), and expressed pluripotent stem cell markers SSEA4 (bar 100 μm), TRA1-60 (bar 100 μm), and TRA1-81 (bar 100 μm). (E) Normal karyotype of PS1 iPSC. (F) Quantitative real-time PCR analysis of hESC marker gene of PS1 iPSC (n = 3 per group). (G) Gross morphology and hematoxylin-eosin staining of representative teratomas generated from PS1-4 iPSCs (bars, 50 μm). (H) Estimation of neural differentiation from control and PS1-4 iPSCs. Representative images of immunocytochemical staining the -III tubulin after neural differentiation (bars, 50 μm). (I) The amount of A42 secreted from control iPSC-derived neuron and PS1 iPSC-derived neuron (n = 5 per group). (J) Characterization of ASM activity in the control and PS1 iPSC and iPSC-derived neurons (n = 6 per group). (K) Western blot analyses for LC3, beclin-1, p62, TFEB, and Lamp1 in the control and PS1-4 iPSC–derived neuron after ASM siRNA treatment (n = 5–6 per group). (L) EM images and quantification data of control and PS1 iPSC-derived neurons. Higher magnification of boxed area shows detail of AVs (arrow; n = 4 per group; bars: [low magnification] 1 μm, [high magnifica-tion] 500 nm). (M) Quantitative real-time PCR analysis of TFEB-target gene expression in iPSC-derived neurons after ASM siRNA treatment (n = 5–6 per group). Data are representative of two (A, D–G, I, and L), or three (B, C, H, J, K, and M) independent experiments. A, C, F, I, J, and M, Student’s t test. B, K, and L, one-way ANOVA, Tukey’s post hoc test. *, P 0.05; **, P 0.01; ***, P 0.001. A–K, error bars indicate SEM. L and M, Error bars indicate SD. 1566 Role of ASM in the pathogenesis of AD | Lee et al.
- 97. JEM Vol. 211,No. 8 Ar t icle 1567 enhanced cathepsin D level in our AD mice induced by in-creased ASM is more likely a compensatory response to an impaired lysosome system. The present study also provides the first evidence of increased ASM activity and autophagic dys-function in living human (iPSC-derived) neurons derived from AD patients and that restoring normal levels of ASM in AD neurons effectively blocks abnormal autophagy. Overall, the data presented here show that increased ASM activity in AD contributes to the abnormal lysosomal/au-tophagic process by leading to dysfunction of ALP. This results in an inability to break down appropriate substrates during the autophagy process. Restoration of ASM effectively blocks AD progression by increasing autophagic degradation. Al-though the involvement of other ASM-related mechanisms in AD remains to be explored, the data in this study demonstrate that inhibition of ASM improves A clearance and rescues impaired memory in a validated mouse model of AD, suggest-ing this as a potential therapy for AD patients in the future. MATERIALS AND METHODS Mice. Transgenic mouse lines overexpressing the hAPP695swe (APP) and presenilin-1M146V (PS1) mutations, respectively, were generated at GlaxoSmithKline by standard techniques as previously described (Howlett et al., 2004). In brief, a Thy-1–APP transgene was generated by inserting the 695 aa isoform of human cDNA (APP695) harboring the Swedish double familial mutation (K670N; M671L) into a vector containing the murine Thy-1 gene. The Thy-1–PS-1 transgene was generated by inserting the coding sequence of human PS-1 cDNA harboring the M146V familial mutation into a vector containing the murine Thy-1 gene. Transgenic lines were generated by pro-nuclear microinjection into fertilized oocytes from either C57BL/6xC3H mice in the case of Thy-1-APP transgene, or into fertilized oocytes from pure C57BL/6 mice in the case of Thy-1–PS-1 transgene. Thy-1 APPswe mice were generated and backcrossed onto a pure C57BL/6 background before crossing with TPM (PS-1 M146V) mice to produce heterozygote double mutant mice. ASM+/ mice (C57BL/6 background; Horinouchi et al., 1995) were bred with APP/PS1 mice to generate APP/PS1/ASM+/ mice. Because APP/PS1 mice show sex difference in disease progression, we used only male mice. Data analysis of APP/PS1 mice was done at 5 or 9 mo old. Block randomization method was used to allocate the animals to experimental groups. To eliminate the bias, we were blinded in experimental progress such as data collection and data analysis. SCID Beige mice (Charles River) were used for teratoma forma-tion assay. Mice were housed at a 12 h day/12 h night cycle with free access to tap water and food pellets. Mouse studies were approved by the Kyungpook National University Institutional Animal Care and Use Committee (IACUC). Plasma collection. Human plasma samples were obtained from individuals with AD, PD, and age-matched, non-AD controls from Yonsei University Severance Hospital (Table S1). Informed consent was obtained from all sub-jects according to the ethics committee guidelines at the Yonsei University Severance Hospital. Cell culture. Human fibroblast lines (normal, PS1, ApoE4, and PD) ac-quired from the Coriell Institute were maintained in DMEM with 15% FBS. The human cortical neuronal cell line HCN-2 was acquired from ATCC. Cells with a passage number 10–15 were used in this study. To obtain CM containing ASM, 5 × 105 Chinese hamster ovary cells overexpressing human ASM (He et al., 1999) were cultured in DMEM for 2 d. Cells were washed with PBS and changed with new media. 24 h later, the CM was collected, centrifuged, and filtered using a 0.22 μm filter. We isolated WT, APP/PS1, APP/PS1/ASM+/, and ASM+/ mouse tail fibroblasts as previously de-scribed (Takahashi et al., 2007a) from 9-mo-old mice. For some experiments, cells were treated with purified, recombinant ASM or ASM siRNA to mea-sure autophagy regulation. NH4Cl was used to inhibit the autophagic flux. For the inhibition of lysosomal ASM uptake, 10 mM M6P or M6P receptor siRNA were added to the fibroblast culture media at the same time as ASM. Drug or CM treatments. 4-mo-old APP/PS1 mice received 100 μg/g body weight AMI (Sigma-Aldrich) per os in their drinking water for 4 mo, and a control group received water without drug. 3-mo-old C57BL/6 mice were treated with ASM-CM via i.v. (100 μl) or i.c. (3 μl) injections on 10 consecutive days. Immunofluorescence. Thioflavin S staining was done according to previ-ously described procedures (Lee et al., 2012). We used anti-20G10 (mouse, 1:1,000, provided by D.R. Howlett, GlaxoSmithKline, Harlow, Essex, UK) for A 42, anti-G30 (rabbit, 1:1,000, provided by D.R. Howlett) for A40, rabbit anti–Iba-1 (1:500; Wako), rabbit anti-GFAP (1:500, Dako), mouse anti–-SMA (1:400; Sigma-Aldrich), rabbit anti-AT8 (1:500; Thermo Fisher Scientific), and rabbit anti–active caspase3 (1:50; EMD Millipore). The sec-tions were analyzed with a laser-scanning confocal microscope (FV1000; Olympus) or with a BX51 microscope (Olympus). MetaMorph software (Molecular Devices) was used to quantification. Ab ELISA. For measurement of A40 and A42, we used commercially available ELISA kits (BioSource). Hemispheres of mice were homogenized in buffer containing 0.02M guanidine. ELISA was then performed for A40 and A42 according to the manufacturer’s instructions. Behavioral studies. We performed behavioral studies to assess spatial learn-ing and memory in the Morris water maze as previously described (Lee et al., 2012). Animals were given four trials per day for 10 d to learn the task. At 11 d, animals were given a probe trial in which the platform was removed. Fear conditioning was conducted as previously described techniques (Kojima et al., 2005). On the conditioning day, mice were individually placed into the con-ditioning chamber. After a 60-s exploratory period, a tone (10 kHz, 70 dB) was delivered for 10 s; this served as the conditioned stimulus (CS). The CS co-terminated with the unconditioned stimulus (US), a scrambled electrical foot-shock (0.3 mA, 1 s). The CS-US pairing was delivered twice at a 20-s intertrial interval. On day 2, each mouse was placed in the fear-conditioning chamber containing the same exact context, but with no administration of a CS or foot shock. Freezing was analyzed for 5 min. On day 3, a mouse was placed in a test chamber that was different from the conditioning chamber. After a 60-s ex-ploratory period, the tone was presented for 60 s without the footshock. The rate of freezing response of mice was used to measure the fear memory. Quantitative real-time PCR. RNA was extracted from the brain homog-enates and cell lysates using the RNeasy Lipid Tissue Mini kit and RNeasy Plus Mini kit (QIAGEN) according to the manufacturer’s instructions. cDNA was synthesized from 5 μg of total RNA using a commercially avail-able kit (Takara Bio Inc.). Quantitative real-time PCR was performed using a Corbett research RG-6000 real-time PCR instrument. Used primers are described in Table S2. EM. Brain tissues and cells were fixed in 3% glutaraldehyde/0.1 M phosphate buffer, pH 7.4, and postfixed in 1% osmium tetroxide in Sorensen’s phosphate buffer. After dehydration in ethyl alcohol, the tissue and cells were embedded in epon (Electron Microscopy Sciences). Samples were cut serially and placed on copper grids and analyzed using a transmission EM (Tecnai). Images were captured on a digital camera and Xplore3D tomography software. Intracellular protein degradation measurement. Total protein degrada-tion in cultured cells was measured by pulse-chase experiments with 48 h pulse with 2 μCi/ml [3H]-leucine for 48 h to preferentially label long-lived proteins (Lee et al., 2010b). Western blotting. Samples were immunoblotted as previously described (Settembre et al., 2011; Lee et al., 2012). Primary antibodies to the following
- 98. in serum-free mediahormone mix media (Okada et al., 2008) for 10–14 d to allow the formation of neurospheres. Neurospheres were passaged repeatedly by dissociation into single cells followed by culture in the same manner. Typi-cally, neurospheres between passages 3 and 8 were used for analysis. For terminal differentiation, dissociated neurospheres were allowed to adhere to poly-l-ornithine– and laminin-coated coverslips and cultured for 10 d. AP, senescence-associated--galactosidase (SA--gal), and immuno-cytochemical staining. AP staining was performed using an ES-AP detec-tion kit (EMD Millipore) according to manufacturer’s recommendations. SA--gal activity was detected using SA--gal staining kit (Cell Signaling Technology) according to manufacturer’s protocol. For immunocytochemical analysis, we used anti-SSEA4, TRA-1-60, TRA-1-81 (mouse, 1:100; EMD Millipore), anti–-III-tubulin (mouse, 1:400; EMD Millipore), rabbit anti–LC- 3B (1:200; Cell Signaling Technology), rabbit anti-ASM (1:1,000, Abcam), mouse anti-LAMP1 (1:100; Abcam), and mouse anti-LBPA (1:500; Echelon). Teratoma formation and histological analysis. Established iPSCs were prepared at 107 cells/ml in PBS. Suspended cells (1–3 × 106) were injected into testes of anesthetized male SCID Beige mice. 8 wk after transplantation, mice were sacrificed and tumors were dissected. Tumor samples were fixed in 10% formalin and embedded in paraffin. Sections were stained with hema-toxylin and eosin. Statistical analysis. Comparisons between two groups were performed with Student’s t test. In cases where more than two groups were compared with each other, a one-way analysis of variance (ANOVA) was used, followed by Tukey’s HSD test. All statistical analysis was performed using SPSS statisti-cal software. P 0.05 was considered to be significant. Online supplemental material. Table S1 shows subjects’ characteristics. Table S2 shows sequences of primer pairs. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20132451/DC1. This work was supported by the Bio Medical Technology Development Program (2010-0020234, 2011-0019356, 2012M3A9C6049913, and 2012M3A9C6050107) of the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT Future Planning, Republic of Korea. The authors declare no competing financial interests. Author contributions: J.K. Lee, H.K. Jin, M.H. Park, B.R. Kim, P.H. Lee, H. Nakauchi, J.E. Carter, and X. He performed experiments and analyzed data, J.K. Lee, H.K. Jin, and J.S. Bae designed the study and wrote the paper. E.H. Schuchman and J.S. Bae interpreted the data and reviewed the paper. All authors discussed results and commented on the manuscript. Submitted: 26 November 2013 Accepted: 20 June 2014 REFERENCES Boland, B., A. Kumar, S. Lee, F.M. Platt, J. Wegiel, W.H. Yu, and R.A. Nixon. 2008. Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer’s disease. J. Neurosci. 28:6926–6937. http://dx.doi.org/10.1523/JNEUROSCI.0800-08.2008 Brewer, G.J., and J.R. Torricelli. 2007. Isolation and culture of adult neu-rons and neurospheres. Nat. Protoc. 2:1490–1498. http://dx.doi.org/10 .1038/nprot.2007.207 Cataldo, A.M., C.M. Peterhoff, S.D. Schmidt, N.B. Terio, K. Duff, M. Beard, P.M. Mathews, and R.A. Nixon. 2004. Presenilin mutations in familial Alzheimer disease and transgenic mouse models accelerate neu-ronal lysosomal pathology. J. Neuropathol. Exp. Neurol. 63:821–830. Cuervo, A.M., L. Stefanis, R. Fredenburg, P.T. Lansbury, and D. Sulzer. 2004. 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We performed densitometric quantification using the ImageJ software (National Institutes of Health). ASM and AC activity assays. We performed the measurements as previ-ously described methods using a UPLC system (Waters; He et al., 2010). Lipid extraction and ceramide/sphingomyelin quantification. We prepared samples for lipid extraction as previously described (Lee et al., 2010a). To quantify the sphingomyelin and ceramide levels, the dried lipid extract was resuspended in 0.2% Igepal CA-630 (Sigma-Aldrich) and the levels of each lipid were determined using the UPLC system. Isolation of neuron and microglia. We isolated neuron and microglia from the mouse brain as previously described (Brewer and Torricelli, 2007). In brief, 9-mo-old mice cerebrum were minced in HibernateA (Brainbits LLC)/B27 (Invitrogen) medium and dissociated using papain solution. After tissue trituration, cells were separated by density gradient centrifugation. Fractionated cells were used for ASM activity assay. LysoTracker labeling and quantification. LysoTracker red (Invitrogen) was used at a final concentration of 75 nM. The cells were trypsinized, resuspended in PBS, and analyzed on a FACSCalibur using FACSDiva software (BD). Cell viability assays. Cell viability was quantified by using MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphen yltetrazoliumbromide; Sigma-Aldrich) re-agent. In brief, human fibroblast was seeded onto 96-well plates at a density of 1.5 × 103 cells per well. 10 μM ASM was added to the culture media, and the cells were incubated for 24 h. MTT stock solution (5 mg/ml) was prepared in PBS (Gibco) and added to the culture media at a final concentration of 1 mg/ml. After 90 min incubation, the media was removed, and the chromogen in the cells was dissolved in DMSO containing 0.01 N NaOH. The absorbance was measured at 570 nm using a 96-well microplate spectrophotometer. Measurement of activity of cathepsin D. Enzyme activity of cathepsin D was determined with cathepsin D activity fluorometric assay kit according to the manufacturer’s protocol (Abcam). Generation of iPSCs. PS1-iPSCs were established from the PS1 patient’s skin fibroblasts (Coriell Institute) as previously described (Okita et al., 2007), with slight modifications. In brief, PS1 fibroblasts were seeded at 3 × 105 cells in 60 mm2 dishes coated with gelatin. On day 1, the VSV-G pseudotyped ret-roviral vector system carrying OCT4, SOX2, KLF4, and c-Myc was added to fibroblast cultures. On day 2, cells were subjected to the same transduction procedures and harvested 24 h later. Transduced cells were replated on mouse embryonic fibroblast (MEF) layers in 100 mm2 dishes containing the fibro-blast medium. On the next day, the medium was changed to complete ES medium with 0.5 mM valproic acid (Sigma-Aldrich), and thereafter changed every other day. After 20 d, ES-like colonies appeared and were picked up to be reseeded on new MEF feeder cells. Cloned ES-like colonies were sub-jected to further analysis. Normal iPSC line (HPS0063) was obtained from the RIKEN Bioresource Center (Takahashi et al., 2007b). In vitro differentiation of human iPSCs. Neural differentiation of iPSCs was performed as described previously (Okada et al., 2008). iPSC colonies were detached from feeder layers and cultured in suspension as embryonic body for 30 d in bacteriological dishes. Embryonic bodies were then enzymati-cally dissociated into single cells and the dissociated cells cultured in suspension 1568 Role of ASM in the pathogenesis of AD | Lee et al.
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- 102. MICROMANIPULATION The mostpowerful aspect of the Multi-Link Position Control™ software is the ability to link and coordinate movement of multiple devices that can be connected to our MPC-200 controller. You can combine manipulators, translators, microscopes (MOM or SOM) and stages to give you complete control over your complex experiments. Bring all devices to memorized positions or bring pipettes to a working location in seconds. Available as a free download on our web site. MICROPIPETTE FABRICATION Sutter Instrument, the recognized leader in micropipette fabrication technology, offers leading edge technology in the P-1000 micropipette puller with an intuitive, full-featured interface. An extensive library of built-in programs is available through the color touch-screen display, taking the guesswork out of pipette pulling. OPTICAL PRODUCTS Our latest products include the Lambda HPX, a new high-powered white light LED and the versatile SOM simple moving microscope optimized to allow in vivo and in vitro experimentation in one set-up. As with our two-photon MOM microscope, the SOM can robotically position the objective over the sample and focus, opening up experimental possibilities. MICROINJECTION The XenoWorks® microinjection system has been designed to meet the needs of a wide variety of applications that require the manipulation of cells and embryonic tissues including ICSI, ES Cell Microinjection, and Adherent Cell Microinjection. Highly-responsive movement and excellent ergonomics intuitively link the user with the micropipette, improving yield – saving time and resources. Precision Instrumentation for the Sciences ONE D I G I T A L D R I V E , N O V A T O , CA. 94949 PHONE: 415.883.0128 | FAX: 415.883.0572 EMAIL: INFO@SUTTER.COM | WWW.SUTTER.COM