cronier 2012

The FASEB Journal article fj.11-201772. Published online June 1, 2012.
The FASEB Journal • Research Communication
Endogenous prion protein conversion is required for
prion-induced neuritic alterations and
neuronal death
Sabrina Cronier,*,1 Julie Carimalo,‡,§ Brigitte Schaeffer,† Emilie Jaumain,*
Vincent Béringue,* Marie-Christine Miquel,‡ Hubert Laude,* and Jean-Michel Peyrin*,‡,1
*UR892, Virologie et Immunologie Moléculaires, and †UR0341, Mathématiques et Informatique
Appliquées, Institut National de la Recherche Agronomique (INRA), Jouy-en-Josas, France,
‡Neurobiologie des Processus Adaptatifs, Unité Mixte de Recherche (UMR) Centre National de la
Recherche Scientifique (CNRS)-7102, Université Pierre et Marie Curie, Paris, France; and §National
Reference Centre for Transmissible Spongiform Encephalopathy Surveillance, Department of
Neurology, Georg August University, Göttingen, Germany
ABSTRACT Prions cause fatal neurodegenerative
conditions and result from the conversion of host-encoded
cellular prion protein (PrPC) into abnormally
folded scrapie PrP (PrPSc). Prions can propagate both
in neurons and astrocytes, yet neurotoxicity mecha-nisms
remain unclear. Recently, PrPC was proposed to
mediate neurotoxic signaling of -sheet-rich PrP and
non-PrP conformers independently of conversion. To
investigate the role of astrocytes and neuronal PrPC in
prion-induced neurodegeneration, we set up neuron
and astrocyte primary cocultures derived from PrP
transgenic mice. In this system, prion-infected astro-cytes
delivered ovine PrPSc to neurons lacking PrPC
(prion-resistant), or expressing a PrPC convertible
(sheep) or not (mouse, human). We show that interac-tion
between neuronal PrPC and exogenous PrPSc was
not sufficient to induce neuronal death but that effi-cient
PrPC conversion was required for prion-associ-ated
neurotoxicity. Prion-infected astrocytes markedly
accelerated neurodegeneration in homologous cocul-tures
compared to infected single neuronal cultures,
despite no detectable neurotoxin release. Finally, PrPSc
accumulation in neurons led to neuritic damages and
cell death, both potentiated by glutamate and reactive
oxygen species. Thus, conversion of neuronal PrPC
rather than PrPC-mediated neurotoxic signaling ap-pears
as the main culprit in prion-induced neurodegen-eration.
We suggest that active prion replication in
neurons sensitizes them to environmental stress regu-lated
by neighboring cells, including astrocytes.—
Cronier, S., Carimalo, J., Schaeffer, B., Jaumain, E.,
Béringue, V., Miquel, M.-C., Laude, H., Peyrin, J.-M.
Endogenous prion protein conversion is required for
prion-induced neuritic alterations and neuronal death.
FASEB J. 26, 000–000 (2012). www.fasebj.org
Key Words: neurodegeneration primary culture astrocyte
scrapie
Transmissible spongiform encephalopathies (TSEs)
include Creutzfeldt–Jakob disease in humans, bovine
spongiform encephalopathy, sheep scrapie, and chronic
wasting disease in cervids. They feature neuronal loss,
spongiosis, and pronounced astrogliosis. These fatal
disorders are caused by prions, a class of unconven-tional
agents that preferentially target the central ner-vous
system (CNS). Prions are essentially composed of
aggregated misfolded scrapie prion protein (PrPSc)
derived from the host-encoded cellular PrP (PrPC).
Prion propagation within and between species is be-lieved
to stem from the ability of PrPSc seeds to promote
the conformational transition from PrPC to PrPSc,
through a nucleated polymerization process (for re-view,
see refs. 1, 2). At the molecular level, cell-free
assays have suggested 2 sequential steps: initial binding
of PrPC to pathological PrP species and conversion into
PrPSc (3).
Although the essential role of PrPC in prion propa-gation
is beyond any doubt (4, 5), its involvement in
neurotoxicity mechanisms remains controversial, and
the nature of PrP toxic species themselves are still
unknown (6). Exposure of primary neuronal cultures
expressing or lacking PrPC to purified PrPSc, aggre-
1 Correspondence: S.C., INRA, UR892, Virologie et Immunolo-gie
Moléculaires, 78352 Jouy-en-Josas, France. E-mail: sabrina.
cronier@jouy.inra.fr; J.-M.P., Neurobiologie des Processus Adapta-tifs,
UMR CNRS-7102, Université Pierre et Marie Curie–Paris 6,
75005 Paris, France. E-mail: jean-michel.peyrin@snv.jussieu.fr
doi: 10.1096/fj.11-201772
Abbreviations: CAS, cerebellar astrocyte; CGN, cerebellar
granule neuron; DAPI, 4=,6-diamidino-2-phenylindole; DIV,
day in vitro; DPE, days postexposure; GFAP, glial fibrillary
acidic protein; GndSCN, guanidine thiocyanate; MAP2, mi-crotubule-
associated protein 2; NeuN, neuronal nuclei; PK,
proteinase K; PrP, prion protein; PrPC, cellular prion protein;
PrPres, proteinase K-resistant prion protein; PrPSc, scrapie
prion protein; ScCAS, scrapie-exposed cerebellar astrocyte;
ScCGN, scrapie-exposed CGN; TSE, transmissible spongiform
encephalopathy
0892-6638/-1900/0026-0001 © FASEB 1

gated full-length recombinant PrP or PrP-derived syn-thetic
fragments provided a number of elements
toward this end (7–8). They were sometimes contradic-tory
(9–11), possibly because supraphysiological doses
used in these acute paradigms did not reflect the “slow”
natural infection process, or because endogenous PrP
conversion is needed to produce toxic species (as we
demonstrate here). In vivo, transmission studies in
transgenic mice have shown that PrP expression at the
neuronal cell surface was necessary to the development
of typical TSE neuropathological changes (4, 12–13).
However, mice expressing PrP specifically in astrocytes
(14) appeared to propagate prion and develop TSE-specific
neuronal lesions, suggesting that astrocytic
prion replication might be sufficient to induce some
neuronal damage (15).
Finally, if PrPC plays a critical role in prion-mediated
neuronal death following interaction with PrPSc, it is
still unclear whether the conversion step is involved.
Indeed, PrPC was recently proposed to mediate toxic
signaling of -sheet-rich conformers, including amyloid
(16–17), and following antibody cross-linking (18–
19), therefore, pointing at a subversion of its putative
neuroprotective functions (20), although these obser-vations
were not consistently reproduced (21–23).
Sheep scrapie prions could efficiently propagate in
primary cultures of either neurons or astrocytes and
induce a late apoptotic neuronal death (24). Here, we
have established a coculture system in which sheep
scrapie-infected astrocytes are in contact with neurons
devoid of PrPC or expressing homologous (i.e., convert-ible)
or heterologous (i.e., nonconvertible) PrPC spe-cies.
We report that prion neurotoxicity dramatically
depends on expression and efficient conversion of
neuronal PrPC into PrPSc. We further show that a
dysfunction of prion-infected astrocytes is unlikely to be
a major determinant of neuronal death initiation but
rather that infected neurons become more susceptible
to various subtoxic stimuli, such as oxidation and
glutamate.
MATERIALS AND METHODS
Transgenic mouse lines
Care of mice was performed according to Institut National de
la Recherche Agronomique and French animal care commit-tee
guidelines. Primary cultures were derived from the follow-ing
homozygous transgenic mouse lines: PrP0/0 (PrP-knock-out
mice; Zurich I; ref. 25), tg338 (ovine PrPVRQ; ref. 26),
tga20 (mouse Prnp-a allele; ref. 27) and tg650 (human Met129
PrP; ref. 28). These PrP-expressing lines were all established
on the same Zurich I mouse PrP0/0 background, thus ensur-ing
a consistent genetic background in the cultures.
Primary cell cultures
Primary cultures of cerebellar granule neurons (CGNs) were
established as described previously (24, 29). Briefly, CGNs
extracted from cerebellum of 6-d-old mice by enzymatic and
mechanical dissociation were plated (400,000 cells/well) in
24-well plates coated with poly-d-lysine (10 g/ml, Sigma; St.
Louis, MO, USA). CGN cultures were maintained at 37°C
with 6% CO2 in DMEM containing glutamax I (Life Technol-ogies,
Paisley, UK), 10% FCS (BioWhittaker, Walkersville,
MD, USA), 20 mM KCl, penicillin, streptomycin (Life Tech-nologies)
and complemented with N-2 and antioxidant-de-pleted
B27 supplements (Life Technologies, Grand Island,
NY, USA). Glucose was maintained at 1 mg/ml by weekly
supplementation, along with antimitotics uridine and fluoro-deoxyuridine
(10 M; Sigma).
Pure cerebellar astrocytes from ovine PrPC-expressing
tg338 mice (CASOv cells) were dissociated similarly to CGNs,
seeded on poly-d-lysine-coated plates (1 g/ml) and cultured
in DMEM-glutamax I containing 10% FCS with antibiotics.
The cell population was fully astrocytic within a week. Astro-cytes
were grown to 70% confluence before use, and the
medium was changed weekly.
Prion infection of cultured cells
Infectious 10% (w/v) brain homogenates were prepared in
PBS from terminally ill tg338 mice inoculated with the 127S
sheep scrapie strain (24, 30). Confluent astrocytes or day in
vitro 2 (DIV2) CGNs were exposed to a final concentration of
0.01% (w/v) infected brain homogenate unless specified, or
not infected for mock infections. Infectivity of astrocyte-conditioned
medium was tested by replacing half of the
culture medium by medium conditioned for 7 d on 21-d
prion-infected astrocytes, supplemented with KCl and N-2.
Following infection, the medium was either left unchanged
for CGN cultures during the whole duration of the experi-ment,
or changed after 7 d and then 1/wk for CAS cultures.
PrP immunoblot
Cell lysates or brain homogenates were treated with protei-nase
K (PK; 7.5 g/mg or 50 g/ml, respectively; Eurome-dex,
Mundolsheim, France), as described previously (29).
After methanol precipitation (1 h, 20°C) and centrifugation
(16,000 g, 10 min), pellets were resuspended in sample buffer
and boiled, and proteins were subjected to SDS/PAGE and
electrotransferred onto nitrocellulose membranes. PK-resis-tant
PrP (PrPres) was detected with anti-PrP monoclonal
antibodies ICSM18 or biotinylated Sha31.
Immunofluorescence
Cells were fixed for 10 min at room temperature in PBS
containing 4% paraformaldehyde and 4% sucrose, and sub-sequently
permeabilized for 5 min with PBS containing 0.1%
Triton X-100. Following treatment for 5 min with 3 M
guanidine thiocyanate (GdnSCN), PrPSc was immunode-tected
with ICSM35 or ICSM33 anti-PrP monoclonal antibod-ies
(31). Neurons were labeled with monoclonal anti-neuro-nal
nuclei (NeuN; 1:100; Chemicon; Temecula, CA, USA)
antibody and astrocytes with polyclonal anti-glial fibrillary
acidic protein (GFAP; 1:400; Dako, Glostrup, Denmark)
antibody. Neuronal processes were stained with either mono-clonal
(1:250; Sigma) or polyclonal (1:500; Chemicon) anti-microtubule-
associated protein 2 (MAP2) antibodies. Cells
were then incubated with appropriate FITC- or Alexa Fluor-conjugated
secondary antibodies and nuclear marker 4=,6-
diamidino-2-phenylindole (DAPI; 2 g/ml; Sigma) and
mounted in Fluoromount (Sigma). Cells were observed un-der
an Axiovert 200 M epifluorescence or an AxioObserver
Z1 microscope (Zeiss, Oberkochen, Germany), images were
acquired with Metaview (Universal Imaging, Downingtown,
PA, USA) and AxioVision (Zeiss) software, respectively, and
2 Vol. 26 September 2012 The FASEB Journal www.fasebj.org CRONIER ET AL.

analysis was performed with ImageJ (U.S. National Institutes
of Health; http://rsbweb.nih.gov/ij/).
Neuron/astrocyte cocultures
Mock- or 127S-infected CASOv cultures were maintained for
21 d. Culture medium was then removed, and freshly disso-ciated
CGNs from tg338, tga20, tg650 or PrP0/0 mice sus-pended
in complete neuronal medium were seeded at a
density of 2000 cells/mm2 on top of CAS cultures. After 7 d,
cells were processed for immunofluorescence. Neuronal sur-vival
rate was determined by counting NeuN-positive cells
with nonpyknotic nuclei. For each experiment, PrPSc immu-nostaining
was performed to confirm that neurons were in
contact with heavily infected astrocytes.
Quantification of astrocyte-conditioned medium toxicity
Fresh culture medium was conditioned for 7 d on mock- or
scrapie-infected astrocytes at 21 days postexposure (DPE) to
brain homogenate, collected, and supplemented with KCl
and N-2 to obtain neuronal medium. At d 7, 25, 50, or 75% of
the culture volume of tg338 CGN (CGNOv) cultures was
replaced by astrocyte-conditioned medium. Cells were moni-tored
daily and processed for immunofluorescence after 4 d.
Neuronal survival rate was determined by counting NeuN-positive
cells with nonpyknotic nuclei.
Glutamate and free-radical treatments
After 14 d exposure to mock- or 127S-infected tg338 brain
homogenate, CGNOv cultures were treated with glutamate
(1–100 M) or hydrogen peroxide (1–10 M) (Sigma) for 48
h. Cells were then processed for immunofluorescence. Neu-ronal
survival was estimated by counting NeuN-positive cells
with nonpyknotic nuclei, and dendritic area was quantified by
measuring MAP2 labeling.
Statistical analysis
Each data set corresponding to glutamate or hydrogen per-oxide
treatment was analyzed using the MIXED procedure of
SAS 1999 (SAS Institute, Cary, NC, USA). The ANOVA model
included a random factor “day” and fixed factors correspond-ing
to the cell state (mock- or scrapie-infected), the treatment
dose, and their interaction. To satisfy homoscedasticity as-sumptions,
logarithm of neuronal survival and square root of
dendritic area were considered. LS means were computed,
and all pair-wise differences were evaluated using Tukey-
Kramer adjustment for P values. For the other sets of exper-iments,
data were analyzed by Student’s t test (2-tailed distri-bution;
2-sample equal variance).
RESULTS
Neurons cocultured with prion-infected astrocytes
rapidly degenerate
First, we investigated the contribution of prion-infected
astrocytes in neurodegeneration. Exposure of CASOv
primary cultures to brain homogenate infected with
127S sheep scrapie strain led to the detection of
neosynthesized PrPres from 7 DPE onward by immuno-blot
(Fig. 1A). PrPres amounts then increased 4-fold by
28 DPE. These data were consistent with those obtained
using cell culture medium instead of brain homogenate
as an infectious source (24). At 21 DPE, a large majority
(80%) of astrocytes exposed to 127S scrapie inocu-lum
(ScCASOv) showed a GdnSCN-dependent, bright
punctated PrP immunostaining specific for prion infec-tion
(Fig. 1B, C), therefore confirming that astrocyte
cultures were heavily infected and laden with PrPSc. At
this time point, we seeded freshly dissociated CGNs
expressing ovine PrPC (CGNOv) on ScCASOv cells or on
mock-infected CASOv cells in a medium depleted of
antioxidants. After 7 d of CGNOv/ScCASOv coculture,
neuronal survival significantly decreased by 40% as
compared to CGNOv/CASOv cocultures (n4 indepen-dent
experiments, Fig. 2A). Colocalization of terminal
deoxynucleotidyltransferase-mediated dUTP nick-end
labeling (TUNEL)-positive cells and pyknotic nuclei
indicated that apoptosis was a prominent feature in
dying neurons (data not shown). Surviving neurons
showed massive dendritic fragmentation (Fig. 2D, top
Figure 1. Efficient prion infection of cerebellar astrocyte cultures. Cerebellar astrocyte cultures from tg338 mice (CASOv) were
exposed to 127S scrapie-infected tg338 brain homogenate. A) Immunoblot of PK-treated lysates probed with anti-PrP mAb
ICSM18, showing PrPres accumulation between 7 and 28 DPE in prion-infected CASOv cultures (ScCASOv). As a control,
nonpermissive CAS0/0 cells were exposed in parallel (ScCAS0/0). B–D) CASOv cells were either mock-infected (B) or
prion-infected (scrapie; C, D), and immunofluorescence was performed 3 wk after infection. PrPSc was detected with mAb
ICSM33 (green) after permeabilization (B, C) or not (D) and GdnSCN denaturation. Red, GFAP; blue, DAPI. In infected
cultures, 80% of cells show a punctate fluorescence assumed to reflect PrPSc accumulation. Scale bars 10 m.
PRION NEURODEGENERATION REQUIRES PRP CONVERSION 3

panels), indicating widespread ongoing neurodegen-eration.
Conditioned medium of prion-infected astrocytes is
not neurotoxic and is poorly infectious
In other proteinopathies, it has been shown that astro-cytes
could contribute to neurodegeneration by releas-ing
toxic factors (32–34). Astrocytes provide trophic
support to neurons and regulate neurotransmitters and
toxin concentrations in the extracellular environment.
To examine whether a possible alteration of such
functions by astrocyte prion replication could contrib-ute
to the neuronal death observed in our 7-d cocul-ture,
the culture medium from mock- or prion-infected
astrocytes was conditioned between 21 and 28 DPE and
subsequently added to differentiated CGNOv cells. No
significant difference in neuronal survival was observed
after up to 4 d of exposure (Fig. 3A). This suggested
Figure 3. Conditioned medium of prion-infected astrocytes is
not toxic to neurons and contains little infectivity. A) Differ-entiated
CGNOv cells were cultured for 4 d with 25% (open
bars), 50% (shaded bars), or 75% (solid bars) of mock-infected
(CASOv) or prion-infected (ScCASOv) astrocyte-con-ditioned
medium. Neuronal survival is expressed as a percent-age
of total living neurons (NeuN-positive cells) in
nontreated cultures. Values are means se (n3). B) PrPres
accumulation in CGNOv cells exposed to a serial dilution of
scrapie-infected tg338 brain homogenate or to ScCASOv-conditioned
medium for 28 d. As a control, CGN0/0 cells were
exposed to conditioned medium. C) Comparison of PrPres
amounts between CASOv cells exposed or not to scrapie-infected
tg338 brain homogenate (0.01% final concentra-tion)
at 21 DPE, and the inoculum used. For quantification
purposes, a serial dilution of the inoculum, ranging from 0.5-
to 2-fold the amount of brain homogenate inoculated per well,
was loaded along with lysates of whole CASOv culture wells. B, C)
Immunoblots were probed with biotinylated mAb Sha31.
Figure 2. Influence of neuronal PrPC expression and primary
sequence in prion-induced neuronal death. Freshly dissoci-ated
neurons expressing ovine (CGNOv), murine (CGNMo),
or human PrPC (CGNHu), or devoid of PrP (CGN0/0), were
seeded on top of mock- or sheep scrapie-infected astrocytes
(CASOv or ScCASOv, respectively) and cocultured for a week.
A, B) Neuronal survival is expressed as a percentage of total
living neurons (NeuN-positive cells) in mock-infected cocul-tures
(control). Values are means se; n 4 (A); n3 (B).
In infected cocultures, survival significantly decreased for
CGNOv, but not for CGN0/0, CGNMo, and CGNHu (N.S., not
significant). **P 0.01; ***P 0.001; Student’s t test. C)
PrPSc was quantified by immunofluorescence in CGNOv,
CGN0/0, and CGNMo cells cocultured with ScCASOv cells
using mAb ICSM33. For each experiment, PrPSc levels in
CGN0/0/ScCASOv cultures were normalized to 1. Mean se
(n4). D) CGNOv or CGN0/0 cells were cocultured with
CASOv or ScCASOv cells and stained with anti-MAP2 mAb
(dendrites, green) and DAPI (blue). Note the intense den-dritic
fragmentation in CGNOv cells cocultured with prion-infected
astrocytes (arrows). Scale bars 10 m.

that ScCASOv culture medium does not contain neuro-toxins,
including possibly neurotoxic PrPSc species.
Relative efficacy of infection of CGNOv cells with ScCASOv-conditioned
medium in comparison with serial dilu-tions
of infectious brain homogenate indicated a low
infectivity titer equivalent to 0.0001% brain homoge-nate
or 103 ID50/ml (Fig. 3B). In contrast, after 21
DPE, astrocyte cell fraction had an infectivity titer
100-fold higher (data not shown), and a PrPres con-tent
equivalent to 0.01% brain homogenate (Fig. 3C).
Immunostaining of nonpermeabilized GndSCN-treated
ScCASOv cells showed intense PrPSc labeling, presum-ably
at the cell surface (Fig. 1D). Together, these results
indicate that prion-infected astrocytes do not exhibit
major dysfunctions and rather suggest that, given the
low infectivity level of the conditioned medium, the
abundant cell-associated PrPSc could be responsible for
neuronal death through direct astrocyte contact in
cocultures.
Expression and efficient conversion of neuronal PrPC
are required for neurotoxicity mediated by
prion-infected astrocytes
To assess whether the rapid death observed in neurons
cocultured with prion-infected astrocytes was PrP me-diated,
neurons derived from mice with identical ge-netic
background but devoid of PrPC (CGN0/0) were
cocultured for 1 wk with either CASOv or ScCASOv cells
in the same culture conditions as CGNOv cells. No
alteration in neuronal survival was observed in prion-infected
cocultures (Fig. 2B), in striking contrast with
the CGNOv situation (Fig. 2A). Moreover, CGN0/0
neurite immunostaining failed to reveal any ongoing
neurodegeneration (Fig. 2D, bottom panels).
As neuronal PrPC expression appeared necessary for
neuronal cell death in our cocultures, we next exam-ined
whether its efficient conversion into PrPSc was
equally crucial. Thus, mock- or scrapie-infected CASOv
cells were cocultured with CGNs expressing either
mouse (CGNMo) or human (CGNHu) PrPC, i.e., heter-ologous
PrP species assumed to be inefficiently con-verted
by ovine PrPSc (35). Indeed, in vivo, 127S prions
exhibit a substantial transmission barrier on transmis-sion
to human and mouse PrP transgenic mice (unpub-lished
results). As a result, neuronal survival slightly
decreased, but not to statistically significant levels,
when CGNHu (P0.29) or CGNMo (P0.10) cells were
cocultured with ScCASOv cells for 7 d (n3 indepen-dent
experiments; Fig. 2B). Abnormal PrP levels in the
cocultures did not vary significantly whether ScCASOv
cells were cocultured with CGN0/0 or CGNMo cells, as
assessed by immunostaining and immunoblot (Fig. 2C
and data not shown). Remarkably, and in contrast,
PrPSc levels were more than doubled on coculture with
CGNOv cells, suggesting active prion replication in
these cells (Fig. 2C).
Altogether, our coculture system shows that neurons
apposed to PrPSc-producing astrocytes undergo a rapid
and significant death only when they express PrPC
molecules that are efficiently converted by nearby PrPSc
into new PrPSc species.
Prion infection of neurons increases their sensitivity
to glutamate and free radical insults
Finally, we investigated whether neuronal neosynthe-sized
PrPSc alone was responsible for CGNOv cell death
observed in cocultures with ScCASOv cells. Infection of
“pure” CGNOv cells with 127S-infected brain homoge-nate
led to specific accumulation of PrPres as early as 7
DPE. Amounts steadily increased 5-fold by 28 DPE
(Fig. 4A). By 14 DPE, ScCGNOv cells accumulated
substantial levels of PrPSc (Fig. 4A), distributed in the
soma and processes (24). However, at this stage, neu-ronal
survival was unaltered (Fig. 4B, see dose 0), and a
Figure 4. Prion-infected neurons display an increased sensi-tivity
to glutamate and H2O2. A) Immunoblot probed with
biotinylated mAb Sha31 showing PrPres accumulation kinetics
in CGNOv cells exposed to 127S scrapie-infected tg338 brain
homogenate along with CGN0/0 (ScCGN0/0). B) CGNOv cells
were either mock-treated (open bars) or prion-infected (solid
bars). At 14 DPE, cells were exposed to increasing concentra-tions
of glutamate or hydrogen peroxide (H2O2) for 48 h.
Neuronal survival is expressed as a percentage of total living
neurons in nontreated cultures. Dendritic area represents the
extent of MAP2 labeling, expressed in pixels. Quantifications
correspond to means se of 3 wells in 1 experiment and are
representative of n 3–4 independent experiments. Statisti-cal
analysis was performed using MIXED procedure in SAS
(see Materials and Methods). **P 0.01; ***P 0.001.

significant increase in neuronal death (40%) actually
occurred at 21 DPE (data not shown).
Thus, the presence of nearby astrocytes seemed to
accelerate neurodegeneration in CGNOv/ScCASOv co-cultures.
Because astrocytes are assumed to regulate
extracellular signals, such as reactive oxygen species
and glutamate, we next subjected 14-d CGNOv or Sc-
CGNOv cultures to subtoxic concentrations of gluta-mate
or hydrogen peroxide for 48 h (Fig. 4B). Prion
infection significantly decreased both viability and den-dritic
area when neurons were exposed to 100 M
glutamate (P0.001). It also significantly decreased
dendritic area following treatment with 10 M H2O2
(P0.01), indicating that ScCGNOv neurons underwent
a degenerative process. Altogether, these data suggest
that PrPSc accumulation in neurons increases their
sensitivity to oxidative stress and glutamate.
DISCUSSION
Our study aimed at deciphering the relative impor-tance
of infected astrocytes and neuronal PrPC expres-sion
and conversion in prion-mediated neurodegenera-tion,
by exposing primary differentiated neurons
expressing or lacking PrPC to a continuous and physi-ological
source of PrPSc delivered by neighboring
prion-infected astrocytes. As a main result, we found
that neuronal PrPC must not only be present but also be
efficiently converted into PrPSc for neurotoxicity to
occur, therefore further extending at the cell level
earlier in vivo observations (4, 13) with another prion
model. We also provide evidence that prion-infected
astrocytes are not detrimental per se but that they
accelerate neurodegeneration.
No significant increase in neuronal cell death was
observed on coculture of ovine PrPSc-producing astro-cytes
with neurons genetically devoid of PrPC or ex-pressing
mouse or human PrPC. In striking contrast,
when neurons expressed ovine PrPC, their survival was
decreased by 40% within a week, the surviving cells
showing widespread ongoing neurodegeneration con-comitantly
with detectable PrPSc accumulation. As dif-ferences
in primary sequence appear to have little
effect on PrPC-PrPSc binding (36, 37), it is likely that
ovine PrPSc bound similarly to sheep, mouse, and
human PrPC, thus dismissing a subversion of PrPC
neuroprotective function by infecting PrPSc as the
major cause of the prominent neuronal death observed
here (38). Instead, the delayed or absence of conver-sion
of mouse and human PrPC by 127S prions in
transgenic mice and the significant PrPSc increase
observed only in homologous cocultures strongly sup-port
the view that the conversion step is critical in the
initiation of neurodegeneration. This sharply contrasts
with the emerging notion that PrP could indifferently
convey toxicity of prions or other abnormally folded
proteic conformers, such as those involved in Alzhei-mer’s
disease. Notably, our results are in apparent
contradiction with the recent findings of Resenberger
et al. (17), in which short-term (16 h) coculture with
minimal cell contact between chronically prion-in-fected
mouse neuroblastoma (ScN2a) cells and immor-talized
SH-SY5Y cells transiently expressing various het-erologous
PrP induced an increased apoptosis rate in
the latter, independent of PrP sequence and with no
apparent conversion. However, in these conditions, the
apoptosis increase appeared marginal (5–10%), and
when they subjected mouse primary cortical neurons to
a 5-d coculture with ScN2a cells, a 40% viability de-crease
was observed, similar to our findings, except that
bona fide prion replication was not assessed, thus recon-ciling
their data with our observation that PrP conver-sion
and accumulation are the major contributors to
neuronal death.
Neurons devoid of PrPC have been reported to
exhibit an increased sensitivity to neurotoxins (39).
The absence of neurodegeneration when they were
cocultured with heavily infected astrocytes indicates
that the latter did not misfunction in a way that would
affect cocultured neurons, nor produce labile non-PrP-related
neurotoxins that could have been diluted or
lacking in astrocyte-conditioned medium. Moreover,
this indicates that extraneuronal, infectious PrPSc par-ticles,
either released (at low levels) by astrocytes in the
extracellular medium or membrane bound, were not
significantly neurotoxic per se. Intriguingly, however,
apoptosis and neuritic alterations appeared consider-ably
earlier in ovine PrPC-expressing neurons infected
on coculture with prion-infected astrocytes than by
scrapie inoculum [respectively, at 7 d (this study) vs. 28
d (24) and 21 d when using antioxidant-depleted
medium (unpublished results)]. As infectivity and
PrPres levels of both infectious sources seemed compa-rable,
this indicates that astrocyte-mediated infection
was able to potentiate neurodegeneration. Neuron/
glia close contacts along with nearly maximal PrPSc
levels—presumably at the astrocyte cell surface—are
likely to favor ignition of prion infection in neurons
through efficient cell-to-cell transfer of PrPSc particles,
as previously shown in immortalized cell models (31).
This is further supported by findings that prion infec-tion
of cells occurs very rapidly and that cell mem-branes
probably constitute the primary site of prion
conversion (40). Whether PrPSc produced by long-term
infected astrocytes not only promotes conversion but
also catalyzes the formation of neurotoxic species from
neuronal PrPC, as recently suggested in mice (41), will
be examined in the future.
Interestingly, we show here that PrPSc-accumulating
neurons are more sensitive to exogenous stimuli, such
as glutamate and hydrogen peroxide, suggesting an
altered response to excitotoxicity and oxidative stress,
as reported with other misfolded proteins and peptides
(42–44). Although our results suggest that infected
astrocytes do not release toxic species, we cannot
exclude that they might no longer help neurons cope
with stresses in their environment, including intracel-lular
protein aggregates. In other neurodegenerative
diseases, accumulation of misfolded protein aggregates

and/or changes in lipid raft content or interactions
have both been shown to be detrimental to neurons, by
inducing massive dendritic degeneration and axonal
damage due to microtubule disruption (45–46).
Whether similar alterations are responsible for the
toxicity observed in our study remains to be deter-mined.
Overall the massive neurodegeneration ob-served
in our cocultures would, therefore, result from
both a particularly efficient initiation of prion infection
in neurons by infected astrocytes and a subsequent
increased neuronal vulnerability.
In neurons, the cellular form of the prion protein
PrPC appears to be involved in a number of possibly
independent neurodegenerative pathways, some being
acute following exposure to extraneuronal PrPSc or
other -sheet-rich conformers (17, 47) and some ne-cessitating
efficient PrPC conversion, acting putatively
on a downstream cascade or physiological process (48).
In this regard, our ex vivo model may help to further
dissect these neurodegenerative mechanisms that
could be relevant to other brain disorders and devel-opment
of rational therapies.
This project was supported by grants from the French
government (GIS-Infections a` Prion) and from the European
Union (Neurodegeneration–QLG3CT2001). S.C. was a recip-ient
of a French Ministry of Research and Education (MRE)
fellowship. J.C. was funded by an MRE fellowship and the
French Foundation France Alzheimer. The authors thank S.
Hawke (Imperial College, London, UK; now at University of
Sydney, Sydney, NSW, Australia) and G. S. Jackson (Medical
Research Council Prion Unit, London, UK) for kindly pro-viding
the antibodies ICSM18 and ICSM35, and ICSM33,
respectively; J. Grassi and S. Simon (Commissariat a` l’énergie
Atomique et aux énergies Alternatives, Saclay, France) for
Sha31 antibody; C. Weissmann (Scripps Research Institute,
Jupiter, FL, USA) for authorizing the inclusion of tga20 and
PrP0/0 mice in this study. The authors thank T. Szeto for
careful reading of the manuscript and R. Young for prepara-tion
of the figures.
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Received for publication February 28, 2012.
Accepted for publication May 21, 2012.

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