This document summarizes a study investigating whether delivering the neuroprotective protein progranulin (PGRN) to motor neurons via lentivirus-mediated gene therapy can prevent motor neuron loss in a mouse model of ALS. PGRN has shown neuroprotective effects in other studies. The researchers injected PGRN-expressing or control lentiviruses into the leg muscles of transgenic ALS mice and wildtype mice before disease onset. Preliminary results show PGRN protects neurons in cell culture and may reduce motor neuron loss when delivered late in disease in mice. Ongoing work will assess effects on neuroinflammation and behavior as disease progresses. The goal is to determine if PGRN gene therapy can prophyl

1. Pierre Zwiegers1,2, Grace Lee1, & Christopher A. Shaw1
1NeuralDynamics Research Group, Univ. of British Columbia
2Department of Medicine, Univ. of British Columbia
2-C-55 Neural Dynamics Research Group
Objective We hypothesize that the secreted neuroprotective Previous Results B) Active Caspase 3 ICC
Primary Antibody:
A) Choline acetyltransferase (ChAT) labeling in murine lumbar spinal cord
Rb α- active caspase 3 WT G37R G37R/Progranulin
protein, Progranulin (PGRN), delivered to motor neurons by lentiviral- A) MPTP Cytotoxicity in PC-12 Cells (Promega)
1:250
Secondary Antibody:
mediated translocation from the injection site will ameliorate motor neuron Gt α -Rb AF546 (Mol.
Probes)
1:200
loss in a genetic model of ALS. Using G37R mSOD mice, we aim to determine
whether LV-PGRN delivered before the onset of major behavioural deficits can
prevent, halt, or even reverse/stabilize mSOD-induced motor neuron death. [MPTP] µM
Figure 1. (A) Cell survival assay (MTT
colorimetric assay) in PC-12 cells evaluating the B) ChAT quantification of lumbar spinal cord Figure 2. Progranulin-mediated neuroprotective
Introduction protective properties of 4nM PGRN in the C) TDP-43 ICC
Primary Antibodies: GFP effect. Transgenic G37R animals at a later stage
presence of 100-1000 µM of MPTP. There was a Rabbit anti-TDP43 PGRN just prior to disease phenotype expression (~ 11
- Encoded as an 8kb genomic fragment on chromosome 17 significant increase in the percentage of Dilution: 1:250
Chicken anti-β-tubulin months of age) as well as wild-type
- Secreted protein derived from 13 exons and comprised of 7.5 tandem repeats of a surviving cells when PGRN was present for Dilution: 1:5 WT mSOD G37R conspecifics, received bilateral injections of
each of the four doses of MPTP treatment. Secondary Antibodies: either Progranulin or GFP-containing lentiviral
conserved 12 cysteinyl granulin motif with a MW of 90kDa in its glycosylated form Mean ± SEM and Student t-test results are Goat anti-rabbit AF546
constructs directly into the gastrocnemius
Dilution: 1:200
shown. (B) Qualitatively, PGRN Goat anti-chicken FITC
muscles (8.2x107 IU/mL).
Dilution: 1:200
Para- neuroprotective action did not involve (A) Choline acetyltransferase (ChAT) immuno-
grnG grnF grnB grnA grnC grnD grnE prevention of caspase-3 activation in PC-12 labeling in the ventral horn of lumbar spinal cord
grn cells treated with 1000µM MPTP. (C) sections. (B) Motor neuron counts revealed a
Similarly, ICC assays demonstrated that the significant neuroprotective effect of progranulin
in the fALS group. Progranulin cDNA injected animals expressing mutant SOD1 exhibit more
- Proteolytic cleavage yields several 6kDa granulin peptides that exhibit a plethora of observed MPTP toxicity did not involve the surviving neurons compared to the conspecific group receiving control GFP injections. Ongoing
extranuclear translocation and/or aggregation
effects on cellular physiology of TDP-43. 20µm; arrows point at active caspase 3 positive cells.
histological studies aim at assessing stress markers and glial markers of inflammation, and will be
Scale bar is communicated in the future.
- Putatively crucial in the long-term survival of neuronal cells
- Additional roles include: Preliminary Results Figure 3. Current ongoing measures of behavioural indices employed to assess the extent of
· wound repair · male-specific brain differentiation long-term neuronal protection conveyed by lentivirus-mediated Progranulin (PGRN) cDNA
upregulation in the lumbar spinal cord delivered intramuscularly at approximately 3.5 months
· neovascularization · anti-inflammatory properties A) B) of age. Due to the expected sex differences in diseased phenotype expression, comparisons
- PGRN expression in an murine model of Alzheimer’s disease showed decreased were made within male (m) and female (f) groups distinguishing between wild-type (WT) and
transgenic (G37R) animals receiving 5x2 µL (4.0x108 TU/mL) injections of either the lentivirus
neuroinflammation, reduced plaque burden and preservation of synapses containing Progranulin (PGRN) or GFP constructs in each gastrocnemius muscle. To
- In a zebrafish model, PGRN overexpression has been shown to rescue mutant TDP-43 date, animals have yet to fully express the expected disease phenotype. In both (A) Weight
induced axonopathy and (B) the Leg Extension reflex score, no significant trend within either sex has yet become
evident. In measures of (C) Rotarod and (D) Wire Hang performance, male animals
- Neuroprotective for cortical and spinal neurons against serum deprivation performed demonstrably poorer than females, however no significant temporal differences
- Nulll mutations implicated as a genetic basis for tau-negative frontotemporal dementia Avg
65 80 92 129 149 177 205 233 262 290 318 346 374 Avg
65 80 92 129 149 177 205 233 262 290 318 346 374
were found when intrasex comparisons were made. Mean +/- SEM shown, intra-sex analysis
age (d) age (d) by one-way ANOVA.
- PGRN mutations or polymorphisms can be a modifier of disease course in amyotrophic
lateral sclerosis in that an ALS phenotype presents at an earlier age of onset and is C) α-GFP NEG Control
100x α-GFP
coupled with a more rapid decline of survival 10x
- Post-mortem analysis of ALS tissue indicates increased PGRN expression in
degenerating motor tracts in the spinal cord & brainstem
- Sortilin has been identified as the receptor mediating an endocytic pathway targeting
extracellular PGRN to lysosomal localization Avg
65 80 92 129 149 177 205 233 262 290 318 346 374 Avg
65 80 92 129 149 177 205 233 262 290 318 346 374
age (d) age (d)
He & Bateman Van Kampen & Kay Ahmed et al Cruts & Van Broeckhoven Hu et al
J Mol Med 81, 2003 J Alz Assoc 7(4), 2011 J Neuroinlammation 4(7), 2007 Trends in Genetics 24(4), 2008 Neuron 68(4), 2010 D)
Cruts et al Sleegers et al Van Damme et al Laird et al Irwin & Russo
Nature 442, 2006 Neurology 71, 2008 J. Cell Biol. 181, 2008 PLoS one 5(10), 2010 J. NeuroSci 276(1-2), 2008 Figure 4. Immunohistological assessment for green fluorescent protein (GFP) following
lentiviral-mediated delivery. Male CD-1 animals received bilateral administration of a GFP-
expressing lentivirus into both gastrocnemius muscles (5x2µL lentiviral vector 4.0x108 TU/mL
per muscle). One-month following transfection, transduction efficiency was assessed via anti-
Experimental Design GFP activity in lumbar cord motor neurons thus establishing the applicability of specific
G37R mSOD or retrograde targeting of motor neurons via intramuscular delivery of the viral vector.
C57BL/6 WT Mice Avg
65 80 177 205 233 262 374 Avg
92 129 149 290 318 346 65 80 92 129 149 177 205 233 262 290 318 346 374
age (d) age (d)
5 weeks baseline behavioural data collection
Progranulin-expressing lentivirus GFP-expressing lentivirus Results Summary Discussion
bilateral intra-gastrocnemius bilateral intra-gastrocnemius -Exogenous administration of full-length PGRN protein in vitro, has The search for putative means in prophylactically ameliorating motor neuron
injections* injections*
shown to protect PC12 cells against a MPTP cytotoxic insult cell death in ALS is of crucial importance as current treatment modalities are
-Late-stage delivery of the PGRN lentiviral construct in G37R mSOD typically administered following disease onset at a stage where significant
♀ G37R mSOD n=12 ♀G37R mSOD n=11 ♂G37R mSOD n=11
♂G37R mSOD n=13
animals has been shown to ameliorate motor neuron loss motor neuron loss may already have been established. If the current aims are
♀ C57BL/6 WT n=12 ♀ C57BL/6 WT n=12 ♂ C57BL/6 WT n=12
♂ C57BL/6 WT n=12 achieved, PGRN-mediated genetic therapy may prove to be an effective tool in
-Early targeting of lumbar cord motor neurons in mSOD animals has not
ameliorating motor neuron degeneration. Previous work by our group has
*5x2µL lentiviral vector 4.0x108 TU/mL per gastrocnemius muscle established any significant effect on behavioural changes as an ALS
(Invitrogen) shown preliminary evidence for the cytoprotective actions of PGRN. The
Behavioural phenotype has yet to become established. current behavioural observations are considered preliminary as the animals
monitoring Histological +
Biochemical
have yet to reach a point at which hind-limb paralysis becomes evident. It is
Leg Extension Rotorod Wire Hang Analysis Future Directions expected, however, that the effect of progranulin in the measured behavioural
indices will be marginal since (i) only lower motor neurons targeting the
Retrograde Transport of Vector Sites of Injection Progranulin Vector Construct -Histological assessment following onset of an ALS phenotype to gastrocnemius muscle are transduced and (ii) postural and behavioural
discern motor neuron viability and markers of neuroinflammation adjustments by additional muscle groups may compensate for localized deficits
-Currently, additional work by our group is assessing whether prior to phenotypic ALS expression. Histological and biochemical examination
of lumbar spinal cord will provide more conclusive evidence of neuroprotective
lentiviral-mediated delivery of progranulin will exhibit activity.
Kaspar et al 2003 Injection site Antal et al 2007 Invitrogen
neuroprotective activity in an environmental model of ALS-PDC,
- Male B6.Cg-Tg(SOD1*G37R)29Dpr/J (Jackson Laboratory) animals were crossed with work that will explore neuronal rescue in the context of a non-
Acknowledgements
congenic C57BL/6 females to generate Tg (+)ve and Tg (-)ve progeny, confirmed via mSOD mediated toxic insult, which may be more telling for cases
genotyping of sporadic ALS Special thanks to ALS Canada for making the current research
- At approx 3.5 months of age, following isoflurane anesthesia, a GFP or PGRN lentiviral -Additional work would need to explore the prophylactic efficacy endeavour possible. Additionally, thanks for the support and
vector was administered to each gastrocnemius muscle with 5 x 2µL injections at in targeting multiple neuronal populations and the subsequent encouragement of members from Neural Dynamics Research
4.0x108 TU/mL via Hamilton syringe
effect on ALS phenotypic outcomes in genetic models. Group as well as Drs D. Kay & J. Van Kampen from Neurodyn
- Bi-weekly behavioral assays (Rotorod, Wire Hang, and Leg Extension reflex score) are
Inc. for their invaluable insights and troubleshooting advice.
employed to monitor the onset of an ALS phenotype If we knew what we were doing, it wouldn't be called research, would it? -Einstein