Pierre Zwiegers 1,2 , Grace Lee 1 , & Christopher A. Shaw 1

1. B) Active Caspase 3 ICC Pierre Zwiegers1,2, Grace Lee1, & Christopher A. Shaw1 Targeted upregulation of Progranulin as a putative means in ameliorating motor neuron death in a genetic model of amyotrophic lateral sclerosis 1Neural Dynamics Research Group, Univ. of British Columbia 2Department of Medicine, Univ. of British Columbia C) TDP-43 ICC Para- grn grnF grnD grnB grnA grnC grnE grnG S7 Objective Introduction Neural Dynamics Research Group Previous Results A) Choline acetyltransferase (ChAT) labeling in murine lumbar spinal cord We hypothesize that the secreted neuroprotective protein, Progranulin (PGRN), delivered to motor neurons by lentiviral-mediated translocation from the injection site will ameliorate motor neuron 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. Primary Antibody: Rbα- active caspase 3 (Promega) 1:250 Secondary Antibody: Gtα -Rb AF546 (Mol. Probes) 1:200 WT G37R G37R/Progranulin GFP PGRN A) MPTP Cytotoxicity in PC-12 Cells B) ChAT quantification of lumbar spinal cord mSOD G37R WT Figure 2.Progranulin-mediated neuroprotective effect. Transgenic G37R animals at a later stage just prior to disease phenotype expression (~ 11 months of age) as well as wild-type conspecifics, received bilateral injections of either Progranulin or GFP-containing lentiviral constructs directly into the gastrocnemius muscles (8.2x107 IU/mL). (A) Choline acetyltransferase (ChAT) immuno-labeling in the ventral horn of lumbar spinal cord sections. (B) Motor Figure 1. (A) Cell survival assay (MTT colorimetric assay) in PC-12 cells evaluating the protective properties of 4nM PGRN in the presence of 100-1000 µM of MPTP. There was a significant increase in the percentage of surviving cells when PGRN was present for each of the four doses of MPTP treatment. Mean ± SEM and Student t-test results are shown.(B) Primary Antibodies: Rabbit anti-TDP43 Dilution: 1:250 Chicken anti-β-tubulin Dilution: 1:5 Secondary Antibodies: Goat anti-rabbit AF546 Dilution: 1:200 Goat anti-chicken FITC Dilution: 1:200 [MPTP] µM - Encoded as an 8kb genomic fragment on chromosome 17 - Secreted protein derived from 13 exons and comprised of 7.5 tandem repeats of a conserved 12 cysteinylgranulin motif with a MW of 90kDa in its glycosylated form Antalet al 2007 NEG Control α-GFP 10x α-GFP 100x neuron counts revealed a significant neuroprotective effect of progranulin in the fALS group. ProgranulincDNA injected animals expressing mutant SOD1 exhibit more surviving neurons compared to the conspecific group receiving control GFP injections. Ongoing histological studies aim at assessing stress markers and glial markers of inflammation, and will be communicated in the future. - Proteolytic cleavage yields several 6kDa granulin peptides that exhibit a plethora of effects on cellular physiology - Putatively crucial in the long-term survival of neuronal cells - Additional roles include: · wound repair · male-specific brain differentiation · neovascularization · anti-inflammatory properties - PGRN expression in an murine model of Alzheimer’s disease showed decreased neuroinflammation, reduced plaque burden and preservation of synapses - In a zebrafish model, PGRN overexpression has been shown to rescue mutant TDP-43 induced axonopathy - Neuroprotective for cortical and spinal neurons against serum deprivation - Nulllmutations implicated as a genetic basis for tau-negative frontotemporal dementia - PGRN mutations or polymorphisms can be a modifier of disease course in ALS in that an ALS phenotype presents at an earlier age of onset and is coupled with a more rapid decline of survival - Post-mortem analysis of ALS tissue indicates increased PGRN expression in degenerating motor tracts in the spinal cord & brainstem - Sortilinhas been identified as the receptor mediating an endocytic pathway targeting extracellular PGRN to lysosomal localization Qualitatively, PGRN neuroprotective action did not involve prevention of caspase-3 activation in PC-12 cells treated with 1000µM MPTP. (C) Similarly,ICC assays demonstrated that the observed MPTP toxicity did not involve the extranuclear translocation and/or aggregation of TDP-43. Scale bar is 20µm; arrows point at active caspase 3 positive cells. Preliminary Results Kasparet al 2003 B) 360 332 304 402 73 446 86 472 219 277 247 A) 191 123 499 135 527 164 556 Avg age (d) Histological + Biochemical Analysis C) Cruts & Van Broeckhoven Trends in Genetics 24(4), 2008 He & Bateman J Mol Med 81, 2003 Van Kampen & Kay J AlzAssoc 7(4), 2011 Ahmed et al J Neuroinlammation 4(7), 2007 Hu et al Neuron68(4), 2010 Laird et al PLoS one 5(10), 2010 Irwin & Russo J. NeuroSci 276(1-2), 2008 Cruts et al Nature 442, 2006 Sleegerset al Neurology 71, 2008 Van Damme et al J. Cell Biol. 181, 2008 5 weeks baseline behavioural data collection 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-GFP activity in lumbar cord motor neurons thus establishing the applicability of specific retrograde targeting of motor neurons via intramuscular delivery of the viral vector. Discussion D) Results Summary Future Directions *5x2µL lentiviral vector 4.0x108 TU/mL per gastrocnemius muscle (Invitrogen) Figure 3. Current ongoing measures of behavioural indices employed to assess the extent of long-term neuronal protection conveyed by lentivirus-mediated Progranulin (PGRN) cDNAupregulation in the lumbar spinal cord delivered intramuscularly at approximately 3.5 months of age. Due to the expected sex differences in diseased phenotype expression, comparisons 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 containing Progranulin (PGRN) or GFP constructs in each gastrocnemius muscle. To date, most animals have yet to fully express the expected disease phenotype. In both (A) Weight and (B) the Rotarod performance score, no significant trend within either sex has become evident, with male animals generally larger coupled with a general deficit in performance. In measures of (C) Wire Hang performance, male animals performed demonstrably poorer than females, however at later time points significant differences are seen in the transgenic female groups. Similarly, with the (D) Leg Extension reflex score, later time points are indicating a significant difference in trans-genic animals. Mean +/- SEM shown, intra-sex analysis by one-way ANOVA& Tukey post-test. The search for putative means toprophylactically ameliorate motor neuron cell death in ALS is of crucial importance. Current treatment modalities are typically administered following disease onset at a stage where significant motor neuron loss may already have been established. If the current aims of this study are achieved, PGRN-mediated genetic therapy may prove to be an effective tool in attenuating motor neuron degeneration. Previous work by our group has shown preliminary evidence for the cytoprotective actions of PGRN. The current behavioural observations are considered preliminary as the majority of animals have yet to reach a point at which hind-limb paralysis becomes fully evident. It is expected, however, that the effect of progranulin in the measured behavioural indices may be limited since (i) only lower motor neurons targeting the gastrocnemius muscle are transduced and (ii) postural and behavioural adjustments by additional muscle groups may compensate for localized deficits prior to phenotypic ALS expression. Histological and biochemical examination of lumbar spinal cord will provide more conclusive evidence of any possible neuroprotectiveactivity. Wire Hang Rotorod Leg Extension Experimental Design Acknowledgements Retrograde Transport of Vector Sites of Injection Progranulin Vector Construct Invitrogen - Male B6.Cg-Tg(SOD1*G37R)29Dpr/J (Jackson Laboratory) animals were crossed with congenic C57BL/6 females to generate Tg (+)ve and Tg (-)ve progeny, confirmed via genotyping - At approx 3.5 months of age, following isofluraneanaesthesia, a GFP or PGRN lentiviral vector was administered to each gastrocnemius muscle with 5 x 2µL injections at 4.0x108TU/mL via Hamilton syringe - Bi-weekly behavioral assays (Rotorod, Wire Hang, and Leg Extension reflex score) are employed to monitor the onset of an ALS phenotype Injection site - Exogenous administration of full-length PGRN protein in vitro, has shown to protect PC12 cells against a MPTP cytotoxic insult -Late-stage delivery of the PGRN lentiviral construct in G37R mSOD animals has been shown to ameliorate motor neuron loss -Early targeting of lumbar cord motor neurons in mSOD animals has not established any significant effect on behavioural measures. This may be due to the fact that an ALS phenotype has yet to become fully established in all experimental groups. -Histological assessment following onset of an ALS phenotype to discern motor neuron viability and markers of neuroinflammation -Currently, additional work by our group is assessing whether lentiviral-mediated delivery of progranulin will exhibit neuroprotective activity in an environmental model of ALS-PDC, work that will explore neuronal rescue in the context of a non- mSOD mediated toxic insult, which may be more telling for cases of sporadic ALS -Additional work would need to explore the prophylactic efficacy in targeting multiple neuronal populations and the subsequent effect on ALS phenotypic outcomes in genetic models. Special thanks to ALS Canada for making the current research endeavour possible. Additionally, thanks for the support and encouragement of members from Neural Dynamics Research Group as well as Drs D. Kay & J. Van Kampen from Neurodyn Inc. for their invaluable insights and troubleshooting advice. If we knew what we were doing, it wouldn't be called research, would it? -Einstein