To measure the effect of C9ORF72 activity on endogenous DPR levels in human motor neurons, we quantified endogenous PR+ puncta in C9-ALS iMNs with or without C9ORF72 overexpression. Using a validated anti-PR antibody 10,50, we found that the majority of PR+ punctae were localized in the nucleus (Fig. 5f), although we also detected cytoplasmic PR+ punctae to a larger extent than we had previously observed with exogenous PR(50) 10. C9-ALS iMNs (n=2 patients) had higher levels of nuclear PR+ puncta than controls (n=2 controls)(Fig. 5f, g) and overexpression of C9ORF72 isoform B significantly reduced the number of PR+ puncta in C9-ALS iMNs (Fig. 5f, h).
To determine if PIKFYVE inhibition rescued patient iMN survival by reversing phenotypic changes caused by C9ORF72 haploinsufficiency, we measured glutamate receptor levels with and without PIKFYVE inhibitor treatment. PIKFYVE inhibition significantly lowered NR1 (NMDA receptor) and GLUR1 (AMPA receptor) levels in patient (n=4 patients) and C9ORF72+/− iMNs (Supplementary Fig. 15p-s). PIKFYVE inhibition also reduced electrophysiological activity in patient motor neurons (C9-ALS1) during glutamate treatment (Supplementary Fig. 15t). To determine if small molecule inhibition of Pikfyve rescues C9ORF72 disease processes in vivo, we first established an NMDA-induced hippocampal injury model in C9orf72-deficient mice. In control mice, hippocampal injection of NMDA caused neurodegeneration after 48 hrs as we have shown previously 57 (Supplementary Fig. 17a, b). Consistent with C9orf72-deficient mice having elevated NMDA receptor levels (Fig. 4h, i and Supplementary Fig. 11a-d), injection of NMDA caused significantly greater neurodegeneration in C9orf72+/− and C9orf72−/− mice than in controls (Fig. 6g, h). Importantly, co-administration of Apilimod rescued the NMDA-induced neurodegeneration in C9orf72-deficient mice (Fig. 6g, h).
Total RNA was extracted from sorted iMNs at day 21 post-transduction with Trizol RNA Extraction Kit (Life Technologies) and reverse transcribed with an Oligo dT primer using ProtoScript® II First Strand Synthesis Kit (NEB). RNA integrity was checked using the Experion system (Bio-Rad). Real-time PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad) using primers shown in Supplementary Data Table 4.
Yingxiao Shi,#1,2,3 Shaoyu Lin,#1,2,3 Kim A. Staats,1,2,3 Yichen Li,1,2,3 Wen-Hsuan Chang,1,2,3 Shu-Ting Hung,1,2,3 Eric Hendricks,1,2,3 Gabriel R. Linares,1,2,3 Yaoming Wang,3,4 Esther Y. Son,5 Xinmei Wen,6 Kassandra Kisler,3,4 Brent Wilkinson,3 Louise Menendez,1,2,3 Tohru Sugawara,1,2,3 Phillip Woolwine,1,2,3 Mickey Huang,1,2,3 Michael J. Cowan,1,2,3 Brandon Ge,1,2,3 Nicole Koutsodendris,1,2,3 Kaitlin P. Sandor,1,2,3 Jacob Komberg,1,2,3 Vamshidhar R. Vangoor,7 Ketharini Senthilkumar,7 Valerie Hennes,1,2,3 Carina Seah,1,2,3 Amy R. Nelson,3,4 Tze-Yuan Cheng,8 Shih-Jong J. Lee,8 Paul R. August,9 Jason A. Chen,10 Nicholas Wisniewski,10 Hanson-Smith Victor,10 T. Grant Belgard,10 Alice Zhang,10 Marcelo Coba,3,11 Chris Grunseich,12 Michael E. Ward,12 Leonard H. van den Berg,13 R. Jeroen Pasterkamp,7 Davide Trotti,6 Berislav V. Zlokovic,3,4 and Justin K. Ichida1,2,3,†
Post mortem tissues were kindly provided by Neil Shneider (Columbia) and were collected from the following individuals: Sample 1 – age: 64, diagnosis: ALS, genotype: positive for C9ORF72 repeat expansion, Sample 2 – age: 55, diagnosis: ALS, genotype: positive for C9ORF72 repeat expansion, Sample 3 – age: 65, diagnosis: ALS, genotype: positive for C9ORF72 repeat expansion, Sample 4 – age: 65, diagnosis: control, genotype: negative for C9ORF72 repeat expansion, Sample 5 – age: 50, diagnosis: control, genotype: negative for C9ORF72 repeat expansion, Sample 6 – age: 50, diagnosis: control, genotype: negative for C9ORF72 repeat expansion, Sample 7 – age: 53, diagnosis: ALS, genotype: negative for C9ORF72 repeat expansion, Sample 8 - age: 64, diagnosis: ALS, genotype: negative for C9ORF72 repeat expansion. All donors except donor 7 (sample 7) were female. For immunofluorescence, 10 µm sections were sliced from flash frozen lumbar spinal cord tissues. Sections were then air dried and fixed with ice cold acetone for 10 minutes, and blocked with 10% normal goat serum/1% BSA/0.3% Triton-X/PBS at room temperature for 1 hour followed by incubation with NR1 antibody (1:200, BD Bioscience) in blocking buffer overnight at 4 ºC. Sections subsequently were blocked using avidin/biotin kit (Vector Lab), and washed with PBS. Then, sections were incubated with goat anti-rabbit IgG Biotin conjugate secondary antibody (1:750, Invitrogen) or with goat anti-mouse IgG Biotin conjugate secondary antibody (1:750, Invitrogen) for 1 hour at room temperature, washed and incubated with streptavidin-Alexa Fluor 488 conjugate (1:500, Invitrogen) in dark for 1 hour at room temperature. Sections were washed and blocked again in blocking buffer for 1 hour at room temperature. For neuronal marker staining, sections were incubated with Tu-20 antibody (1:1000, Abcam) or NeuN antibody (1:500, Abcam) at 37 ºC for 1 hour. Sections were washed with PBS and incubated with goat anti-mouse Alexa Fluor 546 (1:500, Invitrogen) or goat anti-rabbit Alexa Fluor 546 (1:500, Invitrogen) for 1 hour at room temperature. Lipofuscin autofluorescence was quenched by immersing sections in autofluorescence eliminator reagent (Millipore) for 4 minutes following manufacture’s instruction. Sections were then counterstained and mounted with Prolong Gold antifade mounting medium with DAPI (Invitrogen).