Removal of TTX and TEA during glutamate receptor agonist treatment revealed additional increases in Gcamp6 activation in C9ORF72+/− iMNs compared to controls, suggesting that C9ORF72+/− iMNs also fire action potentials more frequently than controls (Supplementary Fig. 13a), although we did not detect large changes in sodium or potassium current amplitudes in C9ORF72+/− iMNs (Supplementary Fig. 13b, c). To determine if increased neuronal activity due in part to elevated glutamate receptor levels contributes to neurodegeneration in C9ORF72 patient and C9ORF72+/− iMNs, we measured iMN survival in the presence or absence of retigabine. Retigabine is approved by the U.S. Food and Drug Administration for the treatment of epilepsy and reduces neuronal excitability by activating Kv7 potassium channels 48. In the glutamate treatment assay, retigabine increased the survival of C9ORF72 patient (n=2 patients) and C9ORF72-deficient iMNs, but not controls (n=2 controls)(Supplementary Fig. 13d-g).
iPSC motor neurons were generated as described previously28, with slight modifications. On day 0, iPSCs were dissociated with Accutase (Life Technologies) and 300,000 iPSCs were seeded into one Matrigel (Corning)-coated well of a 6-well plate in mTeSR medium (Stem Cell Technologies) with 10 μM Rock Inhibitor (Selleck). On day 1, the medium was changed to Neural Differentiation Medium (NDM) consisting of a 1:1 ratio of DMEM/F12 (Genesee Scientific) and Neurobasal medium (Life Technologies), 0.5x N2 (Life Technologies), 0.5x B27 (Life Technologies), 0.1 mM ascorbic acid (Sigma), 1x Glutamax (Life Technologies). 3 μM CHIR99021 (Cayman), 2 μM DMH1 (Selleck), and 2 μM SB431542 (Cayman) were also added. On day 7, cells were dissociated with Accutase and 4.5 million cells were seeded into Matrigel coated 10cm dishes in NDM plus 1 μM CHIR99021, 2 μM DMH1, 2 μM SB431542, 0.1 μM RA (Sigma), 0.5 μM Purmorphamine (Cayman), and 10 μM Rock Inhibitor. Rock inhibitor was removed on day 9. On day 13, cells were dissociated with Accutase and seeded at a density of 40 million cells per well in a non-adhesive 6 well plate (Corning) in NDM plus 0.5 μM RA, 0.1 μM Purmorphamine, and 10 μM Rock Inhibitor. On day 19, the media was changed to NDM plus 1 μM RA, 1 μM Purmorphamine, 0.1 μM Compound E (Cayman), and 5 ng/ml each of BDNF, GDNF and CNTF (R&D Systems). Cells were used for experiments between days 25–35 of differentiation.

The GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for about 10% of each disease worldwide 1–4. In the central nervous system (CNS), neurons and microglia express the highest levels of C9ORF72 5, suggesting that C9ORF72 acts in part cell autonomously and effects in neurons are a key source of disease etiology. Studies showing that the repeat expansion generates neurotoxic species including nuclear RNA foci 6–8, RNA/DNA G-quadruplexes 9, and dipeptide repeat proteins (DPRs) 10–12 have oriented the field towards a therapeutic focus on blocking the toxicity of these products 6–8,13,14. However, these strategies have not fully rescued the degeneration of patient-derived neurons 7,13. Moreover, tandem GGGGCC repeats are transcribed from over 80 other genomic locations within human spinal motor neurons (Supplementary Tables 1 and 2), yet genetic studies have not linked repeat expansions in these regions to ALS/FTD. In addition, hexanucleotide repeat-mediated toxicity in mice requires supraphysiological expression levels or a specific genetic background 14–16. These observations suggest that there are additional pathogenic triggers caused by repeat expansion within C9ORF72.
Whole cell membrane potential and current recordings in voltage- and current-clamp configurations were made using an EPC9 patch clamp amplifier controlled with PatchMaster software (HEKA Electronics). Voltage- and current-clamp data was acquired at 50 kHz and 20 kHz, respectively, with a 2.9 kHz low-pass Bessel filter, while spontaneous action potential recordings were acquired at 1 kHz sampling frequency. For experiments, culture media was exchanged with warm extracellular solution consisting of (in mM): 140 NaCl, 2.8 KCl, 10 HEPES, 1 MgCl2, 2 CaCl2, and 10 glucose, with pH adjusted to 7.3 and osmolarity adjusted to 305 mOsm. Glass patch pipettes were pulled on a Narishige PC-10 puller and polished to 5–7 MΩ resistance. Pipettes were also coated with Sylgard 184 (Dow Corning) to reduce pipette capacitance. The pipette solution consisted of (in mM): 130 K-gluconate, 2 KCl, 1CaCl2, 4 MgATP, 0.3 GTP, 8 phosphocreatine, 10 HEPES, 11 EGTA, adjusted to pH 7.25 and 290 mOsm. Pipettes were sealed to cells in GΩ-resistance whole cell configuration, with access resistances typically between 10–20 MΩ, and leakage currents less than 50 pA. Capacitance transients were compensated automatically through software control. For voltage clamp, cells were held at −70 mV. For Current-voltage traces, a P/4 algorithm was used to subtract leakage currents from the traces. Measurements were taken at room temperature (approximately 20–25 °C). Data was analyzed and plotted in Igor Pro 6 (WaveMetrics) using Patcher’s Power Tools plug-in and custom programmed routines. Current density was obtained by dividing the measured ion channel current by the cell capacitance. For control iMNs, 10/10 tested fired action potentials. For C9-ALS iMNs, 9/10 tested fired action potentials.

To provide a quantitative measure of (GGGGCC)n hexanuceotide expansion in C9ORF72, 100 ng of genomic DNA was amplified by touchdown PCR using primers shown in Supplementary Data Table 4, in a 28-µl PCR reaction consisting of 0.2 mM each of 7-deaza-2-deoxyguanine triphosphate (deaza-dGTP) (NEB), dATP, dCTP and dTTP, 7% DMSO, 1X Q-Solution, 1X Taq PCR buffer (Roche), 0.9 mM MgCl2, 0.7 µM reverse primer (four GGGGCC repeats with an anchor tail), 1.4 µM 6FAM-fluorescently labeled forward primer, and 1.4 µM anchor primer corresponding to the anchor tail of reverse primer (Supplementary Data Table 4). During the PCR, the annealing temperature was gradually decreased from 70 ºC and 56 ºC in 2 ºC increments with a 3 min extension time for each cycle. The PCR products were purified using the QiaQuick PCR purification kit (Qiagen) and analyzed using an ABI3730 DNA Analyzer and Peak Scanner™ Software v1.0 (Life Technologies).
Consistent with previous studies 3,4,6–8, patient iMNs (n=5 patients) had reduced C9ORF72 expression compared to controls (n=3; Fig. 2a and Supplementary Fig. 4a, 5b). While previous studies have linked low C9ORF72 levels to changes in vesicle trafficking or autophagy 18,20,30–33, it remains unknown if loss of C9ORF72 protein directly contributes to degeneration. Thus, we re-expressed C9ORF72 (isoform A or B) in iMNs using a retroviral cassette (Supplementary Fig. 4b) and found that both isoforms rescued C9ORF72 patient iMN survival in response to glutamate treatment (n=3 patients Fig. 2b and Supplementary Fig. 4c). This effect was specific for C9ORF72 iMNs, as forced expression of C9ORF72 did not rescue SOD1A4V iMN survival (Fig. 2c), nor did it improve the survival of control iMNs (n=2 controls Fig. 2d and Supplementary Fig. 4d).
Reprogramming was performed in 96-well plates (8 × 103 cells/well) or 13mm plastic coverslips (3.2 × 104 cells/coverslip) that were sequentially coated with gelatin (0.1%, 1 hour) and laminin (2–4 hours) at room temperature. To enable efficient expression of the transgenic reprogramming factors, iPSCs were cultured in fibroblast medium (DMEM + 10% FBS) for at least 48 hours and either used directly for retroviral transduction or passaged before transduction for each experiment. 7 iMN factors or 5 iDA factors were added in 100–200 µl fibroblast medium per 96-well well with 5 μg/ml polybrene. For iMNs, cultures were transduced with lentivirus encoding the Hb9::RFP reporter 48 hours after transduction with transcription factor-encoding retroviruses. On day 5, primary mouse cortical glial cells from P1 ICR pups (male and female) were added to the transduced cultures in glia medium containing MEM (Life Technologies), 10% donor equine serum (HyClone), 20% glucose (Sigma-Aldrich), and 1% penicillin/streptomycin. On day 6, cultures were switched to N3 medium containing DMEM/F12 (Life Technologies), 2% FBS, 1% penicillin/streptomycin, N2 and B27 supplements (Life Technologies), 7.5 µM RepSox (Selleck), and 10 ng/ml each of GDNF, BDNF, and CNTF (R&D). The iMN and iDA neuron cultures were maintained in N3 medium, changed every other day, unless otherwise noted.

We also found that Reduced C9ORF72 activity also induces iMN hypersensitivity to DPRs by impairing their clearance. This uncovers a more direct form of cooperative pathogenesis between gain- and loss-of-function mechanisms in C9ORF72 ALS/FTD. Through a potentially similar mechanism, reduced C9orf72 levels can also facilitate cytoplasmic TDP-43 accumulation in mouse neurons 20.
Immunostaining revealed that C9ORF72+/− and C9ORF72−/− iMNs contained elevated levels of NMDA (NR1) and AMPA (GLUR1) receptors on neurites and dendritic spines compared to control iMNs under basal conditions (Fig. 4a, c, d and Supplementary Fig. 5b and 10a, c-e, g, h, j, k). In addition, control iMNs treated with C9ORF72-specific ASOs displayed increased numbers of NMDA and AMPA receptors in their neurites (Supplementary Fig. 10l, m). C9ORF72 patient iMNs (n=3 patients) also showed elevated NR1 and GLUR1 levels compared to controls (n=3 controls), and forced expression of C9ORF72 isoform B reduced glutamate receptor levels in patient iMNs (n=3 patients) to that of controls (n=3 controls) (Fig. 4a-c and Supplementary Fig. 10a-h). mRNA levels of NR1 (GRIN1) and GLUR1 (GRIA1) were not elevated in flow-purified C9ORF72+/− iMNs, indicating that increased transcription could not explain the increased glutamate receptor levels (Supplementary Fig. 10n).

Samples were first fixed in 4% PFA (1x PBS) overnight at 4°C and were subsequently washed three times with 1x PBS. Next, cells were permeabilized with 0.3% Triton X-100 (1x PBS) for 10 min at room temperature, followed by three washes with 1x PBS for 10 min each. After permeabilization, the samples were equilibrated in 1x SSC buffer for 10 min at room temperature and then transferred into 50% formamide (2x SSC) for 10 min at 60°C. The repeat expansion-targeting probe and the negative control probe were ordered from Exiqon 58. During this step, the probe mixture (1µl salmon sperm (10 µg/µl), 0.5 µl E. coli tRNA (20 µg/µl), 0.4 µl probe (25 µM), 25 µl 80% formamide/per sample) was made and placed at 95°C for at least 10 min. The samples were submerged in 200 µl of hybridization buffer (4ml 100% formamide, 0.5 ml 20x SSC, 1 ml BSA fraction V, 0.5ml RVC (20 mM), 1ml NaPO4 (0.1 M), 3 ml nuclease-free water) and 27 µl of the probe mixture was added to each sample and incubated for 1 hour at 60°C. After probe hybridization, the samples were washed twice with 50% formamide (2x SSC) for 20 min each at 65°C and once more with 40% formamide (1x SSC) for 10 min at 60°C. The remaining formamide was removed by briefly washing with 1x SSC three times. A final crosslinking step was performed by first incubating the samples with 1x Tris-Glycine for 5 minutes followed by a 5 min incubation in 4% PFA. Samples were washed three times with 1x PBS, stained with DAPI, and imaged using a Zeiss LSM 800 confocal microscope.
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).
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,†