Live imaging of iMNs expressing a M6PR-GFP fusion protein that localizes to M6PR+ vesicles 44 confirmed that C9ORF72 patient and C9ORF72-deficient iMNs possess increased numbers of M6PR+ vesicle clusters, and that overexpression of C9ORF72 isoform A or B rescues this phenotype (Supplementary Fig. 9c-g and Supplementary Videos 5-9). Clusters did not disperse over the time course of the assay, suggesting that they are relatively stable and not in rapid flux (Supplementary Videos 5-9). In addition, M6PR+ puncta moved with a slower average speed in C9ORF72 patient and C9ORF72+/− iMNs than controls (Supplementary Fig. 9h, i). Thus, reduced C9ORF72 levels lead to fewer lysosomes in motor neurons in vitro and in vivo, and this may be due in part to altered trafficking of M6PR+ vesicles.

Therapeutic strategies in development for C9ORF72 ALS/FTD target gain-of-function mechanisms. These include ASOs 6–8 and small molecules 13 that disrupt RNA foci formation. However, these approaches have not fully rescued neurodegeneration in human patient-derived neurons 6–8,13, indicating that replacing C9ORF72 function or new therapeutic targets may be required.


Analysis was performed with the statistical software package Prism Origin (GraphPad Software, La Jolla, USA). Statistical analysis of iMN survival experiments was performed using a two-sided log-rank test to account for events that did not occur (i.e. iMNs that did not degenerate before the end of the experiment). For each line, the survival data from 50 iMNs were selected randomly using Microsoft Excel, and these data were used to generate the survival curve. If all iMNs degenerated in a given experiment, statistical significance was calculated using a two-tailed Student’s t-test. For all other experiments, differences between two groups were analyzed using a two-tailed Student’s t-test, unless the data was non-normally distributed for which two-sided Mann-Whitney testing was used. Differences between more than two groups were analyzed by one way-ANOVA with Tukey correction for multiple testing. Significance was assumed at p < 0.05. Error bars represent the standard deviation unless otherwise stated.
In advanced traditional Chinese kung fu (martial arts), Neijing (Traditional Chinese: 內勁; pinyin: nèijìng) refers to the conscious control of the practitioner's qi, or "life energy", to gain advantages in combat.[1] Nèijìng is developed by using "Neigong" (Traditional Chinese: 內功; pinyin: nèigōng) (內功), or "internal exercises," as opposed to "wàigōng" (外功), "external exercises."
To determine if glutamate receptor accumulation occurs on C9ORF72 patient motor neurons in vivo, we measured glutamate receptor expression in ventral horn neurons in lumbar spinal cord samples from 3 C9ORF72 ALS patients and 3 unaffected controls. We identified motor neurons by size and confirmed that most neurons selected in this manner were CHAT+ and SMI-32+ (Supplementary Fig. 12d). Spinal motor neurons from the C9ORF72 ALS patients displayed higher NR1 levels than control neurons (Supplementary Fig. 12e). In addition, post-synaptic densities isolated from the motor corticesof C9ORF72 patients had higher levels of NR1 and GLUR1 than controls (Fig. 4k, l and Supplementary Fig. 5k).
For experiments other than the comparison of Apilimod and the reduced-activity analog, Apilimod was purchased from Axon Medchem (cat. no. 1369). For the reduced-activity analog assays, Apilimod and the reduced activity analog were synthesized at Icagen, Inc. according to the schemes shown in Supplementary Fig. 16. PIKFYVE kinase inhibition was measured using the ADP-Glo kinase assay from SignalChem according to the manufacturer’s instructions, using purified PIKFYVE kinase (SignalChem cat. no. P17–11BG-05).
Primary chick myoblasts were dissected from D11 chick embryos and plated onto plastic dishes pre-coated with 0.1% gelatin. After 3 days of culture in muscle medium containing F10 (Life Technologies), 10% horse serum, 5% chicken serum (Life Technologies), 0.145 mg/ml CaCl2 (Sigma), and 2% Penicillin/Streptomycin, myoblasts were trypsinized and replated onto iMNs which were at days 15–18 post-transduction. The co-culture was maintained in neuronal medium containing DMEM/F12, 2% B27, 1% GlutaMax and 1% Penicillin/Streptomycin, supplemented with 10ng/ml BDNF, GDNF, and CNTF for 7 days in order to allow neuromuscular junctions to form. Videos were taken using Nikon Eclipse Tis microscope with NIS Element AR software. Light-stimulated contraction shown in Supplementary Figure 2j are representative of contraction observed in 2 biological replicates, with 5 contractile sites per replicate.
Mice were anesthetized with i.p. ketamine (100 mg ⁄ kg) and xylazine (10 mg ⁄ kg), and body temperature kept at 36.9 ± 0.1°C with a thermostatic heating pad. Mice were placed in a stereotactic apparatus (ASI Instruments, USA) and the head is fixed accordingly. A burr hole was drilled, and an injection needle (33 gauge) was lowered into the hippocampus between CA1 and the dentate gyrus (AP −2.0, ML +1.5, DV −1.8). NMDA (20 nmol in 0.3 μl of phosphate-buffered saline, pH 7.4) was infused over 2 min using a micro-injection system (World Precision Instruments, Sarasota, FL, USA). Simultaneously, or independently, Apilimod (0.3 μl of 20 μM in phosphate-buffered saline, pH 7.4) was infused over 2 min using a micro-injection system (World Precision Instruments, Sarasota, FL, USA). The needle was left in place for an additional 8 min after the injection. Animals were euthanized 48 h later. Brains were quickly removed, frozen on dry ice, and stored at −80°C until processing. Thirty-micrometer-thick coronal sections were prepared using a cryostat. Every fifth section 1 mm anterior and posterior to the site of injection was stained with cresyl violet. The lesion area was identified by the loss of staining, measured by NIH ImageJ software and integrated to obtain the volume of injury.
(a) Super-resolution microscopy images of immunofluorescence shows NR1+ puncta on neurites of iMNs overexpressing eGFP or C9ORF72 isoform B-eGFP. Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (b-d) Number of NR1+ puncta per unit area in control (b-d), patient (b), C9ORF72+/− (c), and C9ORF72+/− (d) iMNs. Mean ± s.d. Each grey open circle represents the number of NR1+ puncta per area unit on a single neurite (one neurite quantified per iMN). For (b), n=75 (CTRL + GFP), 84 (C9-ALS + GFP), 95 (C9-ALS + isoA), and 111 (C9-ALS + isoB) iMNs quantified from two biologically independent iMN conversions of 3 CTRL or 4 C9-ALS lines. For (c), n=37 (CTRL + GFP), 37 (C9ORF72+/− + GFP), 25 (C9ORF72+/− + isoA), and 27 (C9ORF72+/− + isoB) iMNs quantified from two biologically independent iMN conversions per condition. For (d), n=37 (CTRL + GFP), 37 (C9ORF72−/− + GFP), 38 (C9ORF72−/− + isoA), and 23 (C9ORF72−/− + isoB) iMNs quantified from two biologically independent iMN conversions per condition. One-way ANOVA with Tukey correction for all comparisons. F-value (DFn, DFd): (3, 360) = 56.63 (b), (3, 122) = 13.42 (c), (3, 131) = 17.11 (d). (e-h) Immunoblotting analysis of surface NR1 after surface protein biotinylation in control (e-h), C9ORF72+/− (e-f), and C9-ALS patient (g-h) iMNs generated with 3 factors (NGN2, ISL1, and LHX3). In (f), n=4 biologically independent iMN conversions from CTRL2 and 2 biologically independent iMN conversions from the C9ORF72+/− line. Mean +/− s.d. In (h), two-tailed Mann-Whitney test. n=11 biologically independent motor neuron cultures from 11 independent control lines and 4 biologically independent motor neuron cultures from 4 independent C9-ALS patient lines. Experiments in (e-h) were repeated twice with similar results. Mean +/− s.e.m. (i-j) Immunoblotting analysis of surface Nr1 and Glur1 in post-synaptic densities (PSDs) from C9orf72 control and knockout mice, two-tailed t-test. t-value: 4.424 (Nr1), 4.632 (Glur1), degrees of freedom: 4 (Nr1), 4 (Glur1). n= 3 control PSD preparations isolated from 3 control mice and 3 C9orf72−/− PSD preparations isolated from 3 C9orf72−/− mice. This experiment was repeated twice with similar results. Mean +/− s.e.m. (k-l) Immunoblotting analysis of surface NR1 and GLUR1 in post-synaptic densities (PSDs) from post mortem control and C9-ALS patient motor cortices, n=3 control and 2 C9-ALS patient PSD preparations isolated from 3 control and 2 C9-ALS patients. This experiment was repeated twice with similar results. Mean +/− s.d. (m) Average Ca2+ flux in the presence of glutamate per minute. n=28 (CTRL1), 15 (CTRL2), 15 (CTRL3), 26 C9-ALS1), 20 (C9-ALS2), 24 (C9-ALS3), and 15 (C9ORF72+/−) iMNs analyzed from two biologically independent iMN conversions for each line. Mean ± s.e.m. One-way ANOVA with Tukey correction between all controls and all patients and C9ORF72+/−. F-value (DFn, DFd): (6, 136) = 11.21.

HEK 293T cells were used to produce retrovirus, lentivirus, and C9ORF72 protein. HEK cells were used for these purposes based on previous published studies using HEK cells in order to produce viral particles and mammalian proteins. HEK cells were obtained from American Type Culture Collection, catalog number CRL-11268. HEK and iPS cells were tested for mycoplasma before, during, and after the study and were negative.
A 241-bp digoxigenin (DIG)-labeled probe was generated from 100 ng control genomic DNA (gDNA) by PCR reaction using Q5® High-Fidelity DNA Polymerase (NEB) with primers shown in Supplementary Data Table 4. Genomic DNA was harvested from control and patient iPSCs using cell lysis buffer (100 mM Tris-HCl pH 8.0, 50 mM EDTA, 1% w/v sodium dodecyl sulfate (SDS)) at 55ºC overnight and performing phenol:chloroform extraction. A total of 25 µg of gDNA was digested with XbaI at 37 ºC overnight, run on a 0.8% agarose gel, then transferred to a positive charged nylon membrane (Roche) using suction by vacuum and UV-crosslinked at 120 mJ. The membrane was pre-hybridized in 25 ml DIG EasyHyb solution (Roche) for 3 h at 47 ºC then hybridized at 47 ºC overnight in a shaking incubator, followed by two 5-min washes each in 2X Standard Sodium Citrate (SSC) and in 0.1% SDS at room temperature, and two 15-min washes in 0.1x SSC and in 0.1% SDS at 68 ºC. Detection of the hybridized probe DNA was carried out as described in DIG System User’s Guide. CDP-Star® Chemilumnescent Substrate (Sigma-Aldrich) was used for detection and the signal was developed on X-ray film (Genesee Scientific) after 20 to 40 min.
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).
Although C9orf72 knockout mice do not show overt neurodegeneration, gain-of-function disease processes may trigger neurodegeneration through mechanisms induced by reduced C9ORF72 levels. For example, DPRs cause mis-splicing of the EAAT2 glutamate transporter in astrocytes, which couldincrease excitotoxicity in neurons with elevated glutamate receptor levels 12. To determine if DPRs alter glutamate uptake by astrocytes, we compared glutamate uptake in human primary astrocytes expressing either GFP or GR50 –GFP. Indeed, GR50 –GFP significantly impaired glutamate uptake by astrocytes (Supplementary Fig. 13h).
Postsynaptic density extraction was done following a protocol published previously 63. Briefly, mouse spinal cord tissue or human cortical tissue was homogenized in cold Sucrose Buffer (320 mM Sucrose, 10 mM HEPES pH 7.4, 2 mM EDTA, 30 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, and protease inhibitor cocktail (Roche)) using a tissue grinder and then spun down at 500 g for 6 min at 4℃. The supernatant was re-centrifuged at 10,000 g for 10 min at 4℃. The supernatant was collected as the “Total” fraction, and the pellet was resuspended in cold Triton buffer (50 mM HEPES pH 7.4, 2 mM EDTA, 50 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, 1% Triton X-100 and protease inhibitor cocktail (Roche)) and then spun down at 30,000 RPM using a Beckman rotor MLA-130 for 40 min at 4℃. The supernantant was collected as the “Triton” fraction and the pellet was resuspended in DOC buffer (50 mM HEPES pH 9.0, 50 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, 20 uM ZnCl2, 1% Sodium Deoxycholate and protease inhibitor cocktail (Roche)) and collected as the “DOC”, PSD-enriched fraction. Collected samples were boiled with SDS-PAGE sample buffer and analyzed by western blot. Purity of the PSD preps was analyzed by immunoblotting for PSD-95 (PSD), p53 (non-PSD), and synaptophysin (non-PSD).
(a-b) Survival of control and CRISPR-mutant iMNs without excess glutamate with overexpression of eGFP or PR(50)-eGFP (a) or GR(50)-eGFP (b). (c-d) Survival of control and C9-ALS iMNs without excess glutamate with overexpression of eGFP or PR(50)-eGFP (c) or GR(50)-eGFP (d). For (a), n=50 (CTRL1 + GFP AND CTRL1 + PR(50)), 49 (C9ORF72+/− + GFP), and 47 (C9ORF72+/− + PR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line. For (b), n=50 (CTRL1 + GFP AND CTRL1 + GR(50)), 49 (C9ORF72+/− + GFP), and 40 (C9ORF72+/− + GR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line. For (c), n=50 (CTRL1 + GFP AND CTRL1 + PR(50)), 50 (from each of two C9-ALS lines + GFP), and 41 (from each of two C9-ALS lines + PR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line per condition. For (d), n=50 (CTRL1 + GFP AND CTRL1 + GR(50)), 50 (from each of two C9-ALS lines + GFP), and 46 and 47 (from two C9-ALS lines + GR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line per condition. All iMN survival experiments in (a-d) were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. Survival curves for the “+GFP” condition were included as a reference, but were not used in statistical analyses. (e) Relative decay in Dendra2 fluorescence over 12 hours in iMNs of specified genotypes. Mean +/− s.e.m. n = 18 (control) and 24 (C9ORF72+/−) iMNs quantified from two biologically independent iMN conversions each, two-tailed t-test with Welch’s correction between data points at each time point, t-value: 2.739, degrees of freedom: 25.62). (f-h) Immunostaining to determine endogenous PR+ puncta in control or C9-ALS iMNs with or without overexpression of C9ORF72 isoform A or B. Scale bar = 2 μm. This experiment was repeated twice with similar results. (g) Mean +/− s.d. n= 4 biologically independent iMN conversions generated from two different iPSC lines per genotype. Quantified values represent the average number of PR+ puncta in 40 iMNs from a single iMN conversion. Two-tailed t-test, t-value: 5.908, degrees of freedom: 6. (h) Mean +/− s.e.m. n= 3 biologically independent iMN conversions per condition. Quantified values represent the average number of PR+ puncta in 40 iMNs from a single iMN conversion. One-way ANOVA with Tukey correction, F-value (DFn, DFd): (2, 6)=10.5. iMN survival experiments in (a-d) were performed in a Molecular Devices ImageExpress.
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