Our results highlight the importance of C9ORF72 protein function, RAB5 activity, PI3P levels, and lysosomeal function as key therapeutic targets for C9ORF72 ALS/FTD. By generating PI3P, RAB5 drives early endosomal maturation and the initial stages of lysosomal biogenesis (Fig. 6f)59. PI3P also plays important roles in autophagosome formation and autophagsome-lysosome fusion. Indeed, a previous study suggests that PIKFYVE inhibition may increase autophagic flux 53, and this should be investigated in the context of motor neurons. Loss-of-function mutations in two other genes whose proteins function to increase PI3P levels, ALS2 and FIG4, also cause ALS 1. ALS2 encodes the RAB5 guanine exchange factor ALSIN 60, while FIG4 converts PI(3,5)P2 into PI3P 55(Fig. 6f). In addition, proteins encoded by several other ALS genes play key roles in lysosomal biogenesis, including CHMP2B, OPTN, and SQSTM1 1. The fact that FIG4 and ALS2 loss-of-function mutations can cause ALS suggests that PIKFYVE inhibition or RAB5 activation may be capable of modulating ALS disease processes in humans.

(a) The levels of C9ORF72 variant 2 mRNA transcript (encoding isoform A). Values are mean ± s.e.m., two-tailed t-test with Welch’s correction. t-value: 5.347, degrees of freedom: 11.08. n= 9 biologically independent iMN conversions from 3 control lines and 12 biologically independent iMN conversions from 5 C9-ALS lines. (b–d) iMN survival in excess glutamate following introduction of C9ORF72 (C9 isoform A or B) into C9ORF72 patient iMNs (b), but not control (b, d) or SOD1-ALS iMNs (c). For (b), n=50 iMNs per line for 2 control and 3 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. For (c), n=50 iMNs per condition, iMNs scored from 3 biologically independent iMN conversions. For (d), n=50 iMNs per line per condition for 2 control lines, iMNs quantified from 3 biologically independent iMN conversions. Each trace includes iMNs from 2–3 donors with the specified genotype (except SOD1-ALS (c)); see full details in Methods. (e) Strategy for knocking out C9ORF72 from control iPSCs using CRISPR/Cas9. (f) Survival of control (CTRL2) iMNs, the isogenic heterozygous (C9+/−) and homozygous (C9−/−) iMNs and C9ORF72 patient (C9-ALS) iMNs in excess glutamate. n=50 biologically independent iMNs per line per condition for one control and two C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions. (g) Control iMN survival in excess glutamate with scrambled or C9ORF72 antisense oligonucleotides (ASO). Each trace includes control iMNs from 2 donors. n=50 biologically independent iMNs per line per condition for 2 control lines, iMNs quantified from 3 biologically independent iMN conversions. All iMN survival experiments were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. iMN survival experiments in (b, d, and g) were performed in a Nikon Biostation, and (e and f) were performed in a Molecular Devices ImageExpress.
We anticipate three key implications of our findings: 1) ALS/FTD caused by the C9ORF72 repeat expansion requires both gain- and loss-of-function mechanisms, 2) increasing C9ORF72 activity in motor neurons should mitigate disease and provides a new therapeutic target, and 3) PIKFYVE inhibition and other approaches that modulate vesicle trafficking may ameliorate C9ORF72 disease processes in both neurons and myeloid cells. The fact that mutations in FIG4 cause ALS, epilepsy, and Charcot-Marie-Tooth 55 illustrates the broad implications of impaired vesicle trafficking within the CNS. The identification of targets that effectively modulate vesicle trafficking in neurons, glia, and myeloid cells could hold tremendous therapeutic value for C9ORF72 ALS/FTD and other CNS disorders.
Our results indicate that haploinsufficiency for C9ORF72 activity triggers neurodegeneration in C9ORF72 ALS, and this occurs by at least two mechanisms. First, reduced C9ORF72 activity causes the accumulation of glutamate receptors and excitotoxicity in response to glutamate. Although C9orf72 knockout mice do not display overt neurodegeneration14,18,22, these mice may be protected from excitotoxicity because they lack gain-of-function disease processes such as DPRs, which induce aberrant splicing and dysfunction of the EAAT2 glutamate transporter in astrocytes in vitro 12 and in C9ORF72 ALS patients 4,27. EAAT2 dysfunction causes glutamate accumulation in the cerebrospinal fluid of ALS patients 27, and consistent with this notion, we found that poly(PR) expression in human astrocytes reduced their rate of glutamate uptake. By using human iMNs, mice, and human post mortem tissue, we show for the first time that reduced C9ORF72 activity modulates the vulnerability of human motor neurons to degenerative stimuli and establish a mechanistic link between the C9ORF72 repeat expansion and glutamate-induced excitotoxicity

The results of an experimental investigation of the effects of container geometry on the recovery of product water from indirectly frozen salt water are presented. Salt water was frozen in containers having circular or rectangular cross-section, then allowed to melt and drain until the residual ice was potable. Thin rectangular cross-sections were found to be more effective than circular ... [Show full abstract]Read more
To determine if C9ORF72 iMNs recapitulate neurodegenerative ALS processes, we examined their survival by performing longitudinal tracking of Hb9::RFP+ iMNs (Fig. 1a). This approach enabled us to distinguish differences in neurogenesis from differences in survival, which could not be addressed using previously-reported cross-sectional analyses6,7,10,26. In basal neuronal medium supplemented with neurotrophic factors, control and C9ORF72 patient iMNs survived equally well (Fig. 1b, Supplementary Fig. 3a, Supplementary Tables 5, 6). As human C9ORF72 ALS patients have elevated glutamate levels in their cerebrospinal fluid (possibly triggered by DPR-mediated aberrant splicing of the astrocytic excitatory amino acid transporter 2 EAAT2 4,27) we stimulated iMN cultures with a high glutamate pulse (12-hour treatment, 10 μM glutamate). This initiated a robust degenerative response in patient, but not control, iMNs (Fig. 1c-e and Supplementary Videos 3, 4) that was consistent across lines from multiple patients (n=6 patients) and controls (n=4 controls)(Fig. 1c, d and Supplementary Fig. 3d, e). While iMN survival varied slightly between live imaging systems, or between independent experiments due to the lengthy time course of neurodegeneration, the relative difference between control and C9-ALS patient iMNs was consistent (Fig. 1c - Nikon Biostation CT and Supplementary Fig. 3b - Molecular Devices ImageExpress). Moreover, iMNs from different iPSC lines derived from the same donor behaved similarly, suggesting genotypic differences accounted for these effects (Supplementary Fig. 3c). Treatment with glutamate receptor antagonists during glutamate administration prevented patient iMN degeneration (Fig. 1f). Alternatively, withdrawal of neurotrophic factors without glutamate stimulation also caused rapid degeneration of patient iMNs (n=3 patients, (Fig. 1g and Supplementary Fig. 3f).

(a) Production of Hb9::RFP+ iMNs and survival tracking by time-lapse microscopy. (b-d) Survival of control (CTRL) and C9ORF72 patient (C9-ALS) iMNs with neurotrophic factors (b) or in excess glutamate (shown with iMNs from all lines in aggregate (b, c) or for each individual line separately (d)). For (b-d), n=50 iMNs per line for 2 control and 3 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. (e) iMNs at day 22 in excess glutamate. This experiment was repeated three times with similar results. (f-g) Survival of control and C9-ALS iMNs in excess glutamate with glutamate receptor antagonists (f) or without neurotrophic factors (g). For (f-g), n=50 iMNs per line for 2 control and 3 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. (h) Survival of induced dopaminergic (iDA) neurons in excess glutamate. n=50 iMNs per line for 2 control and 2 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. Except in (d), each trace includes neurons from at least 2 donors with the specified genotype; see full detail in Methods. Scale bar: 100 μm (e). All iMN survival experiments were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. iMN survival experiments in (b-g) were performed in a Nikon Biostation and experiments in (h) were performed in a Molecular Devices ImageExpress.
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.
Because C9ORF72 activity is required to maintain normal lysosomal function, we measured the effect of C9ORF72 activity on PR50 clearance by monitoring the clearance of PR50-Dendra2 fusion proteins in C9ORF72−/− iPSC-derived fibroblasts with or without exogenous C9ORF72. Dendra2 is a green fluorescent protein that irreversibly converts to red fluorescence when exposed to blue light, enabling quantification of its degradation 49. PR50-Dendra2 formed discrete punctae within cells, indicating that Dendra2 did not prevent intracellular aggregation of PR50 (Supplementary Fig. 14c). Expression of C9ORF72-T2A-GFP in C9ORF72−/− iPSC-derived fibroblasts significantly enhanced the decay of PR50-Dendra2 fluorescence over GFP alone (Supplementary Fig. 14d). To determine if C9ORF72 activity modulates DPR aggregate clearance in human motor neurons, we compared the decay of PR50-Dendra2 in C9ORF72+/+ and C9ORF72+/− iMNs (Fig. 5e and Suppementary Fig. 14e). Consistent with the hypothesis that C9ORF72 activity promotes DPR aggregate clearance, PR50-Dendra2 decayed significantly slower in C9ORF72+/− iMNs (Fig. 5e).
To generate Dox-NIL iMNs, the Dox-NIL construct was integrated into the AAVS1 safe harbor locus of the control, C9+/−, and C9-ALS patient iPSC lines using CRISPR/Cas9 editing (gRNA sequence shown in Supplementary Table 4). Dox-NIL iMNs were generated by plating at ~25% confluency on matrigel coated plates and adding 1 μg/ml of doxycylin in N3 media +7.5 μM RepSox 1 day after plating. Mouse primary mixed glia were added to the cultures at day 6, and doxycyline was maintained throughout conversion. iMN cultures were harvested at day 17.
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.
Our results indicate that haploinsufficiency for C9ORF72 activity triggers neurodegeneration in C9ORF72 ALS, and this occurs by at least two mechanisms. First, reduced C9ORF72 activity causes the accumulation of glutamate receptors and excitotoxicity in response to glutamate. Although C9orf72 knockout mice do not display overt neurodegeneration14,18,22, these mice may be protected from excitotoxicity because they lack gain-of-function disease processes such as DPRs, which induce aberrant splicing and dysfunction of the EAAT2 glutamate transporter in astrocytes in vitro 12 and in C9ORF72 ALS patients 4,27. EAAT2 dysfunction causes glutamate accumulation in the cerebrospinal fluid of ALS patients 27, and consistent with this notion, we found that poly(PR) expression in human astrocytes reduced their rate of glutamate uptake. By using human iMNs, mice, and human post mortem tissue, we show for the first time that reduced C9ORF72 activity modulates the vulnerability of human motor neurons to degenerative stimuli and establish a mechanistic link between the C9ORF72 repeat expansion and glutamate-induced excitotoxicity
The fabrication of composite cathode with boroxine ring for all-solid-polymer lithium cell was described. Composite polymer electrolyte (CPE) was applied between the lithium metal anode and the composite cathode in a coin-shaped cell in order to prepare the solid-polymer electrolyte cell. The CPE films were cast on a flat polytetrafluoroethylene vessel from an acetonitrile slurry containing BaTiO ... [Show full abstract]Read more
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.
Local field potentials (LFPs) were recorded from iPSC-derived motor neurons on days 17–21 in culture in 6-well multielectrode chips (9 electrodes and 1 ground per well) using a MultiChannel Systems MEA-2100 multielectrode array (MEA) amplifier (ALA Scientific) with built-in heating elements set to 37°C. Cells were allowed to acclimate for 5 minutes after chips were placed into the MEA amplifier, and after glutamate addition (10 μM final concentration). For 1 μM Apilimod treatments, chips were incubated for 35 min in a humidified incubator in the presence of the particular drug, then returned to the MEA amplifier and acclimated for 5 min before beginning recordings. For each condition, recordings (5 min baseline, 10 min glutamate and/or drug, 40 kHz sampling rate) were filtered between 1–500 Hz, and average LFP frequency per well was determined using the accompanying MC Rack software.
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).
Amongst four reproducible hit compounds, we identified a PIKFYVE kinase inhibitor (YM201636) that significantly increased C9ORF72 patient iMN survival (n=2 patients) (Fig. 6b, c and Supplementary Fig. 15a). PIKFYVE is a lipid kinase that converts phosphatidylinositol 3-phosphate (PI3P) into phosphtidylinositol (3,5)-bisphosphate (PI(3,5)P2)51(Fig. 6f). PI3P is primarily generated by PI3-kinases recruited to early endosomes by RAB5, and PI3P anchors EEA1 to early endosomes to drive endosomal maturation 52(Fig 6f). Following endosomal maturation into lysosomes, PI3P drives fusion of lysosomes with autophagosomes 53. PIKFYVE regulates PI3P levels by converting PI3P into PI(3,5)P2 52, which disfavors lysosomal fusion with endosomes and autophagosomes 53,54. Therefore, inhibition of PIKFYVEincreases autophagosome-lysosome fusion 53 and may compensate for reduced C9ORF72 activity and other disease processes by increasing PI3P levels to facilitate removal of glutamate receptors or DPRs (Fig. 6f). Interestingly, FIG4 is a phosphatase that opposes PIKFYVE kinase by converting PI(3,5)P2 to PI3P and loss-of-function mutations in FIG4 cause ALS 55. Thus, genetic evidence suggests that PIKFYVE inhibition may be capable of modulating ALS disease processes in humans.
To determine if C9ORF72 iMNs recapitulate neurodegenerative ALS processes, we examined their survival by performing longitudinal tracking of Hb9::RFP+ iMNs (Fig. 1a). This approach enabled us to distinguish differences in neurogenesis from differences in survival, which could not be addressed using previously-reported cross-sectional analyses6,7,10,26. In basal neuronal medium supplemented with neurotrophic factors, control and C9ORF72 patient iMNs survived equally well (Fig. 1b, Supplementary Fig. 3a, Supplementary Tables 5, 6). As human C9ORF72 ALS patients have elevated glutamate levels in their cerebrospinal fluid (possibly triggered by DPR-mediated aberrant splicing of the astrocytic excitatory amino acid transporter 2 EAAT2 4,27) we stimulated iMN cultures with a high glutamate pulse (12-hour treatment, 10 μM glutamate). This initiated a robust degenerative response in patient, but not control, iMNs (Fig. 1c-e and Supplementary Videos 3, 4) that was consistent across lines from multiple patients (n=6 patients) and controls (n=4 controls)(Fig. 1c, d and Supplementary Fig. 3d, e). While iMN survival varied slightly between live imaging systems, or between independent experiments due to the lengthy time course of neurodegeneration, the relative difference between control and C9-ALS patient iMNs was consistent (Fig. 1c - Nikon Biostation CT and Supplementary Fig. 3b - Molecular Devices ImageExpress). Moreover, iMNs from different iPSC lines derived from the same donor behaved similarly, suggesting genotypic differences accounted for these effects (Supplementary Fig. 3c). Treatment with glutamate receptor antagonists during glutamate administration prevented patient iMN degeneration (Fig. 1f). Alternatively, withdrawal of neurotrophic factors without glutamate stimulation also caused rapid degeneration of patient iMNs (n=3 patients, (Fig. 1g and Supplementary Fig. 3f).
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.
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