To examine C9ORF72 function, we determined its localization in iMNs. We first used an HA-tagged C9ORF72 construct to verify that the C9ORF72 antibody specifically recognizes C9ORF72 in cells (Supplementary Fig. 7a). In iMNs, C9ORF72 co-localized to cytoplasmic puncta and ASO-mediated knockdown of C9ORF72 expression reduced the number of antibody-detected cytoplasmic puncta in iMNs, indicating that the antibody specifically recognizes C9ORF72 in these puncta (Supplementary Fig. 7b, c). Super-resolution microscopy and z-stack imaging showed that about 80% of the C9ORF72+ vesicles also expressed the early endosomal proteins RAB5 and EEA1 (Fig. 3a and Supplementary 7d-h). Only rarely did C9ORF72 co-localize with the lysosomal marker LAMP1 (20%)(Supplementary Fig. 7e), and control and patient iMNs showed similar C9ORF72 localization (Supplementary Fig. 7h). We performed density gradient centrifugation on lysates from iPSC-derived motor neurons to separate light (endosomal) and heavy (lysosomal) membrane fractions. C9ORF72 co-segregated with EEA1 and not LAMP1, supporting the notion that C9ORF72 localizes predominantly in early endosomes (Fig. 3b, Supplementary Fig. 5d). In addition, we found that C9ORF72 isoform B bound strongly to an immobilized N-terminal fragment of EEA1 (Supplementary Fig. 7i). C9ORF72 isoform A did not interact as strongly with EEA1 (Supplementary Fig. 7i). The fact that not all EEA1+ vesicles contained high levels of C9ORF72 is consistent with this hypothesis and suggests that C9ORF72 may not localize to all types of EEA1+ vesicles (Fig. 3a).
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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).
Since glutamate receptor activation and neuronal firing both induce calcium influx, we determined their relative contributions to the increased Gcamp6 activation by. using the ion channel inhibitors TTX and TEA to block neuronal firing. C9ORF72+/− iMNs still displayed more frequent Gcamp6 activation than C9ORF72+/+ iMNs (Supplementary Fig. 13a), indicating that part of the hyperexcitability is due to increased glutamate receptor activation. To determine which receptors were responsible for the increased glutamate response, we tested small molecule agonists of specific glutamate receptor subtypes. Activation of NMDA, AMPA, and kainate receptors was higher in C9ORF72+/− iMNs than controls (Supplementary Fig. 13a).
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).
To determine if Pikfyve inhibition rescues gain-of-function processes in vivo, we measured DPR levels in C9-BAC transgenic mice 58 with or without Apilimod treatment. Although it was not previously reported 58, we observed significantly higher levels of GR+ punctae in hippocampal neurons in C9-BAC mice than controls (Fig. 6j) using a previously-validated poly(GR) antibody 11. These data are consistent with findings in another published C9-BAC mouse model 14, suggesting that poly(GR) may be a common feature of C9-BAC mice. We also detected a low level of poly(GR) in neurons from control mice (Fig. 6j), which may be derived from other repeat regions or proteins with short poly(GR) sequences. Nevertheless, GR+ punctae levels were significantly higher in C9-BAC mouse neurons than in controls (Fig. 6j). Importantly, Apilimod treatment significantly reduced the number of GR+ punctae in hippocampal neurons in C9-BAC mice after 48 hrs (Fig. 6i, j). Therefore, small molecule inhibition of Pikfyve rescues both gain- and loss-of-function disease processes induced by C9ORF72 repeat expansion in vivo.
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.

Our iMN survival results (Fig. 1c-e) suggest that the repeat expansion alters iMN glutamate sensing. In cortical neurons, homeostatic synaptic plasticity is maintained through endocytosis and subsequent lysosomal degradation of glutamate receptors in response to chronic glutamate signaling 45,46. Defects in this process lead to the accumulation of glutamate receptors on the cell surface 45,46.

Consistent with PIKFYVE being the relevant target in the iMN survival assay, Apilimod increased C9ORF72 patient, but not control, iMN survival in either neurotrophic withdrawal conditions (Fig. 6d) or excess glutamate (n=4 patients, Supplementary Fig. 15f (n=3 controls, Supplementary Fig. 15g). Automated neuron tracking software independently verified Apilimod efficacy on C9ORF72 patient iMNs (Supplementary Fig. 15h). As further confirmation that PIKFYVE was the active target, ASO-mediated suppression of PIKFYVE also rescued C9ORF72 patient iMN survival (Fig. 6d and Supplementary Fig. 15i). In addition, we synthesized a structural analog of Apilimod with a reduced ability to inhibit PIKFYVE kinase activity in a biochemical assay using purified PIKFYVE protein (Fig. 6b and Supplementary Fig. 15j, 16). The reduced activity analog was significantly less effective at rescuing C9ORF72 patient iMN survival (Fig. 6e). Thus, small molecule inhibition of PIKFYVE can rescue patient motor neuron survival.
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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We wondered if reduced C9ORF72 activity renders motor neurons vulnerable to other insults that might be exacerbated by impaired endosomal-lysosomal system function, such as DPR toxicity. Without excess glutamate treatment, C9ORF72+/−, C9ORF72−/−, and C9ORF72 patient iMN survival was similar to control iMNs (Fig. 5a-d and Supplementary Fig. 14a, b). Exogenous expression of PR50 or GR50 DPRs using cassettes that do not contain the GGGGCC repeat expansion induced control iMN degeneration (Fig. 5a, b). Interestingly, C9ORF72+/−, C9ORF72−/−, and C9ORF72 patient iMNs (n=2 patients) degenerated significantly faster than controls in response to PR50 or GR50 expression (Fig. 5a-d and Supplementary Fig. 14a, b).

Although studies in mice have indicated that C9orf72 activity is important for myeloid cell function 14,18, a report in mouse neurons and zebrafish suggests that C9orf72 isoform A may modulate poly-Q Ataxin-2 toxicity 20, and studies have implicated C9ORF72 in regulating autophagy 20,31,32,38, our study provides the first direct evidence showing that gain- and loss-of-function C9ORF72 mechanisms cooperate to cause the degeneration of human motor neurons. Recent studies have shown that C9ORF72 isoform A, but not isoform B, can form a functional complex with SMCR8 and WDR41 20. However, since both C9ORF72 isoforms rescue C9-ALS iMN degeneration in our assay, other mechanisms or protein interactions may underlie the rescue of patient iMNs.

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.
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,†