CRISPR/Cas9-mediated genome editing was performed in human iPSCs as previously described, using Cas9 nuclease62. To generate loss-of-function alleles of C9ORF72, control iPSCs were transfected with a sgRNA targeting exon 2 of the C9ORF72 gene. Colonies were picked on day 7 after transfection and genotyped by PCR amplification and sequencing of exon 2. Colonies containing a frameshift mutation were clonally purified on MEF feeders and the resulting clones were re-sequenced to verify the loss-of-function mutation in C9ORF72.
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,†

We compared the differential expression results from our data to other transcriptomic datasets in ALS, obtained from the Gene Expression Omnibus (GEO). Raw Affymetrix array data (.CEL files) were downloaded for dataset GSE56504, and preprocessed using a standard exon array pipeline implemented using the R Bioconductor package oligo. For GSE56504, only the laser-capture microdissection samples were included/ Differential expression was calculated using the R Bioconductor package limma. RNA-seq counts data was obtained for dataset GSE67196. For GSE67196, only the frontal cortex samples were included. Normalization and differential expression analysis were performed using DESeq2.
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.
Near the cities Beijing, Tianjin, Shijiazhuang, and Jinan, Wuqiao County has many transportation connections. There are many rail and bus services operating in the town. Wuqiao was the first Chinese city to open up its doors to the world under the "Open Door" policy and over many years development, Wuqiao has become a flourishing city with a favorable investment environment.[citation needed]
iMNs from healthy controls and ALS patients were collected on day 21 post-transduction in RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Roche). Protein quantity was measured by the BCA assay (Pierce) and samples were run on a 10% SDS gel at 4 °C. The gel was transferred onto an Immobilon membrane (Millipore). The membrane was blocked with 5% milk in 0.1% PBS-Tween 20 (PBS-T)(Sigma-Aldrich), incubated with primary antibodies overnight at 4 °C, washed three times with 0.1% PBS-T, then incubated with horseradish peroxidase (HRP)-conjugated (Santa Cruz). After three washes with 0.1% PBS-T, blots were visualized using an Amersham ECL Western Blotting Detection Kit (GE) or the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) and developed on X-ray film (Genesee). The following primary antibodies were used: rabbit anti-C9ORF72 (Proteintech, cat. no. 22637–1-AP), mouse anti-GAPDH (Santa Cruz, cat. no. sc-32233), chicken anti-MAP2 (Abcam, cat. no. ab11267), mouse anti-FLAG (Sigma, cat. no. F1804), rabbit anti-GLUR1 (Millipore, cat. no. 1504), mouse anti-NR1 (Novus, cat. no. NB300118), mouse anti-Transferrin receptor (Thermo, cat. no. 136800), mouse anti-LAMP3 (DSHB, cat. no. H5C6), rabbit anti-LAMP3 (Proteintech, cat. no. 12632), mouse anti-LAMP2 (DSHB, cat. no. H4B4), mouse anti-LAMP1 (Abcam, cat. no. Ab25630), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), mouse anti-EEA1 (BD Biosciences, cat. no. BD610457), mouse anti-TUJ1 (Biolegend, cat. no. MMS-435P), rabbit anti-APP (Abcam, cat. no. ab32136), mouse anti-Tau5 (Thermo, cat. no. AHB0042), mouse anti-PSD-95 (Thermo, cat. no. MA1–045) , mouse anti-p53 (Cell Signaling, cat. no. 2524S), anti-mouse HRP (Cell Signaling, cat. no. 7076S), anti-rabbit HRP (Cell Signaling, cat. no. 7074S). For C9ORF72 western blots, to generate enough motor neurons for C9ORF72 protein detection, we used a directed differentiation method described previously 28.
During lysosomal biogenesis, lysosomal proteins are transported in Mannose-6-Phosphate Receptor (M6PR)+ vesicles from the trans-Golgi Network to early and late endosomes for eventual incorporation into lysosomes 41. Disruption of M6PR+ vesicle trafficking can lead to a reduction in lysosome numbers 42 and altered localization of M6PR+ vesicles 43. In control iMNs (n=3 controls), M6PR+ vesicles were distributed loosely around the perinuclear region and to a lesser extent in the non-perinuclear cytosol (Supplementary Fig. 9a, b). In contrast, C9ORF72 patient (n=4 patients), C9ORF72+/−, and C9ORF72−/− iMNs frequently harbored densely-packed clusters of M6PR+ vesicles (Supplementary Fig. 9a, b). This was not due to a reduced number of M6PR+ vesicles in patient and C9ORF72-deficient iMNs (Supplementary Fig. 9c). Forced expression of C9ORF72 isoform B restored normal M6PR+ vesicle localization in patient (n=4 patients) and C9ORF72-deficient iMNs, confirming that a lack of C9ORF72 activity induced this phenotype (Supplementary Fig. 9a, b).
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."

(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.
We show for the first time that chemical or genetic modulators of vesicle trafficking can fully rescue iMN degeneration caused by the C9ORF72 repeat expansion. Previous studies have implicated several rare ALS or FTD mutations linked to these vesicle trafficking pathways, but by showing that C9ORF72 is haploinsufficient in ALS/FTD and demonstrating that perturbation of vesicle trafficking rescues C9ORF72 neurodegeneration, our findings highlight mechanistic convergence in a large portion of ALS.
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.
(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.
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.
The degree of Li force one can employ in kung fu depends on several variables such as resilience of muscles, strength of bones, speed and timing of attack and so on. An effective way to enhance the Li force is to exercise one's muscles and bones by applying increasing pressure on them (weight training, gym exercises, etc.).[2] The stronger one's muscles and bones become, the more powerful and skillful the level of kung fu is.[3]
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.
We compared the differential expression results from our data to other transcriptomic datasets in ALS, obtained from the Gene Expression Omnibus (GEO). Raw Affymetrix array data (.CEL files) were downloaded for dataset GSE56504, and preprocessed using a standard exon array pipeline implemented using the R Bioconductor package oligo. For GSE56504, only the laser-capture microdissection samples were included/ Differential expression was calculated using the R Bioconductor package limma. RNA-seq counts data was obtained for dataset GSE67196. For GSE67196, only the frontal cortex samples were included. Normalization and differential expression analysis were performed using DESeq2.
(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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