Libraries were prepared from total RNA using Clontech SMARTer Stranded RNA-Seq kit, with Clonetech RiboGone ribodepletion performed ahead of cDNA generation. Amounts of input RNA were estimated using the Bioanalyzer and libraries produced according to Clontech’s protocol. Library generation and sequencing were performed at the Norris Cancer Center Sequencing Core at USC. All FASTQ files were analyzed using FastQC (version 0.10.1), trimmed using the FASTQ Toolkit (v 1.0), aligned to the GRCh37/hg19 reference genome using Tophat (version 2), and transcripts assembled and tested for differential expression using Cufflinks (version 2.1.1). Raw data is available for public download in the NCBI database under accession code PRJNA296854.
To determine if the survival difference between C9ORF72 patient iMNs and controls was specific to our transcription factor-based reprogramming approach, we also measured the survival of Hb9::RFP+ control and C9ORF72 patient motor neurons derived from iPSCs by small molecule activation of the Sonic Hedgehog and retinoic acid signaling pathways 28 (Supplementary Fig. 3g, h). Similarly to iMNs, morphogen-generated motor neurons showed a significant survival difference between C9ORF72 patients and controls (Supplementary Fig. 3i-l).
Wuqiao County (simplified Chinese: 吴桥县; traditional Chinese: 吳橋縣; pinyin: Wúqiáo Xiàn, literally "Wu Bridge") is a county of southeastern Hebei province, China, bordering Shandong province to the southeast. It is the southernmost county-level division of the prefecture-level city of Cangzhou. Wuqiao covers an area of 583 km2 (225 sq mi) with a population of 280,000 and 444 natural villages under its jurisdiction. Over a period of more than 1500 years, Wuqiao is an old county with a vivid and rich history and culture. Wuqiao is situated in the center of the Huabei Plains and has a pleasant climate most of the year round and it is possible to pleasurably visit here at almost any time of the year.
To confirm that glutamate receptor levels were increased on the surface of C9ORF72+/− and C9ORF72 patient iMNs, we used CRISPR/Cas9 editing to introduce a Dox-inducible polycistronic cassette containing NGN2, ISL1, and LHX3 into the AAVS1 safe-harbor locus of control, C9ORF72+/− and C9ORF72 patient iPSCs. This enabled large-scale production of iMNs that expressed motor neuron markers and had transcriptional profiles similar to 7F iMNs (Supplementary Fig. 11). Using this approach, we quantified the amount of surface-bound NR1 by immunoblotting after using surface protein biotinylation to isolate membrane-bound proteins. This confirmed that surface NR1 levels were higher on C9ORF72+/− and C9ORF72 patient iMNs (n=2 patients) than controls (n=3 controls)(Fig. 4e-h, Supplementary Fig. 5g, h).
RNA sequencing output was aligned to the GRCh38 Reference Genome and quantified using the STAR aligner.65 Genes were annotated against the GENCODE version 23 Comprehensive Gene Annotation. Quality control was performed using Picard Tools AlignmentSummaryMetrics. Samples passing quality control and having RNA Integrity Number (RIN) > 5 were used in downstream analysis. To identify differentially expressed genes, the R package DESeq2 was used as previously described.66 The function DESeq was used to estimate size factors, estimate dispersion, fit the data to a negative binomial generalized linear model, and generate differential expression statistics using the Wald test. KEGG enrichment analysis was performed for internal analysis using the R package clusterProfiler.67
We thank the NINDS Biorepository at Coriell Institute for providing the following cell lines for this study: ND12133, ND03231, ND01751, ND11976, ND03719, ND00184, ND5280, ND06769, ND10689, ND12099, ND14954, ND08957, ND12100, and ND014587. We thank Helena Chui and Carol Miller at the University of Southern California Alzheimer’s Disease Research Center and Neil Shneider at the Columbia University Medical Center for control and C9ORF72 patient tissue. We thank the Choi Family Therapeutic Screening Facility for chemical screening support and the Translational Imaging Center at USC for imaging support. We thank Max Koppers, Youri Adolfs, Christiaan van der Meer, and Mark Broekhoven for help with mouse breeding and kainate injection experiments. We thank Prof. Satoshi Waguri for providing the M6PR-GFP construct. We thank Christopher Buser for assistance with electron microscopy. We also thank Sam Alworth (DRVision Technologies, LLC), Katja Hebestreit, and Raj Bhatnagar (Verge Genomics), Bob Baloh, Jacqueline O’Rourke, Christopher Donnelly, Chang Tong, Andrew McMahon and Qing Liu-Michael for reagents, technical support, and discussions. E.Y.S. is a Walter V. and Idun Berry Postdoctoral Fellow. K.A.S. was supported in part by a Muscular Dystrophy Association Development Grant. L.M. was supported by NIH grant T32DC009975–04. This work was supported by NIH grants AG039452, AG023084, and NS034467 to B.V.Z. R.J.P. was supported by grants from ALS Foundation Netherlands (TOTALS), Epilepsiefonds (12–08, 15–05), and VICI grant Netherlands Organisation for Scientific Research (NWO). This work was also supported by NIH grants R00NS077435 and R01NS097850, U.S. Department of Defense grant W81XWH-15–1-0187, and grants from the Donald E. and Delia B. Baxter Foundation, the Tau Consortium, the Frick Foundation for ALS Research, the Muscular Dystrophy Association, the New York Stem Cell Foundation, the USC Keck School of Medicine Regenerative Medicine Initiative, the USC Broad Innovation Award, and the Southern California Clinical and Translational Science Institute to J.K.I. J.K.I. is a New York Stem Cell Foundation-Robertson Investigator.
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.
Complementary DNAs (cDNAs) for the iMN factors (Ngn2, Lhx3, Isl1, NeuroD1, Ascl1, Myt1l, and Brn2) and iDA neuron factors (Ascl1, Brn2, Myt1l, Lmx1a, and Foxa2), were purchased from Addgene. cDNA for C9ORF72 was purchased from Thermo Scientific. Each cDNA was cloned into the pMXs retroviral expression vector using Gateway cloning technology (Invitrogen). The Hb9::RFP lentiviral vector was also purchased from Addgene (ID: 37081). Viruses were produced as follows. HEK293 cells were transfected at 80–90% confluency with viral vectors containing genes of interest and viral packaging plasmids (PIK-MLV-gp and pHDM for retrovirus; pPAX2 and VSVG for lentivirus) using polyethylenimine (PEI)(Sigma-Aldrich). The medium was changed 24h after transfection. Viruses were harvested at 48h and 72 h after transfection. Viral supernatants were filtered with 0.45 µM filters, incubated with Lenti-X concentrator (Clontech) for 24 h at 4 ºC, and centrifuged at 1,500 x g at 4ºC for 45 min. The pellets were resuspended in 300 µl DMEM + 10% FBS and stored at −80 ºC.
Given our observation that iMNs with reduced C9ORF72 levels are hypersensitive to DPR toxicity, we wondered if this might be due to a general disruption of protein turnover by DPRsHowever, PR50-GFP expression did not impair turnover of APP or Tau (Supplementary Fig. 14f, g and Supplementary Fig. 5l). Thus the neurotoxicity caused by DPRs that accumulate rapidly in C9-ALS motor neurons due to reduced C9ORF72 levels is not due to global disruption of protein turnover.
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.). The stronger one's muscles and bones become, the more powerful and skillful the level of kung fu is.
The tomb murals of the Eastern Wei Dynasty (534–550) in the Southern and Northern Dynasties Period (386–581) unearthed from Xiaomachang Village of Wuqiao County in 1958 depict the performances of handstands, plate spinning, deft horsemanship and so on. However, it was after the Yuan Dynasty (1271–1368) that acrobatics of Wuqiao gained much reputation. Before that, acrobatics in Henan Province was much more influential. After the Yuan Dynasty was established, the capital was moved from Kaifeng of Henan to Beijing, and the acrobatics in Wuqiao of Hebei, which neighbors Beijing, began to prosper and was increasingly influential.
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).
The Li force is observable when it is employed. Unlike the Li force, Neijing is said to be invisible. The "pivot point" essential to Li combat is not necessary in Neijing. At the point of attack, one must ‘song’ (loosen) himself to generate all Neijing energy one possesses and direct this energy stream through one's contact point with an opponent. The contact point only represents the gateway to conduct Neijing energy at the point of attack.
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).
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.