Immunostaining revealed that C9ORF72+/− and C9ORF72−/− iMNs contained elevated levels of NMDA (NR1) and AMPA (GLUR1) receptors on neurites and dendritic spines compared to control iMNs under basal conditions (Fig. 4a, c, d and Supplementary Fig. 5b and 10a, c-e, g, h, j, k). In addition, control iMNs treated with C9ORF72-specific ASOs displayed increased numbers of NMDA and AMPA receptors in their neurites (Supplementary Fig. 10l, m). C9ORF72 patient iMNs (n=3 patients) also showed elevated NR1 and GLUR1 levels compared to controls (n=3 controls), and forced expression of C9ORF72 isoform B reduced glutamate receptor levels in patient iMNs (n=3 patients) to that of controls (n=3 controls) (Fig. 4a-c and Supplementary Fig. 10a-h). mRNA levels of NR1 (GRIN1) and GLUR1 (GRIA1) were not elevated in flow-purified C9ORF72+/− iMNs, indicating that increased transcription could not explain the increased glutamate receptor levels (Supplementary Fig. 10n).
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]
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
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
Practitioners of kung fu refer to two separate forms of personal force: Li (Traditional Chinese: 力) refers to the more elementary use of tangible physical (or "external") force, such as that produced by muscles. Neijing (Traditional Chinese:內勁) or Neigong (Traditional Chinese: 內功), in contrast, refer to "internal" forces produced via advanced mental control over psychic energy (the qi).
The kung fu component of Li force is limited by one's physical condition. When a person passes his/her prime age, one's kung fu ability will pass the optimum level, too. The degree of kung fu will decline when muscles and bones are not as strong as they used to be. On the other hand, the kung fu aspect of Neijing is said to continually grow as long as one lives.[7]
(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.
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.[citation needed]
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).

However, C9orf72-deficient mice do not display overt neurodegenerative phenotypes 14,18,19,22. Moreover, no studies have shown that reduced C9ORF72 activity leads to the degeneration of C9ORF72 ALS patient-derived motor neurons, nor have any provided direct evidence identifying a cellular pathway through which C9ORF72 activity modulates neuronal survival. Additionally, a patient homozygous for the C9ORF72 repeat expansion had clinical and pathological phenotypes that were severe but nonetheless did not fall outside the range of heterozygous patients, leaving it uncertain if reductions in C9ORF72 protein levels directly correlate with disease severity 23. Thus, the role of the C9ORF72 protein in C9ORF72 ALS/FTD disease pathogenesis remains unclear.
(a) Super-resolution microscopy images of control iMNs showing colocalization (arrows) of C9ORF72 (green) with EEA1 (red). Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (b) Immunoblot against C9ORF72, EEA1, and LAMP1 on lysates from iPSC-derived motor neurons separated into light (endosomal) and heavy (lysosomal) membrane fractions using percoll gradient centrifugation. This experiment was repeated twice with similar results. (c) Super-resolution microscopy images of LAMP1 immunostaining in iMNs of specified genotypes expressing eGFP or C9ORF72 (isoform A or B)-eGFP. Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (d-f) Number of LAMP1+ vesicles in control (d-f), patient (d), C9ORF72+/− (e), and C9ORF72−/− (f) iMNs overexpressing eGFP or C9ORF72 (isoform A or B)-eGFP. Each grey open circle represents a single iMN, Mean ± s.d. For (d), n=80 (CTRL + GFP), 80 (C9-ALS + GFP), 64 (C9-ALS + isoA), and 61 (C9-ALS + isoB) iMNs quantified from two biologically independent iMN conversions of 3 CTRL or 4 C9-ALS lines. For (e), n=20 (CTRL + GFP), 15 (C9ORF72+/− + GFP), 12 (C9ORF72+/− + isoA), and 13 (C9ORF72+/− + isoB) iMNs f quantified from two biologically independent iMN conversions per condition. For (f), n=20 iMNs quantified from two biologically independent iMN conversions per condition. One-way ANOVA with Tukey correction between CTRL2 and C9ORF72+/− and C9ORF72−/− (e, f), one-way ANOVA with Tukey correction between controls and patient conditions (d). F-value (DFn, DFd): (3, 273)=12.12 (d), (3, 57)=5.64 (e), (3, 77)=6.091 (f). Dotted lines outline iMNs. (g) Representative electron micrographs of control, C9ORF72−/−, and patient iMNs showing lysosomes as electron-dense spherical perinuclear structures (arrows). This experiment was repeated twice with similar results. Scale bar: 1 μm. (h-i) Number of electron-dense spheres per square micron of perinuclear cytosol in control (h-i), C9ORF72−/− (h), and patient iMNs (i) Median ± interquartile range, each data point represents a single cell, Two-sided Mann-Whitney test). For (h), n=20 (CTRL2) and 19 (C9ORF72+/−), and for (i) n=20 (CTRL2) and 26 (C9ORF72 patient) cells quantified from two biologically independent iMN conversions of one line per genotype. (j) Super-resolution microscopy images of Lamp1 immunoreactivity in control and C9-KO mouse spinal neurons. This experiment was repeated twice with similar results. Scale bar: 5 μm. (k) Number of Lamp1+ punctae in Chat+ mouse spinal neurons. Median ± interquartile range, two-tailed t-test. t-value: 3.681. Degrees of freedom: 113. n=59 (CTRL2) and 56 (C9ORF72−/−) cells quantified from sections of two mice per genotype.

Human lymphocytes from healthy subjects and ALS patients were obtained from the NINDS Biorepository at the Coriell Institute for Medical Research and reprogrammed into iPSCs as previously described using episomal vectors61. Briefly, mammalian expression vectors containing Oct4, Sox2, Klf4, L-Myc, Lin28, and a p53 shRNA were introduced into the lymphocytes using the Adult Dermal Fibroblast Nucleofector™ Kit and Nucleofector™ 2b Device (Lonza) according to the manufacturer’s protocol. The cells were then cultured on mouse feeders until iPSC colonies appeared. The colonies were then expanded and maintained on Matrigel (BD) in mTeSR1 medium (Stem Cell Technologies).


To verify that PIKFYVE-dependent modulation of vesicle trafficking was responsible for rescuing C9ORF72 patient iMN survival, we tested the ability of a constitutively active RAB5 mutant to block C9ORF72 patient iMN degeneration. Active RAB5 recruits PI3-kinase to synthesize PI3P from PI and therefore, like PIKFYVE inhibition, increases PI3P levels 56. Constitutively active RAB5 did not improve control iMN survival (n=2 controls)(Supplementary Fig. 15k), but successfully rescued C9ORF72 patient iMN survival (n=3 patients)(Supplementary Fig. 15l). In constrast, dominant negative RAB5, wild-type RAB5, or constitutively active RAB7 did not rescue C9ORF72 patient iMN survival (n=1, 3, 3 patients, respectively)(Supplementary Fig. 14m-o).

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.
However, C9orf72-deficient mice do not display overt neurodegenerative phenotypes 14,18,19,22. Moreover, no studies have shown that reduced C9ORF72 activity leads to the degeneration of C9ORF72 ALS patient-derived motor neurons, nor have any provided direct evidence identifying a cellular pathway through which C9ORF72 activity modulates neuronal survival. Additionally, a patient homozygous for the C9ORF72 repeat expansion had clinical and pathological phenotypes that were severe but nonetheless did not fall outside the range of heterozygous patients, leaving it uncertain if reductions in C9ORF72 protein levels directly correlate with disease severity 23. Thus, the role of the C9ORF72 protein in C9ORF72 ALS/FTD disease pathogenesis remains unclear.
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.

The key to unlock and nurture Neijing is said to be the practice of ‘song’ (Traditional Chinese: 鬆 ). The term ‘song’ can function as a verb which means to keep one's mind and body loose resilient and expanding like the consistency of cotton or clouds or relaxed yet concentrated like the sharp alertness of cats immediately before attack.[8] The term can also be used as an adjective which has the same meaning as described above. The greater the extent one can achieve ‘song’ and minimize the use of Li, the greater the release of Neijing force.[9][10]
The repeat expansion suppresses the production of C9ORF72 protein by inhibiting transcription 3,4,6,7,9,17, raising the possibility that haploinsufficiency for C9ORF72 activity triggers disease pathogenesis. Consistent with this hypothesis, elimination of C9orf72 activity alters myeloid cell behavior in mice 14,18,19 and in vitro studies suggest that C9ORF72 activity may enhance autophagy 20,21.
In myeloid cells, endosomal-lysosomal trafficking regulates inflammatory cytokine release 51 and indeed, C9orf72-deficient macrophages release inflammatory cytokines 18. Interestingly, the PIKFYVE inhibitor Apilimod inhibits the release of pro-inflammatory cytokines IL-12 and IL-23 from human and mouse peripheral blood mononuclear cells 51. If impaired endosomal and lysosomal trafficking in C9ORF72 patients increases the production of pro-inflammatory cytokines that accelerate disease progression 18, PIKFYVE inhibitors or other modulators of this pathway may provide therapeutic benefit by lowering cytokine release.
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.
To measure the effect of dipeptide repeat protein expression on iMN survival, PR50 and GR50 were cloned into the pHAGE lentiviral vector as fusions with GFP to allow tracking of protein expression. iMN cultures were transduced with PR50 and GR50 lentiviruses at day 17 of reprogramming and longitudinal survival analysis was started the same day. 10 ng/ml of GDNF, BDNF, and CNTF was maintained throughout the experiment, and glutamate treatment was not performed. To measure PR50 turnover, PR50 was cloned into the pHAGE lentiviral vector as a fusion with Dendra2 (Addgene). iPSC-derived fibroblasts were generated according to Daley and colleagues64. Briefly, when C9ORF72−/− iPSC cultures reached 80% confluence, the medium was switched from mTeSR1 (Stem Cell Technologies) to human fibroblast medium containing DMEM (Life Technologies), 10% fetal bovine serum (FBS)(Thermo Fisher Scientific), and 1% penicillin/streptomycin (Life Technologies). Cells were passaged 2 to 3 times using Accutase (Life Technologies) before use in experiments. iPSC-derived fibroblasts were transduced with either pMXs-eGFP or pMXs-C9ORF72 isoform B-T2A-eGFP retrovirus and treated with 10 μg/ml mitomycin C for 3 hrs to inhibit cell proliferation. The cells were then transduced with the PR50–Dendra2 lentivirus and exposed to blue light for 1.5 sec using a lumencor LED light source to initiate photoconversion. The amount of decay (as a fraction of the starting level) of the red fluorescent punctae was monitored by longitudinal time lapse imaging in a Molecular Devices ImageExpress and analyzed using SVCell 2.0 (DRVision Technologies). Fluorescence was quantified at t = 0 and 12 hours after photoconversion. Distinct photoconverted punctae were treated as discrete objects for analysis (n = 20 each for +eGFP and +C9ORF72-T2A-eGFP). For each object, background fluorescence was subtracted and fluorescence was normalized according to object size. The fractional decay was statistically analyzed by two-tailed Student’s t-test. ** - p<.01.
Hb9::RFP+ iMNs appeared between days 13–16 after retroviral transduction. RepSox was removed at day 17 and the survival assay was initiated. For the glutamate treatment condition, 10 µM glutamate was added to the culture medium on day 17 and removed after 12 hours. Cells were then maintained in N3 medium with neurotrophic factors without RepSox. For the glutamate treatment condition with glutamate receptor antagonists, cultures were co-treated with 10 μM MK801 and CNQX, and 2 μM Nimodipine during the 12 hour glutamate treatment. The antagonists were maintained for the remainder of the experiment. For the neurotrophic factor withdrawal condition, BDNF, GDNF, and CNTF were removed from the culture medium starting at day 17. Longitudinal tracking was performed by imaging neuronal cultures in a Nikon Biostation CT or Molecular Devices ImageExpress once every 24–72 hours starting at day 17. Tracking of neuronal survival was performed using SVcell 3.0 (DRVision Technologies). Neurons were scored as dead when their soma was no longer detectable by RFP fluorescence. All neuron survival assays were performed at least twice, with equal numbers of neurons from three individual replicates from one of the trials being used for the quantification shown. All trials quantified were representative of other trials of the same experiment. When iMNs from multiple independent donors are combined into one survival trace in the Kaplan-Meier plots for clarity, the number of iMNs tracked from each line can be found in Supplementary Table 5.

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.
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).
Human lymphocytes from healthy subjects and ALS patients were obtained from the NINDS Biorepository at the Coriell Institute for Medical Research and reprogrammed into iPSCs as previously described using episomal vectors61. Briefly, mammalian expression vectors containing Oct4, Sox2, Klf4, L-Myc, Lin28, and a p53 shRNA were introduced into the lymphocytes using the Adult Dermal Fibroblast Nucleofector™ Kit and Nucleofector™ 2b Device (Lonza) according to the manufacturer’s protocol. The cells were then cultured on mouse feeders until iPSC colonies appeared. The colonies were then expanded and maintained on Matrigel (BD) in mTeSR1 medium (Stem Cell Technologies).
To confirm that reduced C9ORF72 protein levels are sufficient to cause neurodegeneration, we used CRISPR/Cas9-mediated genome editing to introduce a frameshift mutation into one or both alleles of C9ORF72 in control iPSCs (Fig. 2e and Supplementary Fig. 4e). qPCR showed that targeting one allele reduced C9ORF72 transcript levels due to nonsense-mediated decay and transcript levels were more severely reduced in homozygous mutant cells (Supplementary Fig. 4f). Frameshift mutations also decreased C9ORF72 protein expression (Supplementary Fig. 4g, 5c). RNA sequencing of flow-purified Hb9::RFP+ iMNs showed that targeting C9ORF72 did not significantly alter the expression of the top 10 genes with predicted off-target sites for the CRISPR guide RNA (Supplementary Fig. 4h and Supplementary Table 7). In addition, expression levels of the 20 genes nearest C9ORF72 on chromosome 9 were largely unperturbed in either the C9ORF72+/− and C9ORF72−/− iMNs, indicating that this approach specifically inactivated C9ORF72 (Supplementary Fig. 4i).
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.
With the four components of a chemical heat pump (two solid-gas reactors, an evaporator and a condenser), a cycle of the double-effect type can be applied to continuous refrigeration. The performance of this process is analysed, allowing the infinite sink temperature and the couples of reactive salts to be used, which depend on the production temperature envisaged, to be selected. The results are ... [Show full abstract]Read more
An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its pathogenic mechanism remains unclear. Here we use human induced motor neurons (iMNs) to show that repeat-expanded C9ORF72 is haploinsufficient in ALS. We show that C9ORF72 interacts with endosomes and is required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduces C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms leads to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescues patient neuron survival and ameliorates neurodegenerative processes in both gain- and loss-of function C9ORF72 mouse models. Thus, modulating vesicle trafficking can rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN, and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS/FTD.

With the four components of a chemical heat pump (two solid-gas reactors, an evaporator and a condenser), a cycle of the double-effect type can be applied to continuous refrigeration. The performance of this process is analysed, allowing the infinite sink temperature and the couples of reactive salts to be used, which depend on the production temperature envisaged, to be selected. The results are ... [Show full abstract]Read more

In myeloid cells, endosomal-lysosomal trafficking regulates inflammatory cytokine release 51 and indeed, C9orf72-deficient macrophages release inflammatory cytokines 18. Interestingly, the PIKFYVE inhibitor Apilimod inhibits the release of pro-inflammatory cytokines IL-12 and IL-23 from human and mouse peripheral blood mononuclear cells 51. If impaired endosomal and lysosomal trafficking in C9ORF72 patients increases the production of pro-inflammatory cytokines that accelerate disease progression 18, PIKFYVE inhibitors or other modulators of this pathway may provide therapeutic benefit by lowering cytokine release.
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.

The following antibodies were used in this manuscript: mouse anti-HB9 (Developmental Studies Hybridoma Bank); 81.5C10. chicken anti-TUJ1 (EMD Millipore); AB9354. rabbit anti-VACHT (Sigma); SAB4200559. rabbit anti-C9ORF72 (Sigma-Aldrich); HPA023873. rabbit anti-C9ORF72 (Proteintech); 25757–1-AP. mouse anti-EEA1 (BD Biosciences); 610457. mouse antiRAB5 (BD Biosciences); 610281. mouse anti-RAB7 (GeneTex); GTX16196. mouse anti-LAMP1 (Abcam); ab25630. mouse anti-M6PR (Abcam); ab2733. rabbit anti-GluR1 (EMD Millipore); pc246. mouse anti-NR1 (EMD Millipore); MAB363. chicken anti-GFP (GeneTex); GTX13970. rabbit anti-Glur6/7 (EMD Millipore); 04–921. mouse anti-FLAG (Sigma); F1804. mouse anti-GAPDH (Santa Cruz); sc-32233. chicken anti-MAP2 (Abcam); ab11267, 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), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), 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).
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.
To measure the effect of dipeptide repeat protein expression on iMN survival, PR50 and GR50 were cloned into the pHAGE lentiviral vector as fusions with GFP to allow tracking of protein expression. iMN cultures were transduced with PR50 and GR50 lentiviruses at day 17 of reprogramming and longitudinal survival analysis was started the same day. 10 ng/ml of GDNF, BDNF, and CNTF was maintained throughout the experiment, and glutamate treatment was not performed. To measure PR50 turnover, PR50 was cloned into the pHAGE lentiviral vector as a fusion with Dendra2 (Addgene). iPSC-derived fibroblasts were generated according to Daley and colleagues64. Briefly, when C9ORF72−/− iPSC cultures reached 80% confluence, the medium was switched from mTeSR1 (Stem Cell Technologies) to human fibroblast medium containing DMEM (Life Technologies), 10% fetal bovine serum (FBS)(Thermo Fisher Scientific), and 1% penicillin/streptomycin (Life Technologies). Cells were passaged 2 to 3 times using Accutase (Life Technologies) before use in experiments. iPSC-derived fibroblasts were transduced with either pMXs-eGFP or pMXs-C9ORF72 isoform B-T2A-eGFP retrovirus and treated with 10 μg/ml mitomycin C for 3 hrs to inhibit cell proliferation. The cells were then transduced with the PR50–Dendra2 lentivirus and exposed to blue light for 1.5 sec using a lumencor LED light source to initiate photoconversion. The amount of decay (as a fraction of the starting level) of the red fluorescent punctae was monitored by longitudinal time lapse imaging in a Molecular Devices ImageExpress and analyzed using SVCell 2.0 (DRVision Technologies). Fluorescence was quantified at t = 0 and 12 hours after photoconversion. Distinct photoconverted punctae were treated as discrete objects for analysis (n = 20 each for +eGFP and +C9ORF72-T2A-eGFP). For each object, background fluorescence was subtracted and fluorescence was normalized according to object size. The fractional decay was statistically analyzed by two-tailed Student’s t-test. ** - p<.01.
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