(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.
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
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
Dirigido a blogueros, personas influyentes, funcionarios de relaciones públicas, personalised de marketing, aspirantes a periodistas o cualquier persona que quiera aprender más sobre el oficio de la escritura, el curso enseña las habilidades básicas de la escritura profesional: la introducción, la pirámide invertida, las 5 W, las 3 C y, lo más importante de todo, la narración de cuentos.

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).
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.
On the other hand, the level of the Neijing force depends on the extent one can exercise over one's will power to release an inner qi energy. Within the framework of Chinese martial arts, every person is believed to possess the inborn energy of qi. Martial artists can harness the force of qi so that it is strong enough to be applied in combat. When qi is being directed by one's will, it is called Neijing.[4]
HEK 293T cells were used to produce retrovirus, lentivirus, and C9ORF72 protein. HEK cells were used for these purposes based on previous published studies using HEK cells in order to produce viral particles and mammalian proteins. HEK cells were obtained from American Type Culture Collection, catalog number CRL-11268. HEK and iPS cells were tested for mycoplasma before, during, and after the study and were negative.

Eliminating C9ORF72 protein expression from one or both alleles reduced iMN survival to levels comparable to patient iMNs (Fig. 2f). Antisense oligonucleotide (ASO)-mediated suppression of C9ORF72 expression levels also reduced control iMN survival (Fig. 2g and Supplementary Fig. 4j), suggesting that reduced iMN survival was not due to an off-target effect of the CRISPR/Cas9 genome editing. Exogenously restoring C9ORF72 expression in C9ORF72+/− and C9ORF72−/− iMNs rescued survival (Supplementary Fig. 4k, l), verifying that depletion of C9ORF72 caused the observed neurodegeneration.


(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.
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).
(a) Phenotypic screening for small molecules that enhance the survival of C9-ALS iMNs. (b) Chemical structure of the PIKFYVE inhibitors YM201636 and Apilimod, and a reduced-activity analog of Apilimod. (c) Live cell images of iMNs at day 7 of treatment with DMSO or YM201636 (scale bar: 1 mm). This experiment was performed 3 times with similar results. (d) Survival effect of scrambled or PIFKVYE ASOs on C9-ALS iMNs in excess glutamate. n=50 iMNs per condition, iMNs quantified from 3 biologically independent iMN conversions per condition. (e) Survival effect of Apilimod and the reduced-activity analog on C9-ALS patient iMNs with neurotrophic factor withdrawal. n=50 iMNs per condition, iMNs quantified from 3 biologically independent iMN conversions per condition. All iMN survival experiments in (d, e) were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. (f) Activities of therapeutic targets in C9ORF72 ALS. (g, h) The effect of 3 μM Apilimod on NMDA-induced hippocampal injury in control, C9orf72+/−, or C9orf72−/− mice. (Mean +/− s.e.m. of n=3 mice per condition, one-way ANOVA with Tukey correction across all comparisons, F-value (DFn, DFd): (3, 8)=43.55, AP = Apilimod, red dashed lines outline the injury sites). (i, j) The effect of 3 μM Apilimod on the level of GR+ puncta in the dentate gyrus of control or C9-BAC mice. Mean +/− s.d. of the number of GR+ puncta per cell, each data point represents a single cell. n=20 (wild-type + DMSO), 20 (wild-type + Apilimod), 87 (C9-BAC + DMSO), and 87 (C9-BAC + Apilimod) cells quantified from 3 mice per condition, one-way ANOVA with Tukey correction for all comparisons, F-value (DFn, DFd): (3, 180) = 16.29. Scale bars = 2 μm, dotted lines outline nuclei, and white arrows denote GR+ punctae (i). (k) Model for the mechanisms that cooperate to cause neurodegeneration in C9ORF72 ALS/FTD. Proteins in red are known to be mutated in ALS or FTD. iMN survival experiments in (d, e) were performed in a Molecular Devices ImageExpress.
On the other hand, the level of the Neijing force depends on the extent one can exercise over one's will power to release an inner qi energy. Within the framework of Chinese martial arts, every person is believed to possess the inborn energy of qi. Martial artists can harness the force of qi so that it is strong enough to be applied in combat. When qi is being directed by one's will, it is called Neijing.[4]
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).
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.
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.
All experiments involving live vertebrates (cortical glial isolation) performed at USC were done in compliance with ethical regulations approved by the USC IACUC committee. All animal use and care at the University Medical Center Utrecht were in accordance with local institution guidelines of the University Medical Center Utrecht (Utrecht, the Netherlands) and approved by the Dierexperimenten Ethische Commissie Utrecht with the protocol number DEC 2013.I.09.069.
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).
To determine if a deletion of C9ORF72 or the C9ORF72 repeat expansion caused changes in endosomal trafficking in motor neurons, we examined the number of early endosomes (RAB5+, EEA1+), late endosomes (RAB7+), and lysosomes (LAMP1+, LAMP2+, LAMP3+) in control, C9ORF72 patient, C9ORF72+/−, and C9ORF72−/− iMNs. We observed the most significant difference in the lysosomal population, with C9ORF72 patient iMNs (n=4 patients) having fewer LAMP1+, LAMP2+, and LAMP3+ vesicles than control iMNs (n=4 controls)(Fig. 3c, d and Supplementary Fig. 8a-d). C9ORF72+/− and C9ORF72−/− also harbored fewer LAMP1+, LAMP2+, and LAMP3+ vesicles than isogenic control iMNs, indicating that reduced C9ORF72 levels alone leads to a loss of lysosomes (Fig. 3c, e, f and Supplementary Fig. 8a-d). ASO-mediated knockdown of C9ORF72 expression also decreased lysosome numbers in iMNs (Supplementary Fig. 8e). Although membrane fractionation showed that control and patient iMNs have similar amounts of LAMP2 in the lysosomal membrane fraction (Supplementary Fig. 8f), analysis of the immunofluorescence intensity of LAMP proteins suggests that this is likely due to the fact that C9ORF72 patient and C9ORF72+/− iMNs have a higher concentration of LAMP proteins in their lysosomal membranes, possibly as a result of fewer lysosomes being present (Supplementary Fig. 8g). Using electron microscopy to identify lysosomes by their high election density 40, we verified that the vesicles reduced in C9ORF72-deficient cells were lysosomes (Fig. 3g-i). Forced expression of either C9ORF72 isoform restored the number of LAMP1+, LAMP2+, and LAMP3+ lysosomes in patient (n=4 patients) and C9ORF72-deficient iMNs (Fig. 3c-f and Supplementary Fig. 8a-h). To determine if loss of C9ORF72 activity reduces lysosome numbers in motor neurons in vivo, we measured the number of lysosomes in spinal motor neurons in Nestin-Cre-Stop-Flox-C9orf72 mice 22. C9orf72−/− motor neurons contained significantly fewer Lamp1+ lysosomes than control motor neurons (Fig. 3j, k).
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.
We also found that Reduced C9ORF72 activity also induces iMN hypersensitivity to DPRs by impairing their clearance. This uncovers a more direct form of cooperative pathogenesis between gain- and loss-of-function mechanisms in C9ORF72 ALS/FTD. Through a potentially similar mechanism, reduced C9orf72 levels can also facilitate cytoplasmic TDP-43 accumulation in mouse neurons 20.
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 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).
GCaMP6 was cloned into the pMXs-Dest-WRE retroviral vector and transduced into reprogramming cultures concurrently with the motor neuron factors. To assess GCaMP6 activity, 1.5 μm glutamate was added to iMN cultures and cells were imaged continuously for 2 minutes at 24 frames per second. GFP flashes were scored manually using the video recording. At least 3 different fields of view from three independent cultures, totalling 50–100 iMNs, were scored per condition.
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).
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).

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]
To study the pathogenic mechanism of the C9ORF72 repeat expansion in human motor neurons, we used the forced expression of the transcription factors Ngn2, Isl1, Lhx3, NeuroD1, Brn2, Ascl1, and Myt1l, to convert control and C9ORF72 ALS/FTD patient induced pluripotent stem cells (iPSCs)(for iPSC characterization, see Supplementary Fig. 1 and Supplementary Tables 3, 4) into iMNs (Supplementary Fig. 2a, b) 10,24. Control and patient iMNs labeled with an Hb9::RFP+ lentiviral reporter construct (Supplementary Fig. 2b-d)25 co-expressed spinal motor neuron markers including TUJ1, HB9, and VACHT; were produced at similar rates amongst different iPSC lines; and possessed electrophysiological properties of motor neurons (Supplementary Fig. 2c-i). Depolarizing voltage steps induced currents characteristic of sodium and potassium channels and iMNs fired single or repetitive action potentials (patient - 90%, n=10; control – 100%, n=10)(Supplementary Fig. 2g-i). When co-cultured with primary chick muscle, channel rhodopsin-expressing control and patient iMNs repeatedly induced myotube contraction upon depolarization with green light, indicating they formed neuromuscular junctions and actuated muscle contraction (Supplementary Fig. 2j and Supplementary Videos 1, 2).

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).
GCaMP6 was cloned into the pMXs-Dest-WRE retroviral vector and transduced into reprogramming cultures concurrently with the motor neuron factors. To assess GCaMP6 activity, 1.5 μm glutamate was added to iMN cultures and cells were imaged continuously for 2 minutes at 24 frames per second. GFP flashes were scored manually using the video recording. At least 3 different fields of view from three independent cultures, totalling 50–100 iMNs, were scored per condition.
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