(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.

J.K.I. and P.A. are co-founders of Acurastem, Inc. P.A. is an employee of Icagen Corporation. J.K.I. and P.A. declare that they are bound by confidentiality agreements that prevent them from disclosing details of their financial interests in this work. S-J.L. is a founder of DRVision Technologies and T-Y.C. is an employee of DRVision Technologies. A.Z. and J.A.C. are co-founders of Verge Genomics and V.H-S., N.W., and T.G.B. are employees of Verge Genomics.
To verify that PIKFYVE is the functional target of the inhibitor, we first confirmed PIKFYVE expression by qPCR in control and patient (n=3 patients) iMNs (Supplementary Fig. 15b). Next, we verified that YM201636 rescued C9ORF72 patient iMN survival in a dose-dependent manner (Supplementary Fig. 15c). We then asked if Apilimod, a structurally distinct PIKFYVE inhibitor, could rescue patient iMN survival 51(Fig. 6b). To verify target engagement by Apilimod in iPSC-derived motor neurons, we administered Apilimod for three hours and measured EEA1+ early endosome size. PIKFYVE inhibition increases PI3P levels, leading to increased recruitment of EEA1 to early endosomes, more homotypic early endosomal fusion, and larger EEA1+ early endosomes 54. As expected, Apilimod treatment increased EEA1+ endosome size in a dose-dependent manner, verifying target engagement in motor neurons (Supplementary Fig. 15d, e).

Whole cell membrane potential and current recordings in voltage- and current-clamp configurations were made using an EPC9 patch clamp amplifier controlled with PatchMaster software (HEKA Electronics). Voltage- and current-clamp data was acquired at 50 kHz and 20 kHz, respectively, with a 2.9 kHz low-pass Bessel filter, while spontaneous action potential recordings were acquired at 1 kHz sampling frequency. For experiments, culture media was exchanged with warm extracellular solution consisting of (in mM): 140 NaCl, 2.8 KCl, 10 HEPES, 1 MgCl2, 2 CaCl2, and 10 glucose, with pH adjusted to 7.3 and osmolarity adjusted to 305 mOsm. Glass patch pipettes were pulled on a Narishige PC-10 puller and polished to 5–7 MΩ resistance. Pipettes were also coated with Sylgard 184 (Dow Corning) to reduce pipette capacitance. The pipette solution consisted of (in mM): 130 K-gluconate, 2 KCl, 1CaCl2, 4 MgATP, 0.3 GTP, 8 phosphocreatine, 10 HEPES, 11 EGTA, adjusted to pH 7.25 and 290 mOsm. Pipettes were sealed to cells in GΩ-resistance whole cell configuration, with access resistances typically between 10–20 MΩ, and leakage currents less than 50 pA. Capacitance transients were compensated automatically through software control. For voltage clamp, cells were held at −70 mV. For Current-voltage traces, a P/4 algorithm was used to subtract leakage currents from the traces. Measurements were taken at room temperature (approximately 20–25 °C). Data was analyzed and plotted in Igor Pro 6 (WaveMetrics) using Patcher’s Power Tools plug-in and custom programmed routines. Current density was obtained by dividing the measured ion channel current by the cell capacitance. For control iMNs, 10/10 tested fired action potentials. For C9-ALS iMNs, 9/10 tested fired action potentials.

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]
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).