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).
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.
To determine if patient iMN degeneration resulted from bona fide ALS disease processes specific for motor neurons, we measured the survival of induced dopaminergic neurons (iDAs) generated by expression of FoxA2, Lmx1a, Brn2, Ascl1, and Myt1l 29. These neurons expressed high levels of tyrosine hydroxylase, indicating they had established a key aspect of the dopamine synthesis pathway and were distinct from iMNs, which do not express this enzyme 24 (Supplementary Fig. 3m, n). Unlike iMN cultures, iDA cultures from C9ORF72 patients (n=2 patients) did not show reduced survival compared to controls (n=2 controls) in either glutamate treatment and neurotrophic factor withdrawal conditions (Fig. 1h and Supplementary Fig. 3o), indicating that the in vitro neurodegenerative phenotype elicited by the C9ORF72 mutation is selective for motor neurons.

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]

For experiments other than the comparison of Apilimod and the reduced-activity analog, Apilimod was purchased from Axon Medchem (cat. no. 1369). For the reduced-activity analog assays, Apilimod and the reduced activity analog were synthesized at Icagen, Inc. according to the schemes shown in Supplementary Fig. 16. PIKFYVE kinase inhibition was measured using the ADP-Glo kinase assay from SignalChem according to the manufacturer’s instructions, using purified PIKFYVE kinase (SignalChem cat. no. P17–11BG-05).
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.
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.
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]
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).
Importantly, our work establishes a new approach for suppressing DPR protein toxicity and blocking C9ORF72 pathogenesis: restoring or replacing C9ORF72 activity. Although high levels of C9ORF72 isoform A may have slightly detrimental effects on control motor neuron survival, we have only observed this in neurons without C9ORF72 repeat expansion. Thus, we would not anticipate a harmful effect of forced C9ORF72 expression in C9ORF72 patients. In addition, a better understanding of the effects of forced C9ORF72 expression could inform safe development of this therapeutic strategy. For example, determining if C9ORF72 accelerates turnover of DPR aggregates by stimulating autophagy could lead help to identify new therapeutic targets.

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.
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,†