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).
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.
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.
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).
In advanced traditional Chinese kung fu (martial arts), Neijing (Traditional Chinese: 內勁; pinyin: nèijìng) refers to the conscious control of the practitioner's qi, or "life energy", to gain advantages in combat.[1] Nèijìng is developed by using "Neigong" (Traditional Chinese: 內功; pinyin: nèigōng) (內功), or "internal exercises," as opposed to "wàigōng" (外功), "external exercises."
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.[5] The contact point only represents the gateway to conduct Neijing energy at the point of attack.[6]
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).
Our results highlight the importance of C9ORF72 protein function, RAB5 activity, PI3P levels, and lysosomeal function as key therapeutic targets for C9ORF72 ALS/FTD. By generating PI3P, RAB5 drives early endosomal maturation and the initial stages of lysosomal biogenesis (Fig. 6f)59. PI3P also plays important roles in autophagosome formation and autophagsome-lysosome fusion. Indeed, a previous study suggests that PIKFYVE inhibition may increase autophagic flux 53, and this should be investigated in the context of motor neurons. Loss-of-function mutations in two other genes whose proteins function to increase PI3P levels, ALS2 and FIG4, also cause ALS 1. ALS2 encodes the RAB5 guanine exchange factor ALSIN 60, while FIG4 converts PI(3,5)P2 into PI3P 55(Fig. 6f). In addition, proteins encoded by several other ALS genes play key roles in lysosomal biogenesis, including CHMP2B, OPTN, and SQSTM1 1. The fact that FIG4 and ALS2 loss-of-function mutations can cause ALS suggests that PIKFYVE inhibition or RAB5 activation may be capable of modulating ALS disease processes in humans.
We show for the first time that chemical or genetic modulators of vesicle trafficking can fully rescue iMN degeneration caused by the C9ORF72 repeat expansion. Previous studies have implicated several rare ALS or FTD mutations linked to these vesicle trafficking pathways, but by showing that C9ORF72 is haploinsufficient in ALS/FTD and demonstrating that perturbation of vesicle trafficking rescues C9ORF72 neurodegeneration, our findings highlight mechanistic convergence in a large portion of ALS.
IPSC-MNs at differentiation D35 were harvested in cold Hypotonic buffer (20 mM HEPES pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1mM EGTA, 1 mM DTT and protease inhibitor cocktail (Roche)) and lysed by passing through G25 needles 25 times and then spun down at 700 x g for 10min at 4℃. The Supernatant was loaded onto pre-made 30% Percoll solution and re-centrifuged at 33,000 RPM using Beckman rotor SWI55 for 50min at 4℃. 300 ul aliquots were taken from top to bottom as fractions and all the collected samples were boiled with SDS-PAGE sample buffer and analyzed by western blot.
Cells were fixed in 6-well culture plates in 2.5 % glutaraldehyde in 0.1M cacodylate buffer, post-fixed in 1% osmium tetroxide for 1 hour and block stained in 1% uranyl acetate in 0.1M acetate buffer pH 4.4 overnight at 4 ˚C. Dehydration was performed in increasing concentrations of ethanol (10%/25%/50%/75%/90%/100%/100%/100%) for 15 minutes each and infiltrated with increasing concentrations of Eponate12 (Ted Pella Inc., Redding, CA, USA), 25% Eponate12 (no catalyst) in ethanol for 3 hours, 50% overnight, 100% for 5 hours, 100% overnight, and polymerized in fresh Eponate12 with DMP-30 for 48 hours at 60 ˚C. Previously marked areas were sawed out, the tissue culture plastic was removed and the selected area sectioned parallel to the substrate at a thickness of 70 nm. Sections at a depth of 3–5 µm were collected on formvar-filmed 50 mesh copper grids and imaged at 80 kV in an FEI 208 Morgagni (FEI is in Hillsboro, OR, USA). Per micrograph, cytosol was used to quantify the number of electron dense spheres that were defined as lysosomes 40.
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.

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).

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.

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 primary astrocytes from Lonza (cat. no. CC-2565) were cultured in Lonza Astrocyte Growth Medium (cat. no. CC-3186) according to the manufacturer’s instructions. To force expression of dipeptide repeat proteins in the astrocytes, lentiviruses were produced in HEK 293T cells and concentrated ~80-fold using Lenti-X Concentrator (Clontech). When astrocytes reached 90% confluency, they were transduced with lentiviruses encoding either GFP or GR(50)-GFP. Fluorescence microscopy analysis was used to confirm that 60–80% of astrocytes were transduced in both conditions. To measure glutamate uptake, GFP- or GR(50)-GFP-expressing astrocytes were incubated with 200 mM glutamate in Astrocyte Growth Medium for 2 hours at 37 degrees Celsius in biological quadruplicate. Media samples were collected after 2 hours and glutamate levels were quantified using the BioVision Glutamate Colorimetric Assay Kit (cat. no. K629) according to the manufacturer’s instructions. Colorimetric substrate levels were quantified at 450 nm using a Molecular Devices SpectraMax i3x Multi-Mode Microplate Reader.

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.
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.
Structured illumination microscopy (SIM) images were acquired using a Zeiss Elyra PS.1 system equipped with a 100X 1.46 NA or 63X 1.4NA objective. Acquisition was performed with PCO edge sCMOS camera and image reconstruction was done with built-in structured illumination model. Confocal microscopy images were acquired using Zeiss LSM800 microcopy with 63X 1.4NA objective or Zeiss LSM780 microcopy with 40X 1.1NA objective. Z stack images were done with a step size of 2.5 um. Further image process was done with Fiji.
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).
Post mortem tissues were kindly provided by Neil Shneider (Columbia) and were collected from the following individuals: Sample 1 – age: 64, diagnosis: ALS, genotype: positive for C9ORF72 repeat expansion, Sample 2 – age: 55, diagnosis: ALS, genotype: positive for C9ORF72 repeat expansion, Sample 3 – age: 65, diagnosis: ALS, genotype: positive for C9ORF72 repeat expansion, Sample 4 – age: 65, diagnosis: control, genotype: negative for C9ORF72 repeat expansion, Sample 5 – age: 50, diagnosis: control, genotype: negative for C9ORF72 repeat expansion, Sample 6 – age: 50, diagnosis: control, genotype: negative for C9ORF72 repeat expansion, Sample 7 – age: 53, diagnosis: ALS, genotype: negative for C9ORF72 repeat expansion, Sample 8 - age: 64, diagnosis: ALS, genotype: negative for C9ORF72 repeat expansion. All donors except donor 7 (sample 7) were female. For immunofluorescence, 10 µm sections were sliced from flash frozen lumbar spinal cord tissues. Sections were then air dried and fixed with ice cold acetone for 10 minutes, and blocked with 10% normal goat serum/1% BSA/0.3% Triton-X/PBS at room temperature for 1 hour followed by incubation with NR1 antibody (1:200, BD Bioscience) in blocking buffer overnight at 4 ºC. Sections subsequently were blocked using avidin/biotin kit (Vector Lab), and washed with PBS. Then, sections were incubated with goat anti-rabbit IgG Biotin conjugate secondary antibody (1:750, Invitrogen) or with goat anti-mouse IgG Biotin conjugate secondary antibody (1:750, Invitrogen) for 1 hour at room temperature, washed and incubated with streptavidin-Alexa Fluor 488 conjugate (1:500, Invitrogen) in dark for 1 hour at room temperature. Sections were washed and blocked again in blocking buffer for 1 hour at room temperature. For neuronal marker staining, sections were incubated with Tu-20 antibody (1:1000, Abcam) or NeuN antibody (1:500, Abcam) at 37 ºC for 1 hour. Sections were washed with PBS and incubated with goat anti-mouse Alexa Fluor 546 (1:500, Invitrogen) or goat anti-rabbit Alexa Fluor 546 (1:500, Invitrogen) for 1 hour at room temperature. Lipofuscin autofluorescence was quenched by immersing sections in autofluorescence eliminator reagent (Millipore) for 4 minutes following manufacture’s instruction. Sections were then counterstained and mounted with Prolong Gold antifade mounting medium with DAPI (Invitrogen).
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.).[2] The stronger one's muscles and bones become, the more powerful and skillful the level of kung fu is.[3]
To determine if reduced C9orf72 levels leads to glutamate receptor accumulation in vivo, we examined spinal motor neurons deleted of C9orf72 in Nestin-Cre-Stop-Flox-C9orf72 mice 22. Immunofluorescence analysis indicated that Nr1 (NMDA) and GluR1 (AMPA) levels were elevated in C9orf72-null motor neurons (Supplementary Fig. 12a, b). To confirm these findings, we isolated post-synaptic densities from the spinal cords of control and C9orf72 knockout mice. Post-synaptic density fractions contained glutamate receptors and PSD-95, but not p53 or synaptophysin, indicating they were enriched for post-synaptic density proteins (Supplementary Fig. 12c, 5i). Immunoblotting showed that post-synaptic densities in C9orf72 knockout mice contained significantly higher levels of Nr1 and Glur1 than in control mice (Fig. 4i, j and Supplementary Fig. 5j).
Consistent with previous studies 3,4,6–8, patient iMNs (n=5 patients) had reduced C9ORF72 expression compared to controls (n=3; Fig. 2a and Supplementary Fig. 4a, 5b). While previous studies have linked low C9ORF72 levels to changes in vesicle trafficking or autophagy 18,20,30–33, it remains unknown if loss of C9ORF72 protein directly contributes to degeneration. Thus, we re-expressed C9ORF72 (isoform A or B) in iMNs using a retroviral cassette (Supplementary Fig. 4b) and found that both isoforms rescued C9ORF72 patient iMN survival in response to glutamate treatment (n=3 patients Fig. 2b and Supplementary Fig. 4c). This effect was specific for C9ORF72 iMNs, as forced expression of C9ORF72 did not rescue SOD1A4V iMN survival (Fig. 2c), nor did it improve the survival of control iMNs (n=2 controls Fig. 2d and Supplementary Fig. 4d).