An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced 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 led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to 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 and FTD.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Renton, A.E., Chiò, A. & Traynor, B.J. State of play in amyotrophic lateral sclerosis genetics. Nat. Neurosci. 17, 17–23 (2014).
Majounie, E. et al. Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol. 11, 323–330 (2012).
DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).
Renton, A.E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268 (2011).
Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014).
Sareen, D. et al. Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci. Transl. Med. 5, 208ra149 (2013).
Donnelly, C.J. et al. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80, 415–428 (2013).
Lagier-Tourenne, C. et al. Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration. Proc. Natl. Acad. Sci. USA 110, E4530–E4539 (2013).
Haeusler, A.R. et al. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507, 195–200 (2014).
Wen, X. et al. Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. Neuron 84, 1213–1225 (2014).
Mori, K. et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339, 1335–1338 (2013).
Kwon, I. et al. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science 345, 1139–1145 (2014).
Su, Z. et al. Discovery of a biomarker and lead small molecules to target r(GGGGCC)-associated defects in c9FTD/ALS. Neuron 84, 239 (2014).
Jiang, J. et al. Gain of toxicity from ALS/FTD-linked repeat expansions in C9ORF72 is alleviated by antisense oligonucleotides targeting GGGGCC-containing RNAs. Neuron 90, 535–550 (2016).
Chew, J. et al. Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss and behavioral deficits. Science 348, 1151–1154 (2015).
Liu, Y. et al. C9orf72 BAC mouse model with motor deficits and neurodegenerative features of ALS/FTD. Neuron 90, 521–534 (2016).
Liu, E.Y. et al. C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD. Acta Neuropathol. 128, 525–541 (2014).
O'Rourke, J.G. et al. C9orf72 is required for proper macrophage and microglial function in mice. Science 351, 1324–1329 (2016).
Burberry, A. et al. Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease. Sci. Transl. Med. 8, 347ra93 (2016).
Sellier, C. et al. Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death. EMBO J. 35, 1276–1297 (2016).
Webster, C.P. et al. The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy. EMBO J. 35, 1656–1676 (2016).
Koppers, M. et al. C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits. Ann. Neurol. 78, 426–438 (2015).
Fratta, P. et al. Homozygosity for the C9orf72 GGGGCC repeat expansion in frontotemporal dementia. Acta Neuropathol. 126, 401–409 (2013).
Son, E.Y. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218 (2011).
Marchetto, M.C. et al. Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells. Cell Stem Cell 3, 649–657 (2008).
Kiskinis, E. et al. Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1. Cell Stem Cell 14, 781–795 (2014).
Lin, C.L. et al. Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron 20, 589–602 (1998).
Du, Z.W. et al. Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat. Commun. 6, 6626 (2015).
Pfisterer, U. et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proc. Natl. Acad. Sci. USA 108, 10343–10348 (2011).
Aoki, Y. et al. C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia. Brain 140, 887–897 (2017).
Sullivan, P.M. et al. The ALS/FTLD associated protein C9orf72 associates with SMCR8 and WDR41 to regulate the autophagy-lysosome pathway. Acta Neuropathol. Commun. 4, 51 (2016).
Farg, M.A. et al. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Hum. Mol. Genet. 23, 3579–3595 (2014).
Prudencio, M. et al. Distinct brain transcriptome profiles in C9orf72-associated and sporadic ALS. Nat. Neurosci. 18, 1175–1182 (2015).
Highley, J.R. et al. Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones. Neuropathol. Appl. Neurobiol. 40, 670–685 (2014).
Levine, T.P., Daniels, R.D., Gatta, A.T., Wong, L.H. & Hayes, M.J. The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs. Bioinformatics 29, 499–503 (2013).
Marat, A.L., Dokainish, H. & McPherson, P.S. DENN domain proteins: regulators of Rab GTPases. J. Biol. Chem. 286, 13791–13800 (2011).
Amick, J., Roczniak-Ferguson, A. & Ferguson, S.M. C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling. Mol. Biol. Cell 27, 3040–3051 (2016).
Neiss, W.F. The electron density of light and dark lysosomes in the proximal convoluted tubule of the rat kidney. Histochemistry 77, 63–77 (1983).
Saftig, P. & Klumperman, J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat. Rev. Mol. Cell Biol. 10, 623–635 (2009).
Ganley, I.G., Carroll, K., Bittova, L. & Pfeffer, S. Rab9 GTPase regulates late endosome size and requires effector interaction for its stability. Mol. Biol. Cell 15, 5420–5430 (2004).
Rodriguez-Gabin, A.G., Yin, X., Si, Q. & Larocca, J.N. Transport of mannose-6-phosphate receptors from the trans-Golgi network to endosomes requires Rab31. Exp. Cell Res. 315, 2215–2230 (2009).
Waguri, S. et al. Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells. Mol. Biol. Cell 14, 142–155 (2003).
Wang, G., Gilbert, J. & Man, H.Y. AMPA receptor trafficking in homeostatic synaptic plasticity: functional molecules and signaling cascades. Neural Plast. 2012, 825364 (2012).
Chen, P., Gu, Z., Liu, W. & Yan, Z. Glycogen synthase kinase 3 regulates N-methyl-D-aspartate receptor channel trafficking and function in cortical neurons. Mol. Pharmacol. 72, 40–51 (2007).
Chen, T.W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
Wainger, B.J. et al. Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell Rep. 7, 1–11 (2014).
Gurskaya, N.G. et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24, 461–465 (2006).
Mackenzie, I.R. et al. Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers. Acta Neuropathol. 130, 845–861 (2015).
Cai, X. et al. PIKfyve, a class III PI kinase, is the target of the small molecular IL-12/IL-23 inhibitor apilimod and a player in Toll-like receptor signaling. Chem. Biol. 20, 912–921 (2013).
Lemmon, M.A. Membrane recognition by phospholipid-binding domains. Nat. Rev. Mol. Cell Biol. 9, 99–111 (2008).
Martin, S. et al. Inhibition of PIKfyve by YM-201636 dysregulates autophagy and leads to apoptosis-independent neuronal cell death. PLoS One 8, e60152 (2013).
Ikonomov, O.C., Sbrissa, D. & Shisheva, A. Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion. Am. J. Physiol. Cell Physiol. 291, C393–C404 (2006).
Chow, C.Y. et al. Deleterious variants of FIG4, a phosphoinositide phosphatase, in patients with ALS. Am. J. Hum. Genet. 84, 85–88 (2009).
Lodhi, I.J. et al. Insulin stimulates phosphatidylinositol 3-phosphate production via the activation of Rab5. Mol. Biol. Cell 19, 2718–2728 (2008).
Zhong, Z. et al. Protein S protects neurons from excitotoxic injury by activating the TAM receptor Tyro3-phosphatidylinositol 3-kinase-Akt pathway through its sex hormone-binding globulin-like region. J. Neurosci. 30, 15521–15534 (2010).
O'Rourke, J.G. et al. C9orf72 BAC transgenic mice display typical pathologic features of ALS/FTD. Neuron 88, 892–901 (2015).
Zeigerer, A. et al. Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature 485, 465–470 (2012).
Topp, J.D., Gray, N.W., Gerard, R.D. & Horazdovsky, B.F. Alsin is a Rab5 and Rac1 guanine nucleotide exchange factor. J. Biol. Chem. 279, 24612–24623 (2004).
Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nat. Methods 8, 409–412 (2011).
Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
Li, J. et al. Long-term potentiation modulates synaptic phosphorylation networks and reshapes the structure of the postsynaptic interactome. Sci. Signal. 9, rs8 (2016).
Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Anders, S. et al. Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nat. Protoc. 8, 1765–1786 (2013).
Yu, G., Wang, L.-G., Han, Y. & He, Q.-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).
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 H. Chui and C. Miller (University of Southern California Alzheimer's Disease Research Center) and N. Shneider (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 M. Koppers, Y, Adolfs, C. van der Meer and M. Broekhoven for help with mouse breeding and kainate injection experiments. We thank S. Waguri (Fukushima Medical University) for providing the M6PR-GFP construct. We thank C, Buser for assistance with electron microscopy. We also thank S. Alworth (DRVision Technologies), K. Hebestreit and R. Bhatnagar (Verge Genomics), B. Baloh (Cedars Sinai Medical Center), J. O'Rourke (Cedars Sinai Medical Center), C. Donnelly, C. Tong, A. McMahon and Q. 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 Organization for Scientific Research (NWO). This work was also supported by NIH grants R00NS077435 and R01NS097850, US 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.
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 A.Z., V.H.-S., N.W. and T.G.B. are employees of Verge Genomics.
Supplementary Figures 1–17 & Supplementary Tables 1–6 (PDF 39872 kb)
RNA sequencing data of Hb9::RFP+ iMNs from CTRL2, C9- ALS1, CTRL2 C9ORF72+/-, and CTRL2 C9ORF72-/- iPSCs. (XLSX 11015 kb)
Channel rhodopsin neuromuscular junction assay: C9-ALS patient iMNs (C9-ALS2). Green light is activated and can be observed by an increase in overall brightness at the following times: 7-13 sec, 18-23 sec, 29-35 sec, 40-45 sec, 51-56 sec, 1:02-1:07. Light-induced myotube contraction can be observed during those intervals. (MOV 11016 kb)
Channel rhodopsin neuromuscular junction assay: control iMNs (CTRL2). Green light is activated and can be observed by an increase in overall brightness at the following times: 7- 12 sec, 18-23 sec, 29-34 sec, 40-45 sec, 52-57 sec, 1:03-1:08. Light-induced myotube contraction can be observed during those intervals. (MOV 16834 kb)
About this article
Cite this article
Shi, Y., Lin, S., Staats, K. et al. Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons. Nat Med 24, 313–325 (2018). https://doi.org/10.1038/nm.4490
Current Opinion in Systems Biology (2020)
The FTLD Risk Factor TMEM106B Regulates the Transport of Lysosomes at the Axon Initial Segment of Motoneurons
Cell Reports (2020)
Frontiers in Molecular Neuroscience (2020)
Frontiers in Neuroscience (2020)
Pluripotent stem cells for neurodegenerative disease modeling: an expert view on their value to drug discovery
Expert Opinion on Drug Discovery (2020)