Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons

Abstract

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.

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Figure 1: C9ORF72 patient iMNs undergo rapid neurodegeneration.
Figure 2: C9ORF72 protein levels determine iMN survival.
Figure 3: Reduced C9ORF72 activity disrupts vesicle trafficking and lysosomal biogenesis in motor neurons.
Figure 4: Low C9ORF72 activity sensitizes neurons to glutamate.
Figure 5: C9ORF72 levels determine dipeptide repeat turnover.
Figure 6: Small molecule modulators of vesicle trafficking rescue neurodegeneration in vitro and in vivo.

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Acknowledgements

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.

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Contributions

Y.S., S.L., Y.L. and J.K.I. conceived the project. Y.S., S.L., E.Y.S., Y.L., L.M., K.A.S., V.R.V., K.S., S.J.J.L., P.R.A., M.C., R.J.P., D.T., B.V.Z. and J.K.I. designed the experiments. Y.S., S.L., W.-H.C., E.Y.S., Y.L., S.-T.H., E.H., G.R.L., T.S., M.H., C.S., A.R.N., T.-Y.C., Y.W., K.K., B.W., L.M., M.J.C., B.G., K.P.S., J. K., N.K., X.W., V.H., A.R.N., K.A.S., V.R.V., K.S., R.J.P. and J.K.I. performed experiments and interpreted data. K.K. performed all electrophysiological studies and P.W., J.A.C., N.H.-S., N.W., T.G.B., A.Z. and K.A.S. performed RNA-Seq analysis. Y.S., S.L., E.Y.S., K.A.S. and J.K.I. prepared the manuscript. C.G. and M.W. developed the method of inducing iMNs using the Dox-NIL construct. All of the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Justin K Ichida.

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Competing interests

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 information

Supplementary Figures & Tables

Supplementary Figures 1–17 & Supplementary Tables 1–6 (PDF 39872 kb)

Life Sciences Reporting Summary (PDF 247 kb)

Supplementary Table 7

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)

Time-lapse video of C9-ALS (C9-ALS3) iMN degeneration. Fluorescent neurons are Hb9::RFP+ iMNs. Frames were captured at 24-hour intervals. (MOV 9143 kb)

Time-lapse video of control (CTRL2) iMN degeneration. Fluorescent neurons are Hb9::RFP+ iMNs. Frames were captured at 24-hour intervals. (MOV 10366 kb)

Time-lapse video of M6PR-GFP+ vesicle trafficking in a control iMN. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The green line outlines the cell soma. (MOV 628 kb)

Time-lapse video of M6PR-GFP+ vesicle trafficking in a C9ORF72+/- (CTRL2, C9+/-) iMN. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The white line outlines the cell soma. (MOV 230 kb)

Time-lapse video of M6PR-GFP+ vesicle trafficking in a C9- ALS iMN. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The white line outlines the cell soma. (MOV 216 kb)

Time-lapse video of M6PR-GFP+ vesicle trafficking in a C9- ALS iMN expressing C9ORF72 isoform A. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The white line outlines the cell soma. (MOV 486 kb)

Time-lapse video of M6PR-GFP+ vesicle trafficking in a C9- ALS iMN expressing C9ORF72 isoform B. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The white line outlines the cell soma. (MOV 487 kb)

Time-lapse video of Gcamp6 fluorescence in control iMNs treated with glutamate. Frames were captured over a 24 second interval and the video was increased to 2x speed to facilitate viewing. (MOV 139 kb)

Time-lapse video of Gcamp6 fluorescence in C9-ALS iMNs treated with glutamate. Frames were captured over a 24 second interval and the video was increased to 2x speed to facilitate viewing. (MOV 233 kb)

Time-lapse video of Gcamp6 fluorescence in C9ORF72+/- iMNs treated with glutamate. Frames were captured over a 24 second interval and the video was increased to 2x speed to facilitate viewing. (MOV 156 kb)

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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

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