An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC‐derived motor neurons, decreased cell survival is correlated with dysfunction in Ca2+ homeostasis, reduced levels of the antiapoptotic protein Bcl‐2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC‐derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063–2078

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

Mislocalization of the predominantly nuclear RNA/DNA binding protein, TDP-43, occurs in motor neurons of ~95% of amyotrophic lateral sclerosis (ALS) patients, but the contribution of axonal TDP-43 to this neurodegenerative disease is unclear. Here, we show TDP-43 accumulation in intra-muscular nerves from ALS patients and in axons of human iPSC-derived motor neurons of ALS patient, as well as in motor neurons and neuromuscular junctions (NMJs) of a TDP-43 mislocalization mouse model. In axons, TDP-43 is hyper-phosphorylated and promotes G3BP1-positive ribonucleoprotein (RNP) condensate assembly, consequently inhibiting local protein synthesis in distal axons and NMJs. Specifically, the axonal and synaptic levels of nuclear-encoded mitochondrial proteins are reduced. Clearance of axonal TDP-43 or dissociation of G3BP1 condensates restored local translation and resolved TDP-43-derived toxicity in both axons and NMJs. These findings support an axonal gain of function of TDP-43 in ALS, which can be targeted for therapeutic development.

TDP-43 dysfunction is common to 97% of amyotrophic lateral sclerosis (ALS) cases, including those with mutations in C9orf72. To investigate how C9ORF72 mutations drive cellular pathology in ALS and to identify convergent mechanisms between C9ORF72 and TARDBP mutations, we analyzed motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs) from patients with ALS. C9ORF72 iPSC-MNs have higher Ca 2+ release after depolarization, delayed recovery to baseline after glutamate stimulation, and lower levels of calbindin compared with CRISPR/Cas9 genome-edited controls. TARDBP iPS-derived MNs show high glutamate-induced Ca 2+ release. We identify here, by RNA sequencing, that both C9ORF72 and TARDBP iPSC-MNs have upregulation of Ca 2+ -permeable AMPA and NMDA subunits and impairment of mitochondrial Ca 2+ buffering due to an imbalance of MICU1 and MICU2 on the mitochondrial Ca 2+ uniporter, indicating that impaired mitochondrial Ca 2+ uptake contributes to glutamate excitotoxicity and is a shared feature of MNs with C9ORF72 or TARDBP mutations.