Targeting RNA Foci in iPSC-Derived Motor Neurons from ALS Patients with a C9ORF72 Repeat Expansion

Abstract

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. Here, we report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS motor neurons. Repeat-containing RNA foci co-localized with hnRNPA1 and Pur-α, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides (ASOs) targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS.

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…A similar pattern was observed in a mouse model of the motor neuron disease spinal muscular atrophy (SMA) [73], suggesting either that these differences might be a secondary phenomenon observed in association with neurodegeneration or that disrupted cell adhesion represents a conserved neurotoxic pathway in ALS and SMA. In support of the latter hypothesis, Sareen et al [66] also noted an enrichment of differentially expressed cellular adhesion genes in iPSC-derived neurons from patients with C9orf72 expansions.…”

“…In addition, C9orf72 knockdown was not associated with adverse effects or neuronal loss in mice receiving ASOs [67]. Knockdown of the mutant C9orf72 allele also restored expression for several affected genes in C9orf72-mutant iPSC-derived neurons [66,68], indicating that this therapeutic strategy may prove effective and safe in humans with disease.…”

“…Pathogenic hexanucleotide expansion mutations in C9orf72 are associated with distinct changes in gene expression that only partially overlap with those induced by TDP43 or FUS dysregulation [66][67][68][69]. Human iPSCs carrying disease-associated C9orf72 expansions exhibited prominent downregulation of several essential genes [68], including those involved in synaptic transmission, cell adhesion, and membrane excitability.…”

“…Importantly, this effect was strikingly muted in mutant C9orf72 fibroblasts [67], suggesting that many of the differentially expressed genes are unique to neurons. The C9orf72 protein is ubiquitously expressed but highly enriched in neurons [66,68,70], with a proposed function in endosomal trafficking [71]. However, targeted knockdown C9orf72 failed to recapitulate the same pattern of gene expression noted in fibroblasts from ALS patient cells carrying C9orf72 mutations, implying that a simple loss of C9orf72 function is unlikely to explain the observed pattern of RNA dysregulation in C9orf72 mutant cells [67].…”

“…1). Many of the proteins bound by G 4 C 2 repeats are required for normal RNA processing, including nucleolin, ADAR, hnRNP A1, hnRNP H, SC35, and serine/ arginine-rich splicing factor 1 and serine/arginine-rich splicing factor 2 [66,68,119,121,122], suggesting that neuronal toxicity in ALS might arise from a functional depletion of these essential factors. If so, then blocking the repeat domain might prevent downstream toxicity.…”