AAGGG repeat expansions trigger repeat-dependent synaptic dysregulation in human CANVAS Neurons [RFC1_Addition]

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a late onset, recessively inherited neurodegenerative disorder caused by a biallelic, non-reference pentameric CCCTT(AAGGG) repeat expansion within the second intron of replication factor complex subunit 1 (RFC1). While rare compound heterozygous CANVAS cases harbor RFC1 loss-of-function mutations alongside monoallelic repeat-expansions, RFC1 expression is maintained at normal levels in the presence of biallelic expanded repeats. To investigate how these pentameric repeats cause disease, we generated CANVAS patient iPSC derived neurons and utilized calcium imaging and transcriptomic analysis to define repeat elicited gain-of-function and loss-of-function contributions to neuronal toxicity. AAGGG repeat expansions do not alter neuronal RFC1 splicing, expression, or DNA repair pathway functions. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins that selectively accumulate in patient brains. However, neither these proteins nor repeat RNA foci were detected in iNeurons and overexpression of these repeats in isolation did not induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded allele. Importantly, these deficits were not replicated by knockdown of RFC1 in control neurons and showed incomplete or no rescue upon over-expression of RFC1. These findings support a repeat-dependent and RFC1-independent mechanism as the underlying cause of neurodegeneration in CANVAS, with important implications for therapeutic development in this currently untreatable condition. Overall design: To assess global dysregulation of gene expression in CANVAS, we generated multiple Control, CANVAS, and Isogenic corrected patient iPSC lines. These were differentiated to forebrain glutamatergic neurons by modified Dual-SMAD inhibition protocol. Neurons were aged to 10-weeks in vitro and were analyzed by paired-end RNASequencing of Control+GFP (n=3), Control+RFC1 (n=3), CANVAS+GFP (n=3), and CANVAS+RFC1 (n=3) iPSC-derived neurons. Neurons were treated with the respective lentivirus particles at DIV5 and continued expressing the integrated shRNA's throughout the 10-week differentiation before analysis. RNASeq data were analyzed for differential expression, differential exon usage, and circRNA expression.