AAGGG repeat expansions trigger <i>RFC1</i>-independent synaptic dysregulation in human CANVAS neurons.

Abstract

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recessively inherited neurodegenerative disorder caused by intronic biallelic, nonreference CCCTT/AAGGG repeat expansions within <i>RFC1</i>. To investigate how these repeats cause disease, we generated patient induced pluripotent stem cell-derived neurons (iNeurons). CCCTT/AAGGG repeat expansions do not alter neuronal <i>RFC1</i> splicing, expression, or DNA repair pathway function. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins. However, these proteins and repeat RNA foci were not detected in iNeurons, and overexpression of these repeats failed to induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded AAGGG allele. These deficits were neither replicated by <i>RFC1</i> knockdown in control iNeurons nor rescued by RFC1 reprovision in CANVAS iNeurons. These findings support a repeat-dependent but RFC1 protein-independent cause of neuronal dysfunction in CANVAS, with implications for therapeutic development in this currently untreatable condition.