AAGGG repeat expansions trigger RFC1-independent synaptic dysregulation in human CANVAS Neurons

Abstract

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a late onset, recessively inherited neurodegenerative disorder caused by biallelic, non-reference pentameric AAGGG(CCCTT) repeat expansions within the second intron of replication factor complex subunit 1 (RFC1). To investigate how these repeats cause disease, we generated CANVAS patient induced pluripotent stem cell (iPSC) derived neurons (iNeurons) 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 CANVAS 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. These phenotypic deficits were not replicated by knockdown of RFC1 in control neurons and were not rescued by ectopic expression of RFC1. These findings support a repeat-dependent but RFC1-independent cause of neuronal dysfunction in CANVAS, with important implications for therapeutic development in this currently untreatable condition.SummaryHuman CANVAS neurons exhibit transcriptional and functional synaptic defects that are corrected by heterozygous repeat deletion but are independent of the gene within which they reside—RFC1.

Structural polymorphism of the nucleic acids in pentanucleotide repeats associated with the neurological disorder CANVAS

“…A recent study reported that pathogenic AAGGG repeat forms a triplex in vitro , and stalls replication occurring only when AAGGG serves as the template strand ( 57 ), indicating non-B DNA-forming potential of the pathogenic repeat in RFC1 during DNA replication results in fork stalling. However, no reductions in RFC1 mRNA and protein levels have been observed in peripheral tissues, postmortem brain samples, and induced pluripotent stem cell–derived neurons from patients with biallelic (AAGGG) exp in the RFC1 ( 8 , 58 ). In the aspect of GOF hypotheses, RNA foci were not detected in patient brains in the initial analysis ( 8 ), but in a recent report, CCTGT- and CCCTT-containing RNA foci were identified in the neuronal nuclei of patients with CANVAS harboring biallelic (ACAGG) exp and (AAGGG) exp , respectively ( 59 ).…”

“…In the aspect of GOF hypotheses, RNA foci were not detected in patient brains in the initial analysis ( 8 ), but in a recent report, CCTGT- and CCCTT-containing RNA foci were identified in the neuronal nuclei of patients with CANVAS harboring biallelic (ACAGG) exp and (AAGGG) exp , respectively ( 59 ). Both AAGGG repeat-RNA foci and CCCTT repeat-RNA foci were detected in control and CANVAS patient induced pluripotent stem cell–derived neurons, but the overall abundance of foci was low and the specificity was incomplete ( 58 ). In RAN translation, AAGGG repeat–derived KGREG peptide products were selectively detected within cerebellar granule cells in CANVAS patients, with no signal detected in controls ( 58 ).…”

“…Both AAGGG repeat-RNA foci and CCCTT repeat-RNA foci were detected in control and CANVAS patient induced pluripotent stem cell–derived neurons, but the overall abundance of foci was low and the specificity was incomplete ( 58 ). In RAN translation, AAGGG repeat–derived KGREG peptide products were selectively detected within cerebellar granule cells in CANVAS patients, with no signal detected in controls ( 58 ). In the relationship between the nucleic acid conformation formed by pathogenic repeats and CANVAS pathology, lower motor neurons are more susceptible in patients exhibiting (ACAGG) exp than (AAGGG) exp ( 60 ), indicating that lower motor neurons may be more susceptible to ACAGG-forming hairpin structure than AAGGG-forming G4.…”

RFC1regulates the expansion of neural progenitors in the developing zebrafish cerebellum

“…Recent studies propose that RFC1 loss-of-function could be at the basis of the repeat expansion pathogenicity 20-23 , as the recessive inheritance pattern also suggests. However, other studies showed no specific neurodegenerative phenotype in RFC1 -depleted neuronal cultures in vitro 24 . This questions the direct involvement of the RFC1 gene in so-called RFC1 -related pathologies.…”

Enhanced Detection and Genotyping of Disease-Associated Tandem Repeats Using HMMSTR and Targeted Long-Read Sequencing

“…Cells were regularly passed and the media replenished every 3 days. Dermal fibroblasts were obtained from consenting patients clinically diagnosed with CANVAS spectrum disorder and genetically confirmed to possess biallelic RFC1 expansions and iPSCs were cultured as described in Maltby et al (35). ALS patient fibroblasts originated from a 66 yo male Michigan Medicine patient and obtained as skin biopsy punch under local IRB approval (HUM00030934).…”