New paper from my postdoc work at @UMichResearch is now out in @ScienceAdvances 🎉 very proud to share the culmination of nearly 4 years of hard work with a fantastic group of colleagues and collaborators. A 🧵 of what we found out about RFC1/CANVAS 👉.

Big thanks to all who contributed to getting this work out, it’s been a pleasure investigating this mysterious disease. @realToddLab @BarmadaLab @DrAnkeDijkstra @AP_Boyle.

While we don’t have all the answers about how these repeat expansions fully elicit the pathology observed in CANVAS, these data will hopefully provide some guidance for future research into the exact chain of toxicity, and hopefully one day a viable therapeutic for CANVAS.

Using reporter constructs containing upstream AluSx3 sequence with expanded AAGGG repeats, we saw selective translation of a poly-KGREG peptide from the +2 frame. Remarkably, we also observed accumulation of these same peptides in the granule cells of patient cerebellar cortices.

So we took a less RFC1 centric view of CANVAS, and began looking at the complementary DNA strand where the AAGGG repeat resides at the 3’ end of an AluSx3 element.

Sustained knockdown of RFC1 failed to recapitulate these phenotypes in control neurons, and sustained reprovision of RFC1 in CANVAS neurons did not correct these phenotypes, further suggesting a potential RFC1-independent mechanism of pathology.

This is especially apparent when watching the spontaneous firing of control (left) and CANVAS neurons (right).

These transcriptomic differences correlated with synaptic activity, where in CANVAS neurons we saw diminished network formation and synchronous firing by calcium imaging across a 12 week maturation timeline, which wasn’t observed in either of the heterozygous states.

Interestingly, this dysregulation was largely corrected when in the heterozygous state of only a single expanded allele, whether that was from CRISPR deletion of a single expanded allele or from a naturally occurring heterozygous state, showing a 3rd distinct transcriptomic state

So we took a more unbiased approach to look for any potential pathways that may be dysregulated by RNASeq of control and patient iPSC-derived neurons. Here, we saw significant downregulation of synapse associated genes at both the mRNA and protein level in CANVAS.

Likewise, we saw no evidence for any aberrant splicing of RFC1 transcripts. No stabilized lariat species, exon skipping, intron retention, or non-canonical mRNA isoforms. We also observed no formation of repeat-containing RNA foci deriving from either DNA strand.

As others have shown before, we saw no evidence for a loss of RFC1 mRNA/protein expression, atypical for a recessively inherited condition. We also saw no evidence for a loss of RFC1 function in DNA damage repair pathways in iPSC-derived neurons.

We generated iPSC lines from patients diagnosed with CANVAS possessing biallelic RFC1 AAGGG expansions, one heterozygous non-affected family member, and a heterozygous CRISPR-corrected line. We differentiated these to neurons and began investigating potential mechanisms…

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New @AP_Boyle and @realToddLab collaboration is now up on @medrxivpreprint 🥳 we used targeted long read sequencing and HMMSTR to successfully identify and genotype known or suspected large tandem repeat expansions at many loci in patient derived samples.

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Some gorgeous iPSC-derived neuronal rosette clumps with some lil baby NPCs and neurons migrating out after 3 weeks of SMAD-inhibition 🧠

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