Length-dependent RNA foci formation and RAN translation are associated with SCA12 disorder

Abstract

Spinocerebellar ataxia type-12 (SCA-12) is a neurodegenerative disease that exhibits a unique progressive tremor/ataxia syndrome caused by a triplet (CAG) repeat expansion in the 5' UTR region of PPP2R2B. So far, no study has been done to investigate the pathological hallmarks using the appropriate disease model. Therefore, we aimed to establish human iPSC-derived SCA12 neuronal cell lines to study the cellular pathological mechanisms induced by CAG expansion. We found that expanded CAG in PPP2R2B transcript causes the formation of RNA foci inside the nucleus of the patient-derived neural stem cells, which in turn may sequester many nuclear proteins, and inhibit their necessary functions. The ectopic expression of this expanded CAG transcript from PPP2R2B accelerates non-canonical Repeat Associated Non-AUG (RAN) translation in multiple frames in HEK293T cells, which was further validated in patient-derived neural stem cells using novel antibodies. Next, an RNA-pull down assay followed by mass-spectrometric-based protein detection analysis of patient and control-derived neural stem cells identified crucial proteins involved in protein homeostasis, vesicular trafficking, ion channels signaling, etc. shedding light on the mechanistic relationship between RAN translation and RNA foci, and their relative contributions to cellular dysfunction. We also performed the transcriptome sequencing of the diseased and control neurons which identified a network of crucial transcription factors affecting neural fate, in addition to alteration of various signaling pathways affecting neuronal and nervous system development and function. Altogether, this study identifies the molecular signatures of spinocerebellar ataxia type-12 disorder using patient-derived neuronal cell lines, wherein RNA foci and RAN protein accumulation impact the functioning of crucial intracellular pathways. Furthermore, overlapping of these pathways in the same cell lines demonstrates the critical partaking of two modifiers in disease progression emphasizing the new therapeutic strategies that target both processes in repeat expansion disorders.