Research Projects

Spinocerebellar ataxia type 12 (SCA12) is a rare, autosomal dominant neurodegenerative disorder caused by expanded CAG repeats in the 5′ UTR of the PPP2R2B gene. Unlike classic polyglutamine diseases, SCA12 pathology is primarily driven by RNA-mediated toxicity, where the expanded CAG repeat RNA (eCAGr) forms nuclear RNA foci that sequester essential RNA-binding proteins (RBPs), disrupt RNA processing, and impair neuronal function. Recent findings show eCAGr RNA undergoes liquid–liquid phase separation (LLPS), forming nuclear condensates with unique biophysical properties, which may mature into gel-like states, exacerbating RBP sequestration and neurodegeneration. Additionally, eCAGr RNA can serve as a template for repeat-associated non-AUG (RAN) translation, producing toxic proteins. Rationale: Current understanding of the structure-function relationship of eCAGr RNA and its interaction with RBPs in SCA12 is limited. Targeting these pathogenic RNA structures and their phase behavior with small molecules represents a novel therapeutic strategy. Cyclic mismatch binding ligands (CMBLs), developed to bind nucleic acid repeats, have shown promise in modulating RNA structure and function in related disorders. Objectives: 1. To examine the impact of CMBL binding on the assembly, dynamics, and material properties of eCAGr RNA condensates in vitro and in SCA12 cellular models, focusing on the role of key RBPs. 2. To evaluate the inhibitory effect of CMBLs on RAN translation and the reduction of toxic protein aggregates in SCA12 models. 3. To assess whether CMBL treatment restores normal RNA metabolism, gene expression, and neuronal phenotypes in SCA12 patient-derived neurons. Hypothesis: CMBLs, by binding specifically to expanded CAG repeats in PPP2R2B RNA, will disrupt toxic RNA foci, release sequestered RBPs, inhibit RAN translation, and restore normal RNA metabolism and neuronal function in SCA12 cellular models. Experiments: In vitro characterization: EMSA, SPR, and thermal melting assays to confirm CMBL binding; SHAPE-MaP to analyze RNA structural changes. Phase separation studies: Reconstitution of eCAGr RNA condensates and assessment of CMBL effects on phase separation, gelation, and condensate heterogeneity using advanced microscopy and FRAP. Cellular studies: Treatment of patient-derived neurons with CMBLs, assessment of RNA foci (RNA-FISH), RBP sequestration (IHC), RAN translation products (antibodies), transcriptomic changes (RNA-seq), and neuronal health (viability, morphology, synaptic markers). Significance: Achieving these objectives will provide the first comprehensive proof-of-concept for targeting the physical and functional properties of pathogenic CAG repeat RNA condensates and RAN translation using synthetic small molecules in SCA12. This dual-action approach may fundamentally advance our understanding of RNA-mediated neurodegeneration and open new therapeutic avenues for SCA12 and related trinucleotide repeat expansion disorders.