Single-Cell RNA Sequencing Reveals Impaired CHIP-Mediated Heat Stress Response in SCA3 Pathogenesis

Nine distinct polyglutamine (PolyQ) diseases are caused by CAG repeat expansions in the coding regions of their respective causative genes, among which spinocerebellar ataxia type 3 (SCA3), driven by CAG repeat expansion in the ATXN3 gene, is one of the most common subtypes. SCA3 is characterized by the aggregation of mutant Ataxin-3 (containing an expanded polyglutamine tract, encoded by ATXN3) into neuronal intranuclear inclusions, leading to progressive neurodegeneration. Despite extensive research, the precise molecular mechanisms underlying mutant Ataxin-3-induced neurotoxicity remain elusive, and there is still a lack of effective therapeutic strategies for SCA3. To address these gaps, the present study aimed to elucidate the key pathophysiological cascades driving SCA3 progression and identify potential therapeutic targets by investigating cellular and molecular alterations in SCA3 models. Our results showed that SCA3 cells exhibited significantly reduced viability and increased thermolability. In SCA3 transgenic mice, a large portion of the C terminus of Hsc70-interacting protein (CHIP) was sequestered within neuronal intranuclear inclusions, resulting in a progressive, age-dependent decline in soluble CHIP level. Single-Cell RNA Sequencing (ScRNA-seq) analysis of the cerebellum from these mice revealed dysregulated cellular stress response in SCA3. Native gel electrophoresis revealed that the level of trimerized HSF1 in SCA3 cells was significantly lower than that in wild-type cells, indicating that stress intolerance may be involved in the pathogenesis of SCA3. This further validated and specified results of ScRNA-Seq. Importantly, overexpression of CHIP partially restored HSF1 function, rescued DNAJB1 (homolog of HSP40) and the defective heat stress response, and ameliorated multiple disease-related phenotypes in SCA3 models. This further reveals that the reduction in functional CHIP caused by Ataxin3 mutation may represent an upstream event responsible for stress intolerance in SCA3. Collectively, our findings demonstrate that depletion of soluble CHIP promotes SCA3 progression by disrupting the CHIP-HSF1/DNAJB1 axis. This work not only clarifies a critical pathogenic mechanism of SCA3 but also underscores the therapeutic potential of modulating CHIP activity as a novel intervention strategy for SCA3. These results provide a clearer contextual framework for understanding SCA3 pathogenesis and lay a foundation for the development of targeted therapies.