Fly strains used in this study.

Spinocerebellar ataxia type 36 (SCA36) is a neurodegenerative disease caused by expanded (GGCCTG)n hexanucleotide repeat sequence in the NOP56 gene. While the expanded repeats could transcribe and form toxic RNA foci within neurons, recent evidence indicates that translation of these repeats produces dipeptide repeats (DPR) that contribute to neurotoxicity. The relative impact of hexanucleotide RNA repeats (HRR) and DPR on the neurodegeneration of SCA36 remains unclear. Here, we established a Drosophila SCA36 model to dissect the neurotoxic effects of HRR and DPR. The fly model recapitulates the cellular defects observed in SCA36 patient fibroblasts, validating its relevance for mechanistic study of SCA36. Further engineering the transgenes to express individual DPRs reveal Proline-Glycine-DPR (PG-DPR) as the most potent neurotoxin causing progressive motor and sensory dysfunction. Expressing a series of the SCA36 transgenes with varying HRR lengths demonstrates an age- and length-dependent adult-onset neurodegeneration. Interestingly, sequence modification of the transgenes to exclusively express HRR or DPR alone causes a milder phenotype, indicating both HRR and DPR contribute partially to the pathogenicity of SCA36. Therefore, this model provides a valuable platform for screening drug targeting either HRR- or DPR-mediated toxicity of SCA36. Suppression of the RNA elongation factor SUPT4H1 ortholog reduces RNA foci in cell culture. However, expression level of SUPT4H1 was not changed in SCA36 patient cells. Interestingly, knockdown of the Drosophila SUPT4H1 ortholog or 6-azauridine treatment to suppress RNA transcription aggravates the neurodegenerative phenotypes in both the fly models and patient-derived fibroblasts, highlighting the complex interplay of pathomechanisms in SCA36. These results underscore the need for carefully evaluating the potential side effects when designing therapeutic interventions for SCA36.