CHN-1 ubiquitylates hTBP43Q and mediates its proteasomal degradation by collaborating with HSP-1 and UFD-2

Spinocerebellar ataxia type 17 (SCA17) is a neurodegenerative disease caused by poly-glutamine (polyQ) expansion mutation in the TATA-box binding protein (TBP) gene. While ≥47Q are fully-penetrant pathogenic repeats, those between 41 and 46 show reduced penetrance, thus called intermediate alleles. These latter lead to the digenic form of SCA17 (SCA17-DI) only when a concurrent heterozygous pathogenic variant in STUB1 is present. STUB1 encodes the C-terminus of HSP70 interacting protein (CHIP), an E3-ubiquitin ligase with co-chaperone activity, which acts as modifier in several neurodegenerative diseases. To unravel the interplay between TBP and CHIP, we modelled SCA17 in Caenorhabditis elegans by panneuronally expressing an extrachromosomal transgene carrying the cDNA of human TBP with different polyQ lengths: 38 (wild-type, hTBP38Q), 43 (intermediate, hTBP43Q), and 54 (fully-penetrant, hTBP54Q). Only the expression of hTBP54Q compromised animals’ response to gentle touch. Although this response is mediated by several neuronal classes, we identified that the observed alteration is driven by defects occurring primarily in the upstream mechanosensory neurons. Interestingly, while hTBP43Q alone was not sufficient to cause a similar defect, its combination with the knockout of chn-1, worm’s ortholog of STUB1, led to dysfunction of mechanosensory neurons, thus phenocopying SCA17-DI. To corroborate these findings, we analysed this phenotype in single-copy integrated models. These showed a similar defective pattern observed in animals carrying the extrachromosomal transgene, confirming that the functional alteration is mutation-driven rather than a copy-number artefact. In SCA17-DI models, the expression of wild-type hCHIP fully rescued the functional defects. However, hCHIP, carrying mutations associated with SCA17-DI, occurring in the two functional domains of the protein, failed to provide similar results. Both in C. elegans and humans, CHIP/CHN-1 is involved in protein degradation through different pathways. We identified that CHIP degrades hTBP43Q specifically by proteasomal route. To exert its functions, CHN-1 collaborates with several proteins. Among them, two well-known interactors are the molecular chaperone HSP-1 and the E4-ubiquitin ligase UFD-2. In hTBP43Q animals, mutations in these genes led to defects in response to gentle touch similar to what observed in SCA17-DI animals. This indicates that both proteins are involved in hTBP43Q degradation. Regarding the ubiquitination process, we identified that, among the fifteen lysine residues present in hTBP43Q, CHN-1 specifically ubiquitinates Lys195. Overall, we have unveiled the specific degradation pathway of hTBP43Q. CHN-1 mediates the ubiquitination at Lys195, and subsequent proteasomal degradation, of hTBP43Q collaborating with HSP-1 and UFD-2. The modification of one of these components leads to mechanosensory neurons’ alteration, resulting in a defective response to gentle touch. Taking advantage of nematodes’ characteristics and of the generated integrated models, we characterized animals’ behaviour using an automated tracker. SCA17 and SCA17-DI animals showed recognizable behavioural alterations compared to wild-type worms. We are currently using these features to conduct a high-throughput screening for drug-repurposing, which will, hopefully, lead to the identification of novel therapeutic molecules. Fond. Cariplo 2021-1544 to SM.