Identification of the calpain-generated toxic fragment of ataxin-3 protein provides new avenues for therapy of Machado-Joseph disease| Spinocerebellar ataxia type 3

Aims Machado-Joseph disease (MJD) is the most frequent dominantly inherited cerebellar ataxia worldwide. Expansion of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within ataxin-3, which upon proteolysis may lead to MJD. The aim of this work was to understand the in vivo contribution of calpain proteases to the pathogenesis of MJD. Therefore, we investigated (a) the calpain cleavage sites in ataxin-3 protein, (b) the most toxic ataxin-3 fragment generated by calpain cleavage and (c) whether targeting calpain cleavage sites of mutant ataxin-3 could be a therapeutic strategy for MJD. Methods We generated truncated and calpain-resistant constructs at the predicted cleavage sites of ataxin-3 using inverse PCR mutagenesis. Lentiviral vectors encoding these constructs were transduced in the adult mouse brain prior to western blot and immunohistochemical analysis 5 and 8 weeks later. Results We identified the putative calpain cleavage sites for both wild-type and mutant ataxin-3 proteins. The mutation of these sites eliminated the formation of the toxic fragments, namely, the 26-kDa fragment, the major contributor for striatal degeneration. Nonetheless, reducing the formation of both the 26- and 34-kDa fragments was required to preclude the intranuclear localisation of ataxin-3. A neuroprotective effect was observed upon mutagenesis of calpain cleavage sites within mutant ataxin-3 protein. Conclusions These findings suggest that the calpain system should be considered a target for MJD therapy. The identified calpain cleavage sites will contribute to the design of targeted drugs and genome editing systems for those specific locations.