Calpain inhibition mediates autophagy-dependent protection against polyglutamine toxicity

Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington''s disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington''s disease.Huntington''s disease (HD) is a currently incurable, autosomal dominant neurodegenerative disease resulting from the expansion of the trinucleotide (CAG) repeat region of the huntingtin (HTT) IT15 gene, encoding huntingtin protein (Htt). In mutant Htt, the polyglutamine tract encoded by this region contains over 35 glutamines and the length of the tract correlates inversely with the age of disease onset, with longer tracts resulting in earlier onset (reviewed in Imarisio et al.¹). HD is one of the 10 trinucleotide repeat disorders resulting from expansions of polyglutamine tracts in different proteins. These expansions cause disease by conferring toxic gain-of-function properties onto the mutant proteins. Hence, one strategy that has been considered for HD and related diseases is to find ways of decreasing the levels of the mutant protein, for instance by harnessing the cell''s capacity to degrade such aggregate-prone proteins via (macro)autophagy.^{2, 3, 4, 5} Autophagy involves the engulfment of cytoplasmic contents by double-membraned autophagosomes, which then traffic to lysosomes where their contents are degraded. Mutant huntingtin, some other polyglutamine expanded proteins like mutant ataxin 3, and proteins like tau (which mediates toxicity in Alzheimer''s disease and related dementias) are autophagy substrates and their clearance can be enhanced in Drosophila and mouse models by autophagy upregulation, which also reduces their toxicity.^{2, 3, 4,6}Calpains are a family of calcium-activated cysteine proteases (reviewed in Ono and Sorimachi⁷) that inhibit autophagy. Strategies that reduce calpain activity in cell culture increase autophagy and decrease levels of autophagy substrates, like mutant Htt. These effects are likely to be mediated by Gsα, a heterotrimeric G-protein subunit which is activated by calpain cleavage. Similar to calpain inhibition, siRNA knockdown of Gsα, or chemical inhibition by NF449, induces autophagy and decreases the number of aggregates resulting from the overexpression of exon-1 Htt with an expanded polyglutamine repeat region (HttQ74) in cell culture models.⁸ In addition to this mechanism of autophagy upregulation by calpains, the core autophagy protein ATG5 has also been demonstrated to be cleaved and inactivated by calpains,^9,10 suggesting that calpains may act on a number of substrates to negatively regulate autophagy.In mammals, the two most abundantly expressed calpains are μ-calpain and m-calpain, which differ in their affinity for calcium and therefore the calcium concentration required for their activation. As well as being regulated by calcium, they are also controlled by an endogenous inhibitor, calpastatin (CAST). Drosophila have four forms of calpain:¹¹ CalpA and CalpB are the conventional calpains formed by a recent duplication in the Drosophila insect lineage, CalpC is also an evolutionarily recent, but not highly conserved duplication (data not shown) and is thought to be catalytically inactive,¹¹ and CalpD (SOL) is a member of the unconventional family of calpains. Drosophila does not appear to have any obvious orthologs of CAST.A role for calpains in HD has been investigated previously. Following observations that shorter Htt fragments are more toxic than full-length Htt,¹² it was demonstrated that Htt can be cleaved by both caspases¹³ and calpains¹⁴ to generate these toxic, short fragments. Blocking Htt cleavage by calpains by mutating their calpain cleavage sites decreases Htt aggregation and toxicity.¹⁵ In addition, calpain activation has been shown to be increased in HD patients compared with controls.¹⁴In this study, we have investigated a role for calpain activity as a modulator of autophagy in both Drosophila and mouse models of HD. To avoid confounding effects from alterations in cleavage of Htt by calpain, we have used models expressing short fragments of Htt, which do not contain calpain cleavage sites and correspond to the shortest fragments of huntingtin seen in patients.¹⁶ We demonstrate that knockdown of CalpA in Drosophila is sufficient to both reduce the number of Htt aggregates and the toxicity associated with the expression of the mutant protein. Importantly, we show that these effects are autophagy-dependent. Furthermore, we show that overexpression of CAST in mice results in enhanced autophagy and improves locomotor function and delays tremor onset in a mouse model of HD, as well as decreasing the number of Htt aggregates seen in the brain. We extended the analysis of CAST overexpressing mice to investigate the possible adverse effects from long-term calpain inhibition or autophagy upregulation but did not observe any obvious deleterious effects.