Abstract
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.
Highlights
Calpains are a family of calcium-activated cysteine proteases that inhibit autophagy
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 Huntington’s disease (HD), as well as decreasing the number of huntingtin protein (Htt) aggregates seen in the brain
In order to extend this finding in vivo, we investigated the effect of CalpA RNA interference (RNAi) knockdown in Drosophila expressing green fluorescent protein (GFP)-tagged huntingtin exon-1 with an expanded polyglutamine repeat (Httex1-Q46-eGFP)[17] in the eye using GMR-GAL4
Summary
Calpains are a family of calcium-activated cysteine proteases (reviewed in Ono and Sorimachi7) that inhibit autophagy. 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.[14]. 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. 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
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