A23 Calpastatin ablation aggravates the molecular phenotype in cell and animal models of huntington disease

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Abstract

Background

Deciphering the molecular pathology of Huntington disease is of particular importance, not only for a better understanding of this neurodegenerative disease, but also to identify potential therapeutic targets. The disease protein huntingtin was shown to undergo proteolysis, resulting in the accumulation of toxic and aggregation-prone fragments. Amongst several classes of proteolytic enzymes responsible for huntingtin cleavage, the group of calcium-activated calpains has been found to significantly mediate disease protein toxicity.

Aims

To confirm the impact of calpain-mediated huntingtin cleavage in Huntington disease, we analyzed effects of depleting or overexpressing the endogenous calpain inhibitor calpastatin in HEK293T cells transfected with wild-type or mutant huntingtin. Moreover, we crossbred two Huntington disease mouse models with calpastatin knockout mice to assess its effect not only on huntingtin cleavage and aggregation but also additional molecular markers.

Methods

Effects of depleting the endogenous inhibitor of calpains on huntingtin fragmentation and aggregation, as well as on other molecular markers of Huntington disease were investigated via western blotting, filter retardation assays and immunocytochemistry or immunohistochemistry.

Results

We demonstrated that reduced or ablated calpastatin expression triggers calpain overactivation and a consequently increased mutant huntingtin cleavage in cells and in vivo. These alterations were accompanied by an elevated formation of predominantly cytoplasmic huntingtin aggregates. Conversely, calpastatin overexpression in cells attenuated huntingtin fragmentation and aggregation. In addition, we observed an enhanced cleavage of DARPP-32, p35 and synapsin-1 in neuronal tissue upon calpain overactivation.

Conclusions

Our results corroborate the important role of calpains in the molecular pathogenesis of Huntington disease and endorse targeting these proteolytic enzymes as a therapeutic approach.

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