Calpain-mediated ataxin-3 cleavage in the molecular pathogenesis of spinocerebellar ataxia type 3 (SCA3)

Abstract

Spinocerebellar ataxia type 3 (SCA3) is pathologically characterized by the formation of intranuclear aggregates which contain ataxin-3, the mutated protein in SCA3, in a specific subtype of neurons. It has been proposed that ataxin-3 is cleaved by proteolytic enzymes, in particular by calpains and caspases, eventually leading to the formation of aggregates. In our study, we examined the ability of calpains to cleave ataxin-3 in vitro and in vivo. We demonstrated in cell culture and mouse brain homogenates that cleavage of overexpressed ataxin-3 by calpains and in particular by calpain-2 occur and that polyglutamine expanded ataxin-3 is more sensitive to calpain degradation. Based on these results, we investigated the influence of calpains on the pathogenesis of SCA3 in vivo. For this purpose, we enhanced calpain activity in a SCA3 transgenic mouse model by knocking out the endogenous calpain inhibitor calpastatin. Double-mutant mice demonstrated an aggravated neurological phenotype with an increased number of nuclear aggregates and accelerated neurodegeneration in the cerebellum. This study confirms the critical importance of calcium-dependent calpain-type proteases in the pathogenesis of SCA3 and suggests that the manipulation of the ataxin-3 cleavage pathway and the regulation of intracellular calcium homeostasis may represent novel targets for therapeutic intervention in SCA3.

Defects in the CAPN1 Gene Result in Alterations in Cerebellar Development and Cerebellar Ataxia in Mice and Humans

“…Thus, mutation in βIII spectrin in both human and mouse caused increased calpain proteolysis of αII spectrin, which results in ataxic and seizure phenotypes (Stankewich et al, 2010). In addition, enhanced calpain activity aggravated pathogenesis of spinocerebellar ataxia type 3 (SCA3) (Hubener et al, 2013). Together with our findings, those studies suggest that unbalanced calpain activity, either too little or too much, can result in cerebellar dysfunction and ataxia.…”

Calpain 5 Is Highly Expressed in the Central Nervous System (CNS), Carries Dual Nuclear Localization Signals, and Is Associated with Nuclear Promyelocytic Leukemia Protein Bodies

“…Of related interest is that PML associates with nuclear aggregates in several neurodegenerative disorders, particularly polyglutamine disorders including spinocerebellar ataxia, Huntington disease, and dentatorubral-pallidoluysian atrophy as well as amyotrophic lateral sclerosis (69 -76). Several of the aggregated proteins have been demonstrated to be calpain substrates (23,(77)(78)(79)(80), with calpain inhibition attenuating the nuclear aggregation (23,78,81).…”

Toward therapeutic targets for SCA3: Insight into the role of Machado–Joseph disease protein ataxin-3 in misfolded proteins clearance

“…Another hypothesis is that cleavage fragments from ataxin-3 form aggregates that are more toxic in nature than aggregates of fulllength proteins. Both the normal ataxin-3 and the polyQ-expanded ataxin-3 could ostensibly be cleaved by proteases, like caspase, and calpain, thus producing a series of fragments dependent on the locations of the cleavage sites Haacke et al, 2007;Hubener et al, 2011Hubener et al, , 2013Jung et al, 2009;Koch et al, 2011;Liman et al, 2014;Pozzi et al, 2008;Simoes et al, 2012Simoes et al, , 2014. Although full-length ataxin-3 does not readily aggregate on its own, it is susceptible to co-aggregation with polyQ-expanded ataxin-3 fragments.…”

“…Interestingly, interaction with soluble polyQelongated fragments causes a structural distortion of wild-type ataxin-3 prior to the formation of stable co-aggregates . Compared to normal ataxin-3, polyglutamineexpanded ataxin-3 is more sensitive to calpain degradation (Hubener et al, 2013). All the resulting fragments, especially the polyQ-containing fragments, have proven to be highly toxic to cells.…”

“…In contrast, inhibition of calpain by inhibitors like calpastatin and calpeptin, or downregulation of calpain by genetic knockdown, induces autophagy and thus enhances the clearance of these aggregate-prone proteins. Correspondingly, enhancing calpain activity by knocking out the endogenous calpain inhibitor calpastatin in a SCA3 transgenic mouse model suggested an aggravated neurological phenotype including increased numbers of aggregates and accelerated neurodegeneration in the cerebellum (Hubener et al, 2013). On the contrary, a novel calpain inhibitor BDA-410, a low-molecular weight drug, which when orally administered in a mouse model of SCA3, reduces the aggregation of mutant ataxin-3 and prevents cell injury and striatal and cerebellar degeneration.…”