Calpain inhibition reduces ataxin-3 cleavage alleviating neuropathology and motor impairments in mouse models of Machado–Joseph disease

Human Molecular Genetics, 2014, Vol. 23, No. 18 doi:10.1093/hmg/ddu209 Advance Access published on May 9, 2014 4932–4944 Calpain inhibition reduces ataxin-3 cleavage alleviating neuropathology and motor impairments in mouse models of Machado– Joseph disease Ana Teresa Simões1,2, Nélio Gonçalves1,2, Rui Jorge Nobre1, Carlos Bandeira Duarte1,3 and Luı́s Pereira de Almeida1,2,∗ 1 CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3004-517, Portugal, 2Faculty of Pharmacy, University of Coimbra, Coimbra 3000-548, Portugal and 3Faculty of Sciences and Technology, Department of Life Sciences, University of Coimbra, Coimbra 3001-401, Portugal Received March 4, 2014; Revised May 1, 2014; Accepted May 2, 2014 INTRODUCTION Machado – Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is the most prevalent autosomal dominantly inherited cerebellar ataxia (1,2). The diagnosis of MJD relies on the use of molecular genetic testing to detect an abnormal CAG trinucleotide repeat expansion in the respective ATXN3 gene located on chromosome 14q32.1 and is suggested in individuals with progressive cerebellar ataxia and pyramidal signs as well as ophthalmoplegia, dystonia, action-induced facial and lingual fasciculation-like movements and bulging eyes (3,4). Current treatment is symptomatic without preventing neuronal cell death or delaying age of onset. Therefore, identification of molecular pathways of disease is crucial to unravel potential therapeutic targets. The neurotoxicity of MJD has been proposed to be ignited by a proteolytic event, a mechanism commonly designated as the toxic fragment hypothesis. Cleavage of mutant ataxin-3 into smaller toxic fragments, which are prone to aggregation and promote cellular dysfunction (5 – 7), coupled with mutant ataxin-3 localization within the cell nucleus (8,9) seem to be key issues for the induction of neurodegeneration. The aforementioned proteolytic cleavage of mutant ataxin-3 has been associated with the protease calpain activity in different models: (i) in mouse neuroblastoma cells (Neuro2a) (10), (ii) in patient—specific induced pluripotent stem cell (iPSC)derived neurons (11), (iii) by our group in the lentiviral mouse model (12) and (iv) in a MJD transgenic mouse model (13). We have previously shown that calpain-mediated proteolysis promotes (a) ataxin-3 translocation to the nucleus, (b) aggregation, (c) cell injury, (d) neurodegeneration and (e) further contribution to the depletion of the endogenous calpain-specific inhibitor calpastatin in MJD models and human tissue (12). Accordingly, knocking out calpastatin in a MJD transgenic ∗ To whom correspondence address should be addressed at: CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal, Tel: +351 966337482; Fax: +351 239853409; Email: ; # The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: Machado – Joseph Disease (MJD) is the most prevalent autosomal dominantly inherited cerebellar ataxia. It is caused by an expanded CAG repeat in the ATXN3 gene, which translates into a polyglutamine tract within the ataxin-3 protein. Present treatments are symptomatic and do not prevent disease progression. As calpain overactivation has been shown to contribute to mutant ataxin-3 proteolysis, translocation to the nucleus, inclusions formation and neurodegeneration, we investigated the potential role of calpain inhibition as a therapeutic strategy to alleviate MJD pathology. For this purpose, we administered orally the calpain inhibitor BDA-410 to a lentiviral mouse model of MJD. Western-blot and immunohistochemical analysis revealed the presence of N- and C-terminal mutant ataxin-3 fragments and the colocalization of large inclusions with cleaved caspase-3 in the mice brain. Oral administration of the calpain inhibitor BDA-410 decreased both fragments formation and full-length ataxin-3 levels, reduced aggregation of mutant ataxin-3 and prevented cell injury and striatal and cerebellar degeneration. Importantly, in correlation with the preserved cerebellar morphology, BDA-410 prevented motor behavioural deficits. In conclusion, BDA-410 alleviates Machado – Joseph neuropathology and may therefore be an effective therapeutic option for MJD. Human Molecular Genetics, 2014, Vol. 23, No. 18 RESULTS BDA-410 inhibits calpain activity and decreases mutant ataxin-3 levels in vitro Several evidences indicate that calpains cleave ataxin-3 producing proteolytic fragments that trigger MJD pathology (10–13), suggesting that calpain inhibition may provide an effective therapy for MJD. Therefore, we first investigated whether calpains could be inhibited by the novel oral calpain inhibitor BDA-410 (Fig. 1A) (23–26), in cultures of cerebellar granule neurons. When calcium concentration increases in the intracellular compartment, a subset of axonal structural proteins is vulnerable to calpain activity, including aII-spectrin, a potential biomarker for neuronal cell injury (27). Interestingly, levels of the calpaingenerated 150/145 kDa fragments of aII-spectrin were further increased when cerebellar granule neurons were transduced with lentiviral vectors encoding the mutant ataxin-3 (ATX-3 72Q) in opposition to wild-type ataxin-3 (ATX-3 27Q) and noninfected cultures (Ø). Importantly, BDA-410 (50 and 100 nM) mediated a decrease of the aII-spectrin fragments in cultures expressing mutant ataxin-3 (Fig. 1B and D). Furthermore, this calpain inhibition translated into a significant 21% decrease in immunolabeling of full-length mutant ataxin-3 (Fig. 1C and E). Since the concentration of BDA-410 used in this experiment was very low (50 and 100 nM), much below the required to inactivate other proteases than calpains (typically in the mM range; see Materials and Methods for IC50 information), we conclude that the decreased levels of mutant ataxin-3 are due to a specific calpain inhibitory action. As a consequence, a reduced calpainmediated cleavage of ataxin-3 might prevent the full-length protein and its fragments from escaping the cytoplasmic quality control mechanisms. BDA-410 reduces cleavage of mutant ataxin-3 in a lentiviral mouse model of MJD Therefore, in order to investigate whether orally administered BDA-410 would inhibit ataxin-3 cleavage in vivo, we daily administered the compound BDA-410 (30 mg/kg in 1% Tween80 saline in a volume equal to 5 ml/kg) by oral gavage to a lentiviral mouse model, wherein lentiviral vectors encoding for wild-type ataxin-3 (ATX-3 27Q) were injected in the left striatum hemisphere and mutant ataxin-3 (ATX-3 72Q) in the right hemisphere, a strategy that we use to generate models of disease (12,19,28 – 30). As a control, a group of animals received only the vehicle in which BDA-410 was resuspended for the treated group. After 4 weeks of treatment, mice were sacrificed and striatal tissue process (...truncated)