The Role of Calpains in the Molecular Pathogenesis of Spinocerebellar Ataxia Type 17

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URI: http://hdl.handle.net/10900/146435
http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1464354
http://dx.doi.org/10.15496/publikation-87776
Dokumentart: PhDThesis
Date: 2023-10-17
Language: English
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Rieß, Olaf (prof. Dr.)
Day of Oral Examination: 2023-07-26
DDC Classifikation: 570 - Life sciences; biology
610 - Medicine and health
Other Keywords:
SCA17
TBP
spinocerebellar ataxia type 17
calpains
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Abstract:

Spinocerebellar ataxia type 17 (SCA17) is an autosomal-dominantly inherited neurodegenerative disease predominantly characterized with ataxia, dysarthria, chorea, dystonia, pyramidal disorders, dementia, psychiatric disorders, and seizures. These symptoms are consequence of neuronal failure and death that appears most prominently in the cerebellum, but also in many other parts of the central nervous system. So far, no causal treatment for this disease is available. The underlying genetic cause is a trinucleotide repeat expansion of the base triplets CAG and CAA in the gene of TATA box-binding protein (TBP), which is translated into an expanded polyglutamine (polyQ) stretch. This characteristic allocates it to the group of polyQ disorders, together with SCA3 and Huntington disease. Cellular effects of the polyQ expansion in TBP that are suspected to drive the molecular pathology comprise the occurrence of dysfunctional fragments of the mutant protein, the formation of TBP-positive neuronal aggregates containing protein fragments, and functional depletion of TBP in the affected neurons. So far, proteases that are responsible for the generation of TBP fragments inheriting cytotoxic properties were not yet identified. In this dissertation, the role of calcium-dependent calpains in the cleavage of TBP and the related aggregation were investigated focusing on the ubiquitously expressed isoforms calpain-1 and calpain-2. Moreover, it was tested whether a targeted inhibition of these proteases may lead to a reduction of the pathological hallmarks. Cleavage assays were conducted in vitro upon addition of exogenous calpain-1 and calpain-2 to protein extracts derived from cerebellum tissue of a SCA17 rat model and from respective cell models, as well as cell-based by activation of endogenous calpains by cellular calcium increase. The evoked TBP fragmentation pattern corresponded to breakdown products found at physiological in vivo conditions in SCA17 rat cerebellum. The comparison of the immunodetection with different TBP-specific antibodies, the fragment length, and in silico cleavage site prediction suggest amino acids at positions between alanine 96 and glutamine 117 as potential calpain cleavage sites within TBP. Western blot analysis of the baseline calpain activation revealed an enhanced calpain activity in association with increased occurrence of TBP fragments both in cell and animal models of SCA17. Calpain inhibition via calpain inhibitor III or overexpression of the endogenous calpain inhibitor calpastatin reduced the levels of TBP fragments in cell culture experiments. Furthermore, filter trap-based analysis of SDS insoluble TBP consisting predominantly of N-terminal TBP fragments showed diminished load of aggregates upon calpain inhibition. Taken together, for the first time, this study identified TBP as a substrate of calpain-1 and calpain-2 and confirms calpain-mediated cleavage of TBP and calpain overactivation in cell and animal models of SCA17, which may contribute to pathogenesis. Inhibition of calpains ameliorated specific molecular disease hallmarks significantly, namely TBP fragmentation and aggregation. These results provide a starting point for further testing of pharmacological calpain-inhibiting substances in vitro and in vivo, which may represent a promising therapeutic strategy for SCA17 and the related neurodegenerative diseases.

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