Einfluss von VDAC1 auf das Calpain-System als mögliche therapeutische Option für Morbus Huntington

Abstract

Introduction: Huntington disease (HD) is a fatal neurodegenerative disorder which is caused by a CAG repeat expansion in the HTT gene coding for the huntingtin protein (HTT). This mutation is translated into an expanded polyglutamine tract in the mutant HTT (mHTT) protein, leading to deleterious alterations of its normal function. An important characteristic of HD is the proteolytic cleavage of mHTT. Different studies showed that calpains, a calcium-activated class of cysteine proteases, are involved in this processing. Since calcium dyshomeostasis and mitochondrial dysfunction is a further characteristic of HD, calcium alterations may lead to more calpain-mediated mHTT fragmentation as well. The calcium-permeable mitochondrial voltage-dependent anion channel 1 (VDAC1) could play an important role in this vicious circle and influencing this target protein could be a promising therapeutic approach to a treatment for HD.

Material and Methods: Brain samples of six-month-old HD knock-in mice were analysed for calpain activation and expression of proteins involved in calcium homeostasis. Calpain overactivation in HD context was achieved by esiRNA-mediated knock-down of the endogenous calpain inhibitor calpastatin in HEK293T cells overexpressing wildtype (15Q) HTT and polyglutamine-expanded (128Q) mHTT. VDAC1 was influenced genetically and pharmacologically in HTT 15Q/128Q overexpressing HEK293T cells. All protein samples were analysed via western blotting followed by immunodetections. Aggregates of mHTT were analysed via Filter-Retardation-Assay or Immunocytochemistry.

Results: We observed that the calpain system is overactivated significantly in the striatum of six-month old HD knock-in mice and that VDAC1 is downregulated in this specific brain region. Furthermore, a cell-based activity induction of calpains led to an increased mHTT cleavage and accelerated mHTT aggregation. The different approaches to influence VDAC1 expression and function did only have minor effects on the markers of the calpain system and mHTT processing.

Conclusion and Discussion: It was shown that calpains play an important role in the proteolysis of mHTT in vivo and in a cell model of HD. The tested cell model did only show minor changes in calpain activation due to VDAC1-targetting. This could possibly be caused by a low sensitivity to decent changes in cytoplasmatic calcium concentration in HEK293T cells. Since VDAC1 is reduced in the striatum of six-month-old HD knock-in mice and due to the aforementioned possible limitations of the employed cell line, the research on VDAC1’s targetability in HD should continue.