Mitochondrial dysfunction and unfolded protein response in the pathogenesis of SCA3 in primary cell culture experiments

The spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an autosomal dominantly inherited neurodegenerative disorder caused by an expanded CAG repeat in the ataxin-3 gene.

To consider the correlation between mitochondrial dysfunction and the pathogenesis of SCA3, we analysed mouse embryonic fibroblasts (MEF), which derived from SCA3 mutant mice, generated by the gene trap (gt) approach. Heterozygous (wt/gt) and homozygous (gt/gt) SCA3 mutant MEF cells were compared to wildtype controls.

In vivo and in vitro studies in the SCA3 mutant mouse model revealed an effect of the unfolded protein response (UPR) in the pathogenesis of SCA3. ER stress causes mitochondria-associated apoptosis influenced by cytochrome c release and mitochondria dysfunction.

To induce ER stress we treated the MEF cells with ER stress specific drugs, e.g. Tunicamycin (Tm) and Thapsigargin (Tg).

At first, the RNA- and protein-level of CHOP, BIP and cytochrome c were analysed at different time points. These three genes are important key players in the mitochondria-associated apoptosis.

Therefore, CHOP and BIP are counterparts in this ER stress pathway. While CHOP leads cells under stress conditions into apoptosis, BIP has a chaperone function and can bind misfolded proteins in the ER to refold them in their originally structure. Cytochrome c release from the mitochondria forms an apoptosome complex with procaspase-9, which activates different caspases by cleavage and leads into apoptosis.

The analysis of BIP, CHOP and cytochrome c release revealed significant differences in RNA- and protein-level after induction of ER stress by Tm and Tg in the SCA3 mutant MEF cells (wt/gt, gt/gt) compared to wildtype controls.

We investigated mitochondria dysfunction by different methods (Oxygraph and FACS analysis). Therefore, untreated cells of all three genotypes were analysed. The measurements of the cells revealed significant differences between wildtype and SCA3 mutant cells (wt/gt, gt/gt) in the respiration rate and complex I and II dependent oxygen consumption.

In the FACS analysis (TMRE staining) we observed a difference in the intact membrane potential between mutant and wildtype cells.

These findings approve the conclusion that mutant cells are already under stress conditions before treatment with specific drugs.