The role of ER stress for the pathogenesis of SCA3 in primary cell culture experiment

The spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominantly inherited neurodegenerative disorder, which is caused by an expanded CAG repeat in the ataxin-3 gene. After a decade of research the function of ataxin-3 is still unidentified.

Here we investigated, whether there is a causal relationship between the pathogenesis of SCA3 and endoplasmic reticulum (ER) stress. Therefore we analyzed mouse embryonic fibroblasts (Mef's), which derived from SCA3 mutant mice, generated by a gene trap (gt) approach. Heterozygous (wt/gt) and homozygous (gt/gt) mutant Mef's for SCA3 were studied in comparison to their wildtype (wt) control Mef's.

In order to induce ER stress MEF's were treated with ER specific drugs, e.g. tunicamycin and thapsigargin in a defined concentration for a determined time. Afterwards we isolated RNA and proteins and analyzed two genes, which are important for the ER stress pathway, CHOP and BiP. These two genes are counterparts in the ER stress pathway. While CHOP leads cells under ER stress into apoptosis, BiP is able to rescue the cells via it's chaperone function.

We found a different regulation of transcription and translation of CHOP and BiP in treated versus untreated cells, but there were no discrepancy between the different genotypes.

Additionally, we performed electron microscopy of treated cells, where we observed an enhancement of proteins associated to the ER in heterozygous and homozygous mutant Mef's for SCA3 in comparison to their wildtype cells. We could confirm this finding by fluorescence microscopy. For this purpose we prior transfected the Mef's with a pDsRed2-ER vector (Clontech).

At the moment we discuss two different hypotheses for this enhancement of proteins related to the ER. On the one hand ER stress might induce a higher production of these proteins. On the other hand it is a dysfunction in protein transport, which might lead to these formations.

These findings suggest a functional relationship between the pathogenesis of SCA3 and the endoplasmic reticulum stress.