Thione (GSH) an important small anti-oxidant molecule protects cells against oxidative
Thione (GSH) an important small anti-oxidant molecule protects cells against oxidative stress by conjugating with ROS in a reaction catalyzed by GST [24]. During the conjugation reaction GSH become oxidized into glutathione disulfide (GSSH) and is thus depleted. GSH depletion have been shown to occur in neurodegenerative disorders like Parkinson’s disease [25]. In mammals, there are at least three forms of SOD: a cytosolic (CuZnSOD/SOD1), a mitochondrial (MnSOD/SOD2) and an extracellular (ECSOD/SOD3) form. SOD enzymes catalyze the dismutation of superoxide, a primary ROS, into hydrogen peroxide [26]. The hydrogen peroxide is then further converted to water and oxygen by CAT, Prxs or Gpxs [14,27]. In this study, we show that oxidative stress plays a major role in ATXN7-induced toxicity using a new stable inducible PC12 cell model. We found that induction of mutant ATXN7Q65-GFP expression led to a concomitant increase in ROS levels and aggregation of the disease protein followed by Stattic site decreased cell viability a few days later. Analysis of some key anti-oxidant defense enzymes revealed decreased levels of catalase, which could contribute to decreased clearance of ROS. Furthermore, inhibition of NOX complexes prevented the increase in ROS and ameliorated aggregation suggesting that mutant ATXN7 increase the ROS levels byactivating this complex. Moreover, supporting the cells through application of exogenous anti-oxidants or inhibition of NOX complexes ameliorated AXTN7Q65 induced toxicity.ResultsExpression of mutant ATXN7 leads to oxidative stress followed by toxicity in an inducible SCA7 PC12 cell modelTo study the impact of mutant ATXN7 on cellular functions we used two recently generated stable inducible PC12 cell lines expressing N-terminal FLAG- and Cterminal GFP-tagged ATXN7 with 10 (FLQ10 line) or 65 (FLQ65 line) glutamines [28]. In these cell lines the expression of the corresponding transgenic proteins named ATXN7Q10-GFP and ATXN7Q65-GFP is controlled by the Tet-off expression system and induced by removal of doxycycline from the cell culture media. The induction timing, expression levels and sub-cellular localization of the transgenic ATXN7-GFP proteins have previously been extensively characterized and showed not to differ in these two cell lines [28]. Immunoblotting with an ATXN7 antibody revealed weak expression after three days of induction, but clear expression of both constructs from day 6 onwards (Figure 1A and [28]. No ATXN7 aggregation was detected in ATXN7Q10-GFP expressing cells [28]. However, in cells expressing ATXN7Q65-GFP filter trap analysis revealed aggregation from day 3 onwards and from day 9 the level of aggregated ATXN7 material was stable (Figure 1B and [28]). Analysis of cell viability/toxicity revealed a progressive decrease in viability and increased toxicity as expression of ATXN7Q65-GFP was induced, see Figure 1C-D. A significant decrease in viability was observed from day 9 after induction using the WST-1 viability assay (Figure 1C). In accordance with this a statistical increase in toxicity, measured as membrane leakage, could also be observed on day 9 after induction (Figure 1D). In contrast, expression PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26866270 of the ATXNQ10-GFP protein did not result in any significant change in cell viability or toxicity on any day (Figure 1C-D). To establish whether induction of oxidative stress by ATXN7Q65-GFP could play a role in the decreased cell viability, we measured total cellular ROS levels at various time points aft.