TRAP1 Mitigates a-Synuclein Toxicity Figure 4. Alterations in TRAP1 levels influence [A53T]a-Synuclein-induced sensitivity to oxidative stress and mitochondrial effects in HEK293 cells. HEK293 cells were transfected with plasmids promoting [A53T]a-Synuclein or TRAP1 expression. Empty vector transfection served as control. In addition, RNAi-mediated silencing of endogenous TRAP1 was induced (siTRAP1). Cells transfected with indicated plasmid combinations were treated with (A) hydrogen peroxide (100 mm) or (B) rotenone (200 mM) to induce oxidative stress. Cell numbers were analyzed to monitor survival. (C–E) HEK293 without oxidative stress treatment overexpressing the indicated proteins, or with RNAi-mediated silencing of TRAP1 were analyzed for (C) ATP production via Complex I, (D) total ATP content, and (E) mitochondrial membrane potential. Statistical analysis of displayed bar graphs was performed using ANOVA followed by Newman-Keuls Multiple Comparison Test. (A, B) Biologically relevant comparisons are indicated in bar graphs. (C) Differences compared to control are indicated. (A, B, C) A detailed summary of all comparisons is summarized in Figure S8. (D, E) Only cells with [A53T]a-Synuclein expression and TRAP1 reduction displayed significant differences in statistical analysis as indicated in graph. All other comparisons were not significant. *p,0.05; **p,0.01; ***p,0.001; ns = not significant. doi:10.1371/journal.pgen.1002488.g004 within the cells, instead of a functional deficit in the ETC, cell samples were probed for two mitochondrial proteins, VDAC1 and COX4. No major differences were observed for expression of VDAC1 and COX4 (Figure S4A). This suggests the detected decrease in Complex I ATP production resulted from a functional ETC deficit. JC-1 is an excellent dye to measure mitochondrial membrane potential and because of the color switch following depolarization, it makes it easy to normalize to cell density. However, JC-1 has been superseded by other dyes, like TMRM, with respect to the potential artifact of local concentration changes. With regard to this potential problem, we repeated mitochondrial membrane potential measurements using TMRM. In addition, we wanted to exclude potential off-target effects by siRNA treatment. Therefore, we generated HEK293 cells with stable expression of shTRAP1 constructs resulting in a roughly 90% loss of TRAP1 protein levels (Figure S5A). In stable TRAP1- silenced cells, a significant reduction in membrane potential was observed after [A53T]a-Synuclein expression. This effect was absent in cells expressing scrambled shRNA, again indicating that TRAP1-silencing in combination with [A53T]a-Synuclein expres- sion causes opening of mitochondrial PTP (Figure S5B). Effect of TRAP1 Mutation on [A53T]a-Synuclein–Induced Toxicity The human TRAP1 ATPase domain shares high homology with both other HSP90 proteins and TRAP1 orthologs found in other species (Figure S6). Recently, the ATPase domain of yeast HSP90 has been shown to be required for its HSP90 function. The mutation of a specific amino acid within the ATPase domain was sufficient to inhibit ATP binding [50]. This amino acid is highly conserved in both HSP90 and TRAP1 proteins (Figure S6). We PLoS Genetics | www.plosgenetics.org 6 February 2012 | Volume 8 | Issue 2 | e1002488