therefore exchanged the aspartic acid at position 158 for asparagine (TRAP1[D158N]), creating a putative non-functional ATPase domain. Introducing the D158N mutation did not interfere with TRAP1 protein turnover, as expression in HEK293 cells resulted in similar abundance of TRAP1[D158N] and TRAP1[WT] proteins (Figure 5A). Next, we asked if TRAP1[D158N] is as effective as TRAP1[WT] in protecting [A53T]a-Synuclein-expressing cells from oxidative stress. Cells overexpressing [A53T]a-Synuclein treated overnight with rotenone displayed a robust reduction in cell survival, which was rescued by TRAP1[WT] overexpression (Figure 4B). In contrast, overexpression of TRAP1[D158N] was less effective (Figure 5B). Similar results were observed when we tested ATP production by Complex I. In the context of [A53T]a- Synuclein expression without oxidative stress, TRAP1[WT] rescued [A53T]a-Synuclein-induced decrease in Complex I ATP production (Figure 5C), while TRAP1[D158N] showed signifi- cantly lower degree of rescue ability. Finally, cell lysates were again analyzed for abundance of the mitochondrial proteins VDAC1 and COX4. No changes in VDAC1 or COX4 protein levels were observed in cell lysates expressing either TRAP1[WT] or mutant TRAP1[D158N] (Figure S4B). These data thus indicate that mutant TRAP1 expression does not alter the overall mitochon- drial content, arguing in favor of a functional ETC Complex deficit, rather than a deficit due to diminished numbers of mitochondrial/ETC components in the cell. Recent data show that a-Synuclein impairs mitochondrial fusion, leading to fragmented mitochondria. Interestingly, the a- Synuclein-induced mitochondrial fragmentation can be attenuated by co-expression of PINK1, Parkin and DJ-1, but not by PD- linked mutant variants of these proteins [51]. Therefore, we sought to determine if TRAP1 is also able of attenuating a-Synuclein- induced mitochondrial fragmentation in SH-SY5Y cells. The [A53T]a-Synuclein-induced punctate mitochondrial staining was reversed to a tubular mitochondrial network by TRAP1[WT] co- TRAP1 Mitigates a-Synuclein Toxicity expression. In contrast, co-expression of TRAP1[D158N] showed no effect (Figure 6A, 6B). The expression of both TRAP1 variants alone had no impact on mitochondrial integrity. Verification of protein expression levels revealed robust a-Synuclein and TRAP1 expression after transfection with respective plasmids (Figure 6C). Thus, the impaired rescue ability of TRAP1[D158N] in comparison to TRAP1[WT] cannot be explained by the lower abundance of TRAP1[D158N] protein. It is rather the conse- quence of an altered function of the inherent ATPase function of TRAP1[D158N]. In addition, we asked if reduced TRAP1 levels might enhance mitochondrial fragmentation induced by [A53T]a- Synuclein expression. We noticed that TRAP1-silencing increased the number of cells with fragmented mitochondria. Combining TRAP1-silencing with [A53T]a-Synuclein expression enhanced fragmentation of mitochondria even further (Figure 7A, 7B). Effective TRAP1-silencing and [A53T]a-Synuclein expression was confirmed by Western blot analysis (Figure 7C). Localization of [A53T]a-Synuclein to Mitochondria and Protein–Protein Interaction with TRAP1 in HEK293 Cell Culture TRAP1 is defined as a mitochondrial molecular chaperone and has been shown to be protective against oxidative stress-induced cell death via multiple postulated mechanisms including cyto- chrome c release inhibition, interference with caspase-3 activation and attenuation of ROS production [39,46,48,52–54]. We thus hypothesized that TRAP1 might directly antagonize a-Synuclein mitochondrial-related toxicity. Confirming that TRAP1 is indeed found in the mitochondria, co-localization studies in HEK293 cells revealed a strong overlap between TRAP1 staining with ‘‘Mitotracker Orange’’-labeled mitochondria (Figure S7A). There- fore, it was interesting to see if a-Synuclein might also localize with mitochondria, as previously reported [33,37,38]. To determine this, we performed cell fractionation experiments to separate cytoplasmic and mitochondrial enriched fractions. Using Western Figure 5. Effect of TRAP1 mutation on modification of [A53T]a-Synuclein toxicity. (A) Western blot analysis of HEK293 lysates transfected with indicated constructs showed similar expression levels of TRAP1[WT] and TRAP1[D158N] and a reduction of endogenous TRAP1 by siTRAP1. Blot was probed with TRAP1-specific antibody. ß-Actin served as loading control. (B, C) Effect of TRAP1[WT] and TRAP1[D158N] on [A53T]a-Synuclein- induced effects in HEK293 cells. (B) Effect of TRAP1[D158N] on [A53T]a-Synuclein-induced toxicity. Cell survival after rotenone (200 mM) treatment was monitored. Compared to TRAP1[WT], cells expressing TRAP1[D158N] displayed a significant reduction in survival (t-test, **p,0.01). (C) Assessment of ATP production via Complex I in unstressed cells with [A53T]a-Synuclein expression revealed a significant reduction of ATP levels in TRAP1[D158N] versus TRAP1[WT] expressing cells (t-test, ***p,0.001). doi:10.1371/journal.pgen.1002488.g005 PLoS Genetics | www.plosgenetics.org 7 February 2012 | Volume 8 | Issue 2 | e1002488