TRAP1 Mitigates a-Synuclein Toxicity Figure 3. TRAP1 overexpression protects rat cortical neurons from [A53T]a-Synuclein-induced sensitivity to rotenone. (A) Western blot analysis of cells infected with lentivirus promoting either GFP, TRAP1 or [A53T]a-Synuclein expression. For visualization, the blot was probed with either TRAP1- or a-Synuclein-specific antibodies, respectively. ß-Actin was used for normalization. (B) Rat cortical neurons infected with lentivirus promoting GFP expression were stained for neuronal marker NeuN (red) and DNA (Hoechst, blue). A high percentage of cells showed co-localization between GFP and the neuronal marker NeuN, indicative for high infection efficacy. Scale bar indicates 43 mm. (C) Quantification of cell survival after 16 h of rotenone treatment of cells infected with viruses mediating expression of indicated protein. Significant differences compared to control (GFP) are indicated. All other comparisons revealed highly significant differences (p,0.001) in statistical analysis (ANOVA followed by Newman-Keuls Multiple Comparison Test). **p,0.01; ***p,0.001; ns = not significant. doi:10.1371/journal.pgen.1002488.g003 effect of TRAP1 on neurons independent of effects on [A53T]a- Synuclein-induced toxicity. Modification of [A53T]a-Synuclein Toxicity by TRAP1 in Human HEK293 Cell Culture To study the functional role of TRAP1, we used both TRAP1 overexpression and specific knockdown by small interfering RNA (siRNA) in human embryonic kidney cells-293 (HEK293) cells. Two different siRNAs directed against TRAP1 were first compared for efficacy. Both were able to reduce endogenous TRAP1 expression in HEK293 cells (Figure S3). The most efficient siRNA was used for all further investigations. To confirm whether treatment of HEK293 cells mimicked the in vivo fly and in vitro rat neuron data concerning TRAP1 and stress sensitivity, HEK293 cells were treated overnight with a low dose of either hydrogen peroxide (Figure 4A) or the Complex I inhibitor rotenone (Figure 4B). [A53T]a-Synuclein expression enhanced cell sensitivity to both stressors. Reduction in TRAP1 expression further reduced survival in the presence of [A53T]a-Synuclein. For both, rotenone and hydrogen peroxide treatment, overex- pression of TRAP1 in the context of [A53T]a-Synuclein expression attenuated the decrease in cell survival. The magnitude of the rescue effect was greatest when cells were exposed to rotenone (Figure 4A, 4B). In cells without [A53T]a-Synuclein expression, reduction of TRAP1 also caused stress sensitization. These data corroborate the reported function of TRAP1 as a protective mitochondrial chaperone [46–48]. Previous reports have indicated that [A53T]a-Synuclein may interfere with mitochondrial respiration, in particular with Complex I function [34]. Given the noted rescue effect of TRAP1 on rotenone-treated cells with or without [A53T]a-Synuclein expression, we hypothesized that the TRAP1 effect on [A53T]a- Synuclein may in part be related to altered ETC function. Thus, ATP production via Complex I was assayed in cells without oxidative stress, to evaluate the general effects of [A53T]a- Synuclein on ETC in combination with altered TRAP1 levels. Expression of [A53T]a-Synuclein reduced Complex I activity in HEK293 cells (Figure 4C). TRAP1-silencing enhanced this reduction, while TRAP1 overexpression rescued the [A53T]a- Synuclein-induced defect. In light of the defects observed in [A53T]a-Synuclein-induced Complex I ATP production (Figure 4C), total ATP levels in the cell were also investigated. Only cells expressing [A53T]a-Synuclein in combination with siTRAP1 showed a reduction of total ATP levels (Figure 4D). Although [A53T]a-Synuclein alone significantly reduced Complex I activity, overall ATP levels were unchanged. Loss of mitochondrial membrane potential predisposes cells to apoptosis. [A53T]a-Synuclein has been suggested to adopt an alpha-helical conformation that could perforate membranes. At the same time, TRAP1 protection against apoptosis has been suggested to act via inhibition of opening mitochondrial permeability transition pore (PTP) [49]. The mitochondrial membrane potential is thought to indirectly reflect the state of the PTP. Cells were thus assessed for mitochondrial membrane potential using the mitochondrial membrane dye, JC-1. Only cells expressing [A53T]a-Synuclein in combination with siTRAP1 showed a loss of mitochondrial membrane potential (Figure 4E). Finally, to exclude the possibility that altered Complex I ATP production might be due to varying quantites of mitochondria PLoS Genetics | www.plosgenetics.org 5 February 2012 | Volume 8 | Issue 2 | e1002488