Figure 1. [A53T]a-Synuclein expression in fly heads results in age-dependent loss of DA. (A) Western blot showing abundance of human [A53T]a-Synuclein after aminergic neuron (ddc-GAL4)-specific expression in lysates of fly heads. While flies without [A53T]a-Synuclein transgene do not show any detectable signal for [A53T]a-Synuclein in Western blot, an increase of [A53T]a-Synuclein protein levels was observed by increasing the copy number of transgenes. Syntaxin served as a loading control and molecular weight markers are indicated. (B) Whereas ddc.A53T/+ flies displayed no significant difference in longevity as compared to ddc/+ flies, flies homozygous for ddc.A53T showed a significant decrease (p,0.001, Log rank test). (C) Compared to controls (ddc/+), ddc.A53T/+ flies showed a significant age- dependent loss of DA at 3 and 4 weeks post eclosion (*p,0.05 vs. control). (D) Only ddc.A53T flies showed a significant decrease (***p,0.001) in DA concentration in fly heads at 4 weeks. Comparisons of multiple controls were not significant (4 week values as per cent of 1 week values; ANOVA followed by Newman-Keuls Multiple Comparison Test). doi:10.1371/journal.pgen.1002488.g001 TRAP1 Mitigates a-Synuclein Toxicity (hTRAP1). Interestingly, overexpression of hTRAP1 in fly heads was able to provide a rescue effect against [A53T]a-Synuclein- induced DA loss (Figure 2A). The effects of TRAP1 on DA levels were also reflected by its effect on tyrosine hydroxylase (TH)- positive neurons. ddc.A53T expressing flies showed increased loss of TH-positive neurons if TRAP1 levels were reduced (Figure 2B). In contrast, hTRAP1 overexpression was able to restore [A53T]a- Synuclein-induced loss of TH-positive neurons to control levels. Interestingly, ddc-driven overexpression of hTRAP1 did not increase longevity of ddc.A53T heterozygous flies (not shown). In PD patients, a reduction in brain DA content is later followed by neuronal decline. The same seems to hold true for flies. Although no reduction in longevity of ddc.A53T/+ flies is observed, these flies display a significant reduction in DA content. A more pronounced DA reduction (ddc.A53T/ddc.A53T) results in neuronal decline, eventually leading to early death, reflected by a significantly shortened lifespan (Figure 1B). This might explain why TRAP1 will still provide 100% protection against loss of neurons (Figure 2B), even if DA levels have already started to decline (Figure 2A). PD is clinically defined as a movement disorder. Thus, key to an animal disease model recapitulating this phenotype is loss of locomotor ability. Locomotion in flies is measurable using the negative geotaxis assay. In agreement with previous reports, ddc.A53T flies showed an age-related deficit in climbing ability [21,43]. Notably, ddc-driven hTRAP1 expression was able to significantly rescue the locomotion deficit in ddc.A53T flies (Figure 2C). Therefore, taken together, these data indicate that the rescue of head DA content is sufficient to restore motor ability. Our data suggest that TRAP1 protects from toxic effects induced by [A53T]a-Synuclein. However, TRAP1 might also provide protection to any toxic insult. To address this possibility, we examined if TRAP1 provides protection against toxicity induced by the expression of other well-known toxic proteins/ peptides. Eye-specific expression (GMR-GAL4) of either human Tau or SCA3-derived polyglutamine stretches (PolyQ) in the fly eye result in a rough eye phenotype (REP). These REPs are sensitive to genetic modifiers and have successfully been used for screening [44,45]. Interestingly, overexpression of TRAP1 did not show a rescue of the PolyQ-induced REP. Moreover, silencing of TRAP1 by RNAi did not enhance the REP (Figure 2D). Similar results were obtained with Tau-expressing flies (not shown). A general protective role of TRAP1 in any toxic trigger is therefore unlikely but appears to be specific for a-Synuclein toxicity. Modification of [A53T]a-Synuclein Toxicity by TRAP1 in Rat Primary Cortical Neuron Culture In flies, overexpression of hTRAP1 was able to reduce [A53T]a-Synuclein-induced loss of DA in fly heads and loss of DA neurons, respectively. Thus, to additionally confirm that overexpression of hTRAP1 is able to rescue [A53T]a-Synuclein- induced sensitivity in vertebrate neurons, we used terminally- differentiated rat primary neuron cultures. Lentiviral infection specificity and efficacy in these cells was first verified (Figure 3A, 3B). As the neurons did not display robust toxicity upon [A53T]a- Synuclein expression alone, cells were exposed to low doses of the mitochondrial Complex I inhibitor rotenone. Compared to GFP- virus infected cells (control), co-expression of [A53T]a-Synuclein significantly enhanced cell loss (Figure 3C). In agreement with fly data, coincident overexpression of TRAP1 restored survival to control values. Interestingly, expression of TRAP1 alone enhanced survival beyond that of control cells, indicative of a protective PLoS Genetics | www.plosgenetics.org 3 February 2012 | Volume 8 | Issue 2 | e1002488