Author Summary Parkinson’s disease (PD) is a progressive neurodegenera- tive disorder, pathologically characterized by loss of dopaminergic neurons in the substantia nigra pars compacta brain region. Mutations in a-Synuclein or gene duplication or triplication result in autosomal-dominant inherited PD. Indeed, aggregated and insoluble a-Synu- clein is found in Lewy bodies, a pathological hallmark common to both sporadic and hereditary forms of PD. In order to better define a-Synuclein’s pathogenic mecha- nism, we first used a fly genetic screen to search for novel genetic modifiers of mutant human [A53T]a-Synuclein neurotoxicity. We identified the mitochondrial chaperone protein TRAP1 as a novel modifier of the toxicity induced by [A53T]a-Synuclein. [A53T]a-Synuclein–induced toxicity was enhanced when TRAP1 expression was decreased, while overexpression of human TRAP1 (hTRAP1) provided a rescue. Cell culture experiments further demonstrated that [A53T]a-Synuclein directly interferes with a number of mitochondrial functions, including Complex I ATP produc- tion, mitochondrial fragmentation, and sensitivity to oxidative stress. These effects could be blocked by TRAP1 overexpression. As mitochondrial dysfunction has previ- ously been linked to mutations in several other genes associated with genetic PD, these data provide further evidence of a common mitochondrial-centric mechanism of PD pathogenesis. their native unfolded state is linked to cell membrane damage and results in dysfunction of multiple cell systems such as the ubiquitin proteasome system, the endoplasmic reticulum and lysosomes [24– 31]. Recent data also suggests that a-Synuclein plays a role in modulating both mitochondrial function and damage. a-Synu- clein-overexpressing cells exhibit multiple markers of mitochon- drial dysfunction, including increased protein oxidation, increased ROS production, loss of mitochondrial membrane potential and reduced Complex I activity [32–38]. Several groups have demonstrated that a-Synuclein’s entry in mitochondria is mediated via an N-terminal mitochondrial targeting sequence, with localization at the inner membrane [37,38]. Moreover, PD patient brain histology shows a-Synuclein accumulation within mitochondria of the SNc and striatum, a feature absent in control brains [38]. Mitochondrial dysfunction associated with adenosine triphosphate (ATP) depletion and electron transport chain (ETC) defects reduces the cell’s ability to handle oxidative protein damage and cellular tasks, suggesting a possible reason for cell death. In PD patient brains, early DA reduction indicates the withdrawal of SNc striatal projections, finally resulting in DA neuron loss and PD-related symptoms of rigidity and akinesia. In flies, expression of [A53T]a-Synuclein is accompanied by an age- dependent loss of DA and DA neurons, respectively. Thus, fly head DA levels provide an indirect readout for [A53T]a- Synuclein-induced toxicity. To further investigate the mechanism and identify novel modifiers of a-Synuclein toxicity, we performed a genome-wide genetic screen in Drosophila. In this screen, we identified, among other gene products, the mitochondrial chaperone protein TRAP1 (GenBank ID 10131) as a novel modifier of [A53T]a-Synuclein-induced DA loss. TRAP1 has previously been shown to function downstream of the PD-related serine/threonine kinase PINK1 (GenBank ID 65018). PINK1- induced phosphorylation of TRAP1 seems to be necessary for the protein’s protective effects against oxidative stress [39]. In our report we further characterize the functional consequences of TRAP1 Mitigates a-Synuclein Toxicity TRAP1 reduction or overexpression in Drosophila, in primary neurons and dopaminergic cell lines and the effects on mitochondrial morphology and function. Results Expression of Human [A53T]a-Synuclein in Fly Heads and Genetic Screening for Modification of [A53T]a-Synuclein Toxicity Expression of a-Synuclein in Drosophila is established as a useful model of PD [21]. As the fly lacks an a-Synuclein homolog, this model relies on ectopic expression of human a-Synuclein using the UAS/GAL4 system [40]. We have previously analyzed DA neuron number in aged flies, expressing different mutant variants of a- Synuclein. Compared to controls, wild type a-Synuclein did not cause a decline in DA neuron number. Moreover, locomotion was not impaired in aged wild type a-Synuclein-expressing flies [41]. Based on these results, we chose [A53T]a-Synuclein for our screening. With single copy expression of a UAS:[A53T]a-Synuclein transgene (A53T) in aminergic neurons (dopa decarboxylase-GAL4 driver, ddc-GAL4) (Figure 1A), no difference to overall fly fitness, as assessed by longevity, was observed (Figure 1B). In contrast, expression of two transgene copies, resulting in higher expression levels (Figure 1A), caused earlier lethality compared to controls (Figure 1B). However, when we measured DA levels of flies expressing one copy of [A53T]a-Synuclein under control of ddc- GAL4 (ddc.A53T), we noticed a significant decrease in DA levels with aging (Figure 1C). Thus, measuring DA levels using high performance liquid chromatography represents a sensitive system to address DA levels in fly heads. After carefully addressing sensitivity, specificity and reproducibility of our readout marker (Figure 1D, Figure S1), we decided to perform a genome-wide screen to identify modifiers of [A53T]a-Synuclein-induced DA loss in vivo. Thus, flies with expression of [A53T]a-Synuclein in aminergic neurons were crossbred to fly lines carrying chromo- somal deletions (deficiencies), utilizing the ‘‘Bloomington Defi- ciency Kit’’. Progeny were screened for changes in DA loss over time (a summary of the screen results is given in Text S1 and Tables S1, S2, S3, S4). Although detailed single gene analysis is still ongoing, we identified a large number of genes coding for proteins involved in mitochondrial function within the candidate deficiencies. Therefore, we additionally cross-referenced our data with results from a genome-wide RNAi-screen, set to identify modulators of mitochondrial function [42]. Common genes were screened for alteration of [A53T]a-Synuclein-toxicity with respect to viability and DA loss. TRAP1 Modification of [A53T]a-Synuclein Toxicity in the Fly Among the deficiencies screened, Df(2R)nap9 caused the greatest enhancement of [A53T]a-Synuclein-induced DA loss of all non- lethal interacting deficiencies. Of the 153 genes deleted by Df(2R)nap9, we found TRAP1 reduction to enhance [A53T]a- Synuclein-induced DA loss. The loss-of-function allele TRAP1[KG06242] (hereafter referred to as TRAP1[KG], Figure S2) caused a reduction of fly head DA levels similar to those of Df(2R)nap9 (data not shown). However, TRAP1[KG] did not alter DA levels (Figure 1D). Thus, flies with reduced TRAP1 in combination with the ddc-GAL4 driver (TRAP1[KG]/+; ddc/+) served as controls in later analysis. TRAP1 is a mitochondrial chaperone, recently reported as a downstream phosphorylation target of the PD protein PINK1 in rat and human cell lines [39]. As both fly and human TRAP1 share high sequence homology, we generated a UAS-transgenic fly to express human TRAP1 PLoS Genetics | www.plosgenetics.org 2 February 2012 | Volume 8 | Issue 2 | e1002488