TRAP1 Mitigates a-Synuclein Toxicity Figure 6. Inhibition of mitochondrial fusion by [A53T]a-Synuclein is rescued by TRAP1. SH-SY5Y cells were co-transfected with the indicated constructs and mito-DsRed to visualize mitochondria. The mitochondrial morphology of transfected cells was analyzed by fluorescence microscopy. (A) Confocal images of representative cells displaying either an intact tubular mitochondrial network (control) or a fragmentation of this network (A53T). Co-expression of TRAP1[WT] prevented [A53T]a-Synuclein-induced mitochondrial fragmentation, whereas the TRAP1[D158N] mutant did not show rescue activity. Overexpression of either wild type or mutant TRAP1 alone did not influence mitochondrial morphology under steady state conditions. (B) For quantification, the mitochondrial morphology of at least 300 transfected cells per coverslip was determined in a blinded manner. Quantifications were based on triplicates of at least three independent experiments. Shown is the percentage of cells with fragmented mitochondria. (C) Expression levels of [A53T]a-Synuclein and TRAP1 were analyzed by Western blotting. b-Actin served as a loading control. ***p,0.001 (ANOVA). doi:10.1371/journal.pgen.1002488.g006 blotting, these fractions and input control were compared for the content of endogenous, VDAC1 (mitochondrial outer membrane protein), b-Tubulin (cytosol) and a-Synuclein proteins. Whereas the input showed abundance of all tested proteins, the cytosolic fraction displayed expected cytosolic proteins b-Tubulin and a- Synuclein. In the mitochondria enriched fraction, no contaminat- ing protein from b-Tubulin could be detected. Importantly, exogenous [A53T]a-Synuclein protein was found within the mitochondria enriched fraction (Figure S7B). Given the strong rescue effect of TRAP1 on toxicity induced by [A53T]a-Synuclein in various systems (flies, primary rat neurons, and human cells), this implies at least a genetic interaction of these proteins. Whether there is a direct interaction of [A53T]a- Synuclein and TRAP1 awaits further analysis. Discussion a-Synuclein plays an important role in PD pathogenesis. However, the mechanisms that actually lead to a-Synuclein- induced neurotoxicity remain unresolved. To gain insights into the disease mechanisms triggered by a-Synuclein, we performed a genome-wide modifier screen on [A53T]a-Synuclein-induced toxicity in flies. We used [A53T]a-Synuclein for our screen because its overexpression in flies results in a robust Parkinsonian phenotype [21,55–57]. Toxicity induced by a-Synuclein or its mutant variants is rather low and eye-specific expression of A53T does not cause rough eye phenotypes (REPs). Such REPs induced by eye-specific expression of toxic proteins provide an excellent tool for screens and have successfully been used in the past to identify genetic interactions applying alterations in eye morphol- ogy due to photoreceptor degeneration as an endpoint. Given the low toxicity of [A53T]a-Synuclein, such screening approaches could not be conducted with regard to a-Synuclein-induced toxicity in flies. Our genetic screen fulfilled two important requirements: it utilized (i) an age-dependent model of [A53T]a- Synuclein toxicity, and (ii) an endpoint that is relevant to PD, this being the loss of DA. However, apart of being used as a neurotransmitter, DA in flies is also used for cuticle tanning. Thus, we cannot exclude the possibility that cuticle-derived DA might contribute to the overall DA in fly heads. Therefore, the measured decline is not only connected to DA loss in neurons. Nevertheless, secondary readouts like locomotion measurements or DA neuron counts indicate a strong correlation between decreased head DA content and proper function of DA neurons. One of the candidates identified in our screen was the mitochondrial chaperone TRAP1. Consistent with our results, a genetic screen for alteration of a-Synuclein aggregation, conduct- ed in C. elegans, identified R151.7, a homologue to Drosophila and human TRAP1, as a candidate worm gene. Knockdown of R151.7 resulted in premature a-Synuclein aggregation [58]. Although aggregation was not assayed in our screen, this finding acts as an external confirmation that TRAP1 genetically interacts with a- Synuclein in different in vivo systems. In multiple cell culture systems, TRAP1 has been shown to provide anti-apoptotic functions [48,52,53] as high levels of TRAP1 reduce the release of key factors involved in apoptosis, including Apoptosis Inducing Factor-1 and Cytochrome c, and additionally prevents Caspase-3 cleavage [39,46,48,59]. The direct mechanisms by which TRAP1 might inhibit apoptosis were not examined in this study. However, given that overexpression of TRAP1 in both rat primary neurons and HEK293 cells was able to enhance cell survival after rotenone treatment, we hypothesize that anti-apoptotic mechanisms might in part be responsible for rescue of [A53T]a-Synuclein toxicity by TRAP1. This is in agreement with the observation that PD-associated neuronal death involves apoptotic cell death [60–63]. In addition, the effects of TRAP1 modulation on ATP synthesis and activities of the ETC support a mitochondrial function. For more than two decades, biochemical studies, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyr- idine (MPTP) and transgenic animal models have implicated mitochondrial dysfunction in the pathogenesis of PD [5,64–71]. Genetic data, including mutations in PINK-1, Parkin, DJ-1, and HtrA2, have now specifically linked PD to both dysfunction and morphological change of the mitochondria [72–80]. However, the relationship of a-Synuclein pathology and mitochondrial dysfunc- tion has been less clear. Our data are compatible with a localization of [A53T]a-Synuclein either in mitochondria or in mitochondrial membranes. Recent findings, though, have indi- cated that a-Synuclein may be localized to the outer mitochon- drial membrane in pathological conditions and induce morpho- logical changes of mitochondria by inhibiting mitochondrial fusion and enhancing mitochondrial fragmentation [51]. These morpho- logical changes were rescued by overexpression of wild type PINK1, Parkin, and DJ-1 [51]. We show here that TRAP1 overexpression is also able to reverse [A53T]a-Synuclein-induced mitochondrial fragmentation. Interestingly, TRAP1 has been identified as a substrate of the serine/threonine kinase PINK1. Phosphorylation of TRAP1 by PINK1 seems to be required for the protective effects mediated by PINK1. Combining these data with our findings leads to a potential pathogenic model, in which [A53T]a-Synuclein induces mitochondrial stress impairing, most likely, Complex I of the ECT by an as yet unidentified mechanism. Overexpression of TRAP1 counteracts this effect in flies, primary neurons and human neuronal as well as non-neuronal cells. TRAP1[D158N] is less effective in protecting from [A53T]a-Synuclein-induced detri- mental effects. The finding suggests that a functional ATPase domain is required for TRAP1 function. Methods Fly Stocks Flies were raised and maintained on standard cornmeal-yeast- molasses-agar food at 25uC unless otherwise noted. Non-RNAi stocks were obtained from the Bloomington Drosophila Stock Centre, UAS-RNAi stocks either from the Vienna Drosophila RNAi PLoS Genetics | www.plosgenetics.org 9 February 2012 | Volume 8 | Issue 2 | e1002488