macrolide antibiotic that specifically inhibits vacuolar ATPase (V- ATPase) and therefore inhibits the acidification of lysosomes and promotes the accumulation of autophagosomes [38,39]. We found that lysosomal inhibition in MEF expressing physiological DJ-1 led to similar changes in MMP as observed in untreated DJ-1 KO cells (Figure 8B). This correlates with reduced lysosomal activity and reduced basal autophagy observed in untreated DJ-1 KO cells (Figure 4A and Figure 5A–C). Interestingly no further decrease in MMP was observed in DJ-1 KO cells upon treatment with BafA1 (Fig. 8B). Analysis of Mechanisms of Mitophagy Regulation of autophagy and especially the autophagic degradation of mitochondria is an essential tool to maintain mitochondrial function and cellular homeostasis. Recent studies showed that DJ-1 is involved in the regulation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and ERK1/2 was shown to be necessary and sufficient to induce macroautophagy and mitochondrial turnover [40]. Since mitochondrially localized ERK2 was described to be involved in the regulation of macroautophagy [41], we investigated, whether differences in the levels of activated cytoplasmic and mitochondrial ERK1/2 may contribute to the observed accumulation of dysfunctional mitochondria. Our results show a reduction in phosphorylated ERK2 in the mitochondrial fraction of DJ-1 KO MEF, while there was no difference in the phosphorylation of neither ERK1 nor ERK2 in the cytosolic fraction between DJ-1 KO and DJ-1 WT MEF. These results support a possible involvement of DJ-1 mediated ERK2 phosphorylation in the control of autophagic and lysosomal function. Discussion Mitochondria have been early recognized as a critical interface between vital energy metabolism and cell death. Based on biochemical findings in affected brain regions of PD patients a role of mitochondrial dysfunction and oxidative damage has been hypothesized in both ageing and neurodegeneration [42]. This hypothesis was further emphazised by the identification of nuclear genes mutated in familial forms of PD that are directly linked to mitochondrial function, i.e. Parkin, PINK1 and DJ-1 [10,43,44]. In the present study we demonstrate, that the Parkinson’s disease associated protein DJ-1 integrates both mitochondrial and lysosomal function. We have shown that the loss of the mitochondrial protein DJ-1 KO in MEF cells causes multiple mitochondrial impairments including functional and morphological changes: (i) a significant increase of mitochondrial ROS formation in DJ-1 KO cells which could impair the oxidative phosphorylation as indicated by (ii) diminished pyruvate dependent respiration rates and (iii). reduced MMP. Importantly, the decreased MMP as a parameter of mitochondrial dysfunction was restored by complementation of the DJ-1 KO model with physiological active DJ-1 protein (Fig. 2C). These metabolically detectable impairments were paralleled by morphological changes as decreased mitochondrial branching which could be explained by a relative augmentation of fission events. Since there was no change in the steady state levels of the main fusion/fission mediators Drp1, Mfn2, Fis1, and OPA1 (Fig. 3C), we speculate that increased fission could be caused by a reduced energy supply to the energy consuming fusion processes. Indeed regarding our findings on mitochondrial depolarization these results support the notion that relevant reduction in the MMP negatively regulates the prevalence of fusion events (Twig et al., 2008). Disrupted fusion results in mitochondrial heterogeneity DJ-1 in Mitochondrial Dynamics and decreased rates of cellular respiration was recently shown in cellular models of mutations of Mfn1 and Mfn2 [45]. Moreover it seems that cells with impaired fusion/fission dynamics are unable to use mitochondrial redistribution in order to provide increased local ATP supply at cellular sites of particular demand [46]. Loss of DJ-1 shifted the mitochondrial network towards the status of reduced mitochondrial connectivity. Indeed our first study of fibroblasts derived from patients with homozygous mutations in the DJ-1 gene revealed similar phenotype with reduced mitochondrial branching in the human condition (Fig. 3D, 3E) indicating that DJ-1 may interfere with the critical balance between mitochondrial fusion and fission dynamics. First evidence for a potential role of DJ-1 in the regulation of mitochondrial morphology came from comparative studies of different generated DJ-1 mutants that influenced the oxidative modification of a critical cysteine in position 106 [11]. In the same study, functional mitochondrial connectivity was assayed in DJ-1 KO MEF by fluorescence recovery after photobleaching (FRAP) measurement and indicated a loss of connectivity. This is consistent with our present study using direct morphometry as an independent method to assess mitochondrial connectivity. Recently, effects on mitochondrial morphology and a critical role for mitochondrial homeostasis in PD has been attributed to the loss of PINK1 function, and it was suggested that the PD- associated proteins PINK1 and Parkin may act in the same pathway to modulate mitochondrial morphology [44,47,48]. In mammalian cells, loss of PINK1 led to increased fission events causing mitochondrial fragmentation, that were rescued by Parkin [44,49]. However, in invertebrate in vivo models loss of PINK1 function increased mitochondrial fusion events, that was rescued by increasing Drp1 gene dosage [47]. Thus species-specific differences in the regulation of mitochondrial dynamics may exist and may explain the variable deflection of the critical balance between fusion and fission events in different in vivo and in vitro models. In this context it is of note that we observed a similar phenotype in a murine and human cellular model of loss of DJ-1 function. Whether the mitochondrial phenotype caused by loss of DJ-1 (our study here) and PINK1 function reflects converging signaling pathways warrants further studies. However, previous data of our own research as well as in vivo studies in Drosophila models of PINK1 inactivation argue against a functional compensation of PINK1 by DJ-1 [44,50]. Mitochondrial integrity is critically secured by autophagic clearance [51,52]. During the process of autophagy unique vesicles, autophagosomes, are generated that sequester the cytoplasmic cargo, including cellular organelles and long-lived proteins. Subsequently, autophagosomes fuse with lysosomes for cargo degradation. Autophagy occurs on a constitutive level in all eukaryotic cells and as a consequence, cellular homeostasis is secured by constant clearance and recycling of cytoplasmic constituents (basal autophagy). Importantly, due to a variety of cellular insults, including ROS production, autophagy is induced to an extended degree (induced autophagy) [32,53]. In this respect, ROS of mitochondrial origin, depolarization of mito- chondria and increased fission events each have been reported to trigger autophagic activity [41,54]. However, although we measured an increase of mitochondrial ROS production, a prominent depolarization of mitochondria in DJ-1 KO cells, and reduced mitochondrial connectivity, the basal autophagy levels in these cells were reduced compared to WT controls (Fig. 5). Autophagic pathways are an important protection system against ROS-mediated damage of proteins and organelles in the cell [52]. These pathways are compromised during aging and age- PLoS ONE | www.plosone.org 12 February 2010 | Volume 5 | Issue 2 | e9367