related disorders like PD are characterized by the accumulation of oxidized proteins and dysfunctional mitochondria [4,55,56,57]. In support, premature aging, that is hypothesized to be elementary connected to misregulated autophagy [58], represents a known mechanism involved in PD pathogenesis [59]. Importantly, loss of key autophagy proteins, such as Atg5 have been shown to be responsible for reducing basal autophagy levels that led to neurodegenerative symptoms in mouse models [60]. In Atg5- deficient peripheral T cells reduced levels of basal autophagy lead to an impaired removal of damaged mitochondria that resulted in increased mitochondrial mass [61]. Indeed we demonstrated that human and murine cells lacking physiological DJ-1 show evidence for a decrease in mitochondrial degradation (Fig. 7) and an increase in mitochondrial mass (Fig. 8A). In further support, the multilamellar lysosomal structures accumulating in DJ-1 KO cells are highly reminiscent of pigmented vacuoles that represent autophagic responses due to neurodegeneration and support the idea of a mitochondrial-lysosomal axis of aging [31]. Recent studies provided first evidence for a potential role of DJ- 1 in autophagic response to cellular stress. In human osteosarcoma cells an impairment of hypoxia-induced autophagy after DJ-1 knock-down was described by analyzing LC3 [62]. Moreover, a role of DJ-1 in the paradigm of paraquat-induced autophagy was defined in a knock-down model of DJ-1 in human neuroblastoma cells [63]. However, effects on basal autophagy as demonstrated in our DJ-1 KO cellular model, were not described in both siRNA knock-down models, most probably due to the remaining DJ-1 function mediated by residual protein levels of endogenous DJ-1 after siRNA treatment [62,63]. In our model further induction of autophagy by amino acid deprivation or rapamycin treatment showed the presence of autophagic flux in both, DJ-1 KO and WT cells. However levels of autophagic activity remained reduced for the DJ-1 KO condition compared to control cells as demonstrated by LC3 protein monitoring. Based on our findings of a functional role of DJ-1 in preserving basal autophagy the observed changes in mitochondrial connec- tivity due to loss of DJ-1 may be secondary to the accumulation of damaged mitochondria. Indeed, lysosomal storage disorders are characterized by an inefficient autophagic clearence of mitochon- dria leading to loss in mitochondrial connectivity [64]. Mitochon- drial alterations observed in lysosomal storage disorders were replicable by treatment of control cells with lysosomal inhibitors (Jennings et al., 2006). We also found that lysosomal inhibition of control MEF expressing physiological DJ-1 led to similar changes in MMP as observed in DJ-1 KO cells (Fig. 8B). In order to understand the connection between ROS produc- tion and autophagy levels in more detail, it will be of great interest to identify signaling cascades by which ROS triggers autophagy [65]. Here we provide evidence for a correlation of DJ-1 protein function and ERK1/2 signaling pathways that are involved in the regulation of autophagy. Recent studies demonstrated an involvement of ERK2 in the regulation of autophagy [41,66]. It has been shown that ERK1/2 is involved in the modulation of lysosomal degradation and that basal levels of ERK2 kinase activity are required for its effect on autophagy [67]. In this context higher levels of phopho-ERK2 and/or mitochondrial ERK2 localization induced autophagy including mitochondrial clearance [41]. Consistently, we found that DJ-1 KO cells display both, a decrease in mitochondrial ERK2 activation and basal autophagy (Fig. 5 and Fig. 8C). In further support, the neuroprotective role of DJ-1 in terms of oxidative stress was recently found to be related to the activation of the ERK1/2 signaling pathway [40]. DJ-1 in Mitochondrial Dynamics Based on previous findings regarding the function of the DJ-1 protein and our present study, we suggest that DJ-1 might function in regulating cellular organelle homeostasis. Upon mitochondrial ROS production, DJ-1 quenches ROS levels [10,11] thereby preventing cell death and preserving the integrity of mitochondria and lysosomes. Our findings suggest that loss of DJ-1 leads to the accumulation of dysfunctional mitochondria characterized by increased intramitochondrial ROS levels and reduced MMP that under physiological conditions would be compensated by increased housekeeping-degradation of damaged organelles via autophagy. However, due to disturbed autophagic clearance of damaged mitochondria, as demonstrated by lysosomal alterations that include a reduction of lysosomal activity and of basal auophagy levels, and the appearance of multilamellar lysosome- like intracellular bodies, mitochondrial dynamics shift to reduced connectivity. On this basis it will be of great interest to define the regulative signaling cascades, such as the ERK pathway, that modulate the function of DJ-1 in maintaining mitochondria and lysosomal homeostasis. Supporting Information Figure S1 Protein levels of DJ-1 in fibroblasts from carriers of the E64D mutation in the DJ-1 gene and a healthy control. The pedigree of the E64D family shows one affected patient carrying a homozyous E64D mutation (dark grey sign) and two sibling that carry the mutation in the heterozygous state (light grey sign). Western blot analyses using an antibody against human DJ-1 revealed markedly reduced levels of endogenous DJ-1 in the homozygous carrier (Mut/Mut) of the E64D mutation. Physio- logical levels of DJ-1 are represented by an unrelated healthy control (WT/WT; white sign). Heterozygous carriers (Mut/WT) were unaffected and display similar levels of DJ-1 protein as the control. Found at: doi:10.1371/journal.pone.0009367.s001 (0.18 MB TIF) Figure S2 GFP-WIPI-1 puncta-formation analyzes reveals an unaltered induction of autophagy in DJ-1 KO MEF. Quantifica- tion (Supplemental Table S1) of confocal microscopy (Supple- mental Figures S2 A-H) of GFP-WIPI-1 protein puncta as a measure for the onset of autophagy was conducted from three independent sets of experiments (100 cells in each of the experiments) in WT and DJ-1 KO MEF. Found at: doi:10.1371/journal.pone.0009367.s002 (2.53 MB TIF) Table S1 GFP-WIPI-1 puncta-formation analyzes reveals an unaltered induction of autophagy in DJ-1 KO MEF. Quantifica- tion (Supplemental Table S1) of confocal microscopy (Supple- mental Figures S2 A-H) of GFP-WIPI-1 protein puncta as a measure for the onset of autophagy was conducted from three independent sets of experiments (100 cells in each of the experiments) in WT and DJ-1 KO MEF. Found at: doi:10.1371/journal.pone.0009367.s003 (0.66 MB TIF) Acknowledgments We thank the members of the E64D family for their continuous support and interest in the study. Moreover we would like to thank W. Wurst (Helmholtz Center Munich, Institute of Developmental Genetics) for the kind disposal of DJ-1 KO mice and C. Schiesling for technical support. Author Contributions Conceived and designed the experiments: GK TPC RK. Performed the experiments: GK SR NK BM HW ZG. Analyzed the data: GK LFB HW FNG OR TPC RK. Contributed reagents/materials/analysis tools: JW DW PK RK. Wrote the paper: RK. PLoS ONE | www.plosone.org 13 February 2010 | Volume 5 | Issue 2 | e9367