Figure 1. Respirometric detection of mitochondrial dysfunc- tion in intact DJ-1 deficient and DJ-1 WT MEF cells. (A) Mitochondrial oxygen consumption was measured with high resolution respirometry in freshly harvested MEF. Cells (26106 cells/ml) were incubated in a Hank’s medium containing 10 mM pyruvate. Endoge- nous respiration was observed without any further additions. Oligo- mycin resistent respiration was followed in the presence of 10 mg/ml oligomycin. Then uncoupled respiration was measured by stepwise additions of 100 nM FCCP. Data as means 6 SE of 13 (wild type MEF, .) and 11 (KO MEF, N) single incubations performed of 5 different cell preparations. DJ-1 KO MEF showed significantly reduced oxygen consumption compared to controls (#: p,0.05, one-way ANOVA). (B) Mitochondria were investigated by respirometry in cell homogenates of DJ-1 KO cells and WT controls using multiple substrate inhibitor titration. Measurements were performed in homogenates from 36106 cells/ml MMMPK buffer with 10 mM pyruvate and 2 mM malate as substrates (Additions: ADP, 5 mM ADP; Cyt c, 10 mM cytochrome c; NADH, 150 mM NADH; Rot, 1,5 mM rotenone, Suc, 10 mM succinate; Cat, 50 mM carboxyatractylate). Blue lines indicate the oxygen concentration in the oxygraph (left ordinate), red lines represent the first derivative of the oxygen time curve, directly indicating the rate of respiration (right ordinate). Typical respirograms from four independent experiments with similar results are shown. The state 3pyr/mal/state 3suc –ratio was 0.5760,12 for DJ-1KO cells and 0.9360,15% for WT cells (p,0.05). doi:10.1371/journal.pone.0009367.g001 substrate succinate allowed the measurement of state 3suc.respira- tion. In both types of cells the complex II dependent respiration was comparable to each other indicating that the impairment of DJ-1 KO cells is restricted on the complex I-related mitochondrial metabolism. The ratios calculated from complex I- and complex II-dependent state 3 respiration measured in the same sample (state 3pyr/mal/state 3suc) allow the quantification of the complex I- DJ-1 in Mitochondrial Dynamics related mitochondrial impairment [23]. Specifically state 3pyr/mal respiration was significantly reduced in DJ-1 KO cells (239%). By additions of cytochrome c and NADH to respiring mitochondria is it possible to prove whether the permeability of outer or inner membranes for these molecules is pathologically changed [23][26]. However, the similar and only modest increase of state 3pyr/mal respiration in DJ-1 KO and WT cells (Fig. 1B) indicated intact mitochondrial membranes in both types of cells. Finally the nonphosphorylating rate of respiration was measured by inhibition of the ADN-carrier with carboxyatractyloside. As also shown for the measurements in intact cells (Fig. 1A) there was no change of nonphosphorylating rate of respiration in DJ-1 KO cells. All these data show, that the mitochondrial impairment in DJ-1 KO cells is not caused by a general mitochondrial dysfunction but by specific impairments of complex I-dependent oxidative phosphorylation in DJ-1 KO cells which can cause energetic depression of cell metabolism [16,18]. Increase in Intramitochondrial ROS Formation in DJ-1 KO Cells Next, we investigated how depletion of DJ-1 in our KO model interferes with mitochondrial function and integrity. One important pathophysiological property of mitochondria is the generation of ROS. Intramitochondrial ROS production was monitored and quantified by using MitoSOX RedTM (Molecular Probes, USA,), a fluorogenic dye that is targeted to the mitochondria and readily oxidized by superoxide, but not by other ROS- or reactive nitrogen species–generating systems. We found a significant increase in intramitochondrial ROS in MEF from DJ-1 KO mice compared to wild type controls (p = 0.011; Fig. 2A). Treatment of MEF with 5 mM H2O2 further increased intramitochondrial ROS production in both DJ-1 KO MEF and MEF from WT DJ-1 littermates with a higher ROS content in KO cells (data not shown). These results were replicated in 3 independently prepared MEF cell lines from either KO mice or wild type littermates. Interestingly, in the cytoplasmic compart- ment no increase in basal ROS was observed using H2DCFDA [20]. This is in line with loss of the physiological antioxidant function of DJ-1. Decreased MMP in MEF from DJ-1 KO Mice Based on the alterations in oxygen consumption and increased intramitochondrial ROS production we next examined the effects of DJ-1 deficiency on the MMP as assessed by flow cytometry using TMRM (Invitrogen, USA). Analysis of DJ-1 KO MEF revealed a reduction of the MMP when compared to wild type controls, supporting an involvement of DJ-1 in mitochondrial homeostasis (p,0.001; Fig. 2B). In order to determine whether exogenous DJ-1 would be able to rescue the observed mitochondrial dysfunction in DJ-1 KO cells, we stably transfected DJ-1 KO MEF with a plasmid expressing human DJ-1 cDNA or an empty vector control. Using TMRM staining, we found a significant increase in MMP in KO cells stably transfected with DJ-1 compared to empty vector controls (p = 0.027; Fig. 2C). These results indicate that loss of DJ-1 leads to impaired mitochondrial function that can be rescued by reintroduction of physiological DJ-1 protein into the KO background. Changes in Mitochondrial Morphology Due to Loss of DJ- 1 Function Recent data indicate that dynamic morphological alterations of mitochondria are involved in mitochondrial dysfunctions [17]. PLoS ONE | www.plosone.org 4 February 2010 | Volume 5 | Issue 2 | e9367