implicates broad biological functions including modulation of transcription, chaperone-like functions and antioxidant properties [9]. Although present in various subcellular compartments including cytoplasm and nucleus, targeting of DJ-1 to mitochon- dria was reported to contribute to its physiological cytoprotective role [10,11]. In conditions of oxidative stress DJ-1 is converted into an acidic variant allowing it to quench reactive oxygen species (ROS) and to localize to the mitochondria [12]. Cell culture experiments revealed that oxidation of a specific cysteine residue in position 106 of the DJ-1 peptide sequence is responsible for mitochondrial targeting and protection against oxidation-induced cell death [10,11]. Indeed, promoting the mitochondrial localiza- tion of DJ-1 increased DJ-1 dimer formation at the outer mitochondrial membrane and the cytoprotective activity towards oxidative insults [13]. In vivo, loss of DJ-1 function is linked to increased vulnerability to complex I inhibition as shown in fly and mouse models [14,15]. Mitochondrial dysfunction plays a central role in neuronal cell death in PD. Impaired mitochondrial function is critically linked to imbalanced dynamic fusion and fission events of mitochondria, to energetic depression and may subsequently result in the activation of programmed cell death mechanisms [16,17,18]. Hence, selective removal of dysfunctional mitochondria by lysosomal degradation pathways is critical for the maintainance of cellular integrity [17,19]. Although a critical role of DJ-1 for mitochondrial homeostasis has been recognized by different in vitro and in vivo approaches, no effects on mitochondrial dynamics and down- stream lysosomal degradation pathways, such as macroautophagy (hereafter = autophagy) were reported. Here we provide evidence that loss of DJ-1 function causes a prominent disturbance of both, mitochondrial function and morphology, that is linked to decreased basal autophagy and impaired lysosomal degradation. Methods Cell Culture For the functional analysis of the DJ-1 protein experiments were performed in immortalised DJ-1 knockout (KO) and DJ-1 wild- type (WT) mouse embryonic fibroblasts (MEF) that have been described previously [20]. Stably back-transfected DJ-1 MEF were generated by transfecting DJ-1 KO MEF with pcDNA3.1/Zeo (Invitrogen, USA) vector containing a DJ-1 WT construct or the empty control vector, respectively. Transfection was performed using Fugene 6 HD (Roche Diagnostics; Mannheim) according to the manufacturers’ instruction. MEF cells were cultured in a 5% CO2 humidified atmosphere in DMEM medium (Invitrogen, USA) containing penicillin, streptomycin (Gibco, Invitrogen, USA), and 10% fetal calf serum (FCS, Biochrom, Germany). Stable transfected DJ-1 knock-out MEF cells were cultured in DMEM medium (Invitrogen, USA) containing Zeocin (Invitrogen, USA, 400 mg/ml) and 10% fetal calf serum (FCS, Biochrom, Germany) in a 5% CO2 humidified atmosphere. Moreover fibroblasts from members of the family carrying the E64D mutation in the DJ-1 gene and a healthy control were included in our analyses [8]. Skin biopsies were taken from the index patient carrying the homozygous E64D mutation, two unaffected sibs carrying the E64D mutation in the DJ-1 gene in a heterozygous state and a healthy age-matched control individual. The study was approved by the ethics committee of the University of T u¨bingen. All patients and controls gave written and informed consent. Primary fibroblast cells were maintained in RPMI medium with 10% FCS supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin and 1 mM pyruvate. DJ-1 in Mitochondrial Dynamics Respirometry Mitochondrial respiration of MEF cells (26106 cells/ml) was measured with an OROBOROS-oxygraph in Hanks solution at 30uC as described previously [21,22]. Cell homogenates were prepared by gentle homogenisation (107 cells/200 ml HBSS buffer consisting of 132 mM NaCl, 5.4 mM KCl, 0.44 mM KH2PO4, 0.34 mM NaH2PO4, 0.49 mM MgCl2, 0.41 mM MgSO4, 10 mM HEPES, 1 mM CaCl2, 10 mM pyruvate, pH = 7.3). Multiple substrate inhibitor titration of mitochondrial respiration was performed with cell homogenates maintained in MMMPK buffer (5 mM MgCl2, 120 mM mannitol, 40 mM MOPS, 5 mM KH2PO4, 60 mM KCl, pH = 7.4) as described previously [23]. Assessment of Mitochondrial and Lysosomal Parameters by Fluorescent-Activated Cell Sorting (FACS) Analysis of intramitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), mito- chondrial mass and lysosomal activity in DJ-1 KO MEF cells compared to DJ-1 WT MEF cells in living cells was performed by a FACS-based methods. Cells were harvested with 2 mM EDTA solution in PBS and washed with PBS. To analyze the production of intramitochondrial ROS cells were incubated with PBS containing 2 mM MitoSOXH (Invitrogen, USA) for 15 min at 37uC. The MMP was measured by staining with 200 nM tetramethylrhodaminemethylester (TMRM; Invitrogen, USA) in PBS for 15 min at 37uC. Mitochondrial mass and lysosomal activity in MEF were determined by staining the cells with either 50 nM MitoTrackerH Green FM (Invitrogen, USA) or 1 mM LysoTrackerH (Invitrogen, USA), respectively. To determine the relative uptake of MitoTrackerH or LysoTrackerH the threshold was set at about 50% of the WT signal intensity and the percentage of shift in the fluorescence activated cell sorter (FACS) staining was determined with the Summit version 4.2 software (Beckman Coulter). For each sample, at least 30.000 cells were analyzed for the corresponding fluorescence on a CyAnTM ADP apparatus (Beckman Coulter) with a 488-nm or 415-nm laser. Experiments were performed three times with similar results. Immunocytochemistry and Transfection For immunocytochemistry MEF cells were grown on collagen- coated slides and transfected with either GFP-LC3 (kindly provided by Tamotsu Yoshimori to TP-C) or the corresponding empty EGFP vector control. Cells were transfected using Lipofectamine 2000 (Invitrogen, USA) according to the manufac- turer’s introduction. Forty eight hours after transfection cells were washed with PBS, fixed with 4% (wt/vol) PFA in PBS for 10 min and permeabilized with 0.1% (vol/vol) Triton X-100 for 10 min. After washing with PBS cells were incubated for 30 min in 10% (vol/vol) FCS in PBS for blocking. Cells were labelled at 4uC overnight with a specific antibody against the protein of interest. The next day cells were washed with PBS and labelled with a combination of either Cy3-conjugated anti-mouse and FITC- conjugated anti-rabbit or Cy3-conjugated anti-rabbit and FITC- conjugated anti-mouse secondary antibodies (Dianova, Germany). Cell nuclei were stained with the fluorescent chromatin dye Hoechst 33342 (Molecular Probes, USA). Immunofluorescence Microscopy Mitochondrial morphology was analyzed by life cell imaging. MEF cells or human fibroblasts were cultured in Lab-TekHII chambered coverglasses (#155382; Nalge Nunc International). Mitochondria were stained with 200 nM MitoTrackerH Green PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9367