1012 A. Siddiqui et al. been associated with functional loss of the protein’s E3 ligase activity [6]. Many of these mutations appear to alter the solubility of parkin, resulting in the formation of cytoplasmic parkin aggregates that may account for its enzymatic inhibition. In addi- tion to genetic mutations, exposure to oxidative stress has also been reported to result in the forma- tion of insoluble parkin aggregates and decreased E3 ligase activity [11]. Here, we report that induction in human MAO-B levels in vitro to levels associated with sporadic forms of PD results in reductions in parkin’s E3 ligase acti- vity. These reductions did not prevent the protein from being recruited to damaged mitochondria, but did result in an accumulation of damaged mitochon- dria and an overall cellular reduction in mitochon- drial function. Co-treatment of cells with the mTOR inhibitor rapamycin was found to promote clearance of damaged mitochondria and to rescue losses in mitochondrial deﬁcits elicited by MAO-B increase. Methods All chemicals were from Sigma unless otherwise noted. Generation of inducible MAO-B cell lines constitutively-expressing human parkin A stable doxycycline (dox)-inducible human MAO-B PC12 cell line previously generated and characterized by the Andersen laboratory [9] was used for the cur- rent studies. MAO-B cells were transfected with 3X-FLAG vector containing an insertion of human parkin cDNA (gift of Keio University School of Medicine Tokyo, Japan) and a neomycin selection marker (Clontech) using Lipofectamine 2000 reagent (Invitrogen). Parkin-containing clones were selected via growth at 37°C in Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing 10% Tet-FBS (Clon- tech), 5% horse serum (GIBCO), 1% streptomycin- penicillin (GIBCO) and 200 μg/ml of G418 (Cellgro). Transfection efﬁciency was determined via Western blot analysis using an anti-mouse 3X-FLAG antibody (Sigma–Aldrich). Cells were differentiated into neural cells using 50 ng/ml of nerve growth factor (NGF) (Sigma–Aldrich) administered one day prior to dox treatment. Doxycycline, FCCP and rapamycin treatment Oxidative stress conditions were induced via treatment with dox (40 μg/ml, Sigma–Aldrich) for 16 hours to stimulate MAO-B expression [9]. Non-toxic rapamy- cin concentrations used for described studies were initially determined via cell viability analyses using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were pre-treated with rapamycin (Sigma–Aldrich) for 1 hour prior to dox treatment. For experiments involving FCCP, cells were treated with 4 μM of the agent for 1 hour follow- ing dox treatment in the absence or presence of rapamycin prior to cell collection. E3 ligase activity assay via assessment of parkin auto-ubiquitination Immunoprecipitation of transgenic human parkin protein was carried out using Dynabeads® anti- Mouse IgG magnetic beads (Invitrogen). Cellular protein fractions were pre-cleared of non-speciﬁc IgG antibody using non-antibody bound beads for 1 hour at 4°C. While samples were being pre-cleared, 3X-FLAG antibody was bound to magnetic beads via incubation at 4°C for 2 hours. Pre-cleared samples were then immunoprecipitated using 3X-FLAG anti- body for 2 hours. Samples were incubated in E3 ligase cocktail (2 mM ATP, 50 mM Tris-Cl, pH 7.4, 2.5 mM MgCl2, 100 ng E1 ligase and 250 ng E2 ligase) either with or without 10 μg ubiquitinCH5 (UbCH5) for 2 hours. Parkin auto-ubiquitination was analysed by subsequent immunoblot analysis (see below) using parkin or ubiquitin antibodies; actin was used as a loading control. Mitochondrial fractionation Mitochondrial fractionation was carried using the QIAGEN Qproteome™ mitochondria isolation kit. All buffers and solutions used in this protocol were supplemented with protease inhibitor. After collec- tion in PBS, cell suspensions were centrifuged at 4°C at 300 g for 3 minutes, followed by the removal of supernatant. The cell pellet was then re-suspended in ice-cold lysis buffer for 10 minutes at 4°C followed by centrifugation at 1000 g for 10 minutes at 4°C. The resulting supernatant was removed and retained as the cytoplasmic fraction. The cell pellet was re- suspended in ice-cold disruption buffer and then centrifuged at 1000 g for 10 minutes at 4°C. The resulting supernatant was centrifuged at 6000 g for 10 minutes at 4°C.The resulting whole mitochondrial pellet was washed in mitochondrial storage buffer and centrifuged at 6000 g for 20 minutes at 4°C. The resulting supernatant was discarded and the mito- chondrial pellet re-suspended in mitochondrial stor- age buffer for use in subsequent assays. Immunoblot analyses For whole cell westerns, cells were lysed in RIPA buffer [50 mMTris, pH 7.4, 1% Igepal, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and a proteinase inhibitor cocktail (Roche Molecular