1016 A. Siddiqui et al. Figure 5. Z-stack images of parkin (green), LC3 (red), and merged (yellow) were taken using LSM 510 confocal microscopy. (A) MAO-B; (B) MAO-B; (C) MAO-B, 1 μM rapamycin; (D) MAO-B, 1 μM rapamycin. Images were taken at the following parameters: (x) 126.8 μm x (y) 140.6 μm x (z) 7.0 μm. data was further veriﬁed by Western blot analysis of LC3 levels (data not shown). In agreement with data from other laboratories (1, 14, 21), pre-treatment with rapamycin was found to increase LC3 expres- sion, suggesting restoration of mitophagy reduced as a consequence of MAO-B induction. MAO-B induction results in a loss in spare capacity and mitochondrial complex I activity which are restored by rapamycin treatment Reductions in mitophagy could result in subsequent reductions in overall cellular metabolic function. Levels of overall mitochondrial respiration in response to MAO-B in cells expressing human parkin were there- fore analysed using the Seahorse XF Extracellular Ana- lyzer. Basal respiration rates were measured followed by measurement of respiration in the presence of oligomy- cin and FCCP to assess ATP synthesis efﬁciency and maximal respiratory capacity, respectively. Results from these studies (Figure 6) demonstrate that MAO-B induction results in effects on both oligomycin-sensitive respiration and maximal respiratory rates that were reversed in the presence of rapamycin treatment. Human parkin expression alone appeared to have no signiﬁcant effects on either (data not shown). Mitochondrial com- plex I activity, another measure of mitochondrial func- tion, was also found to be inhibited in the presence of MAO-B induction and restored by rapamycin. Discussion and conclusions Major ﬁndings from this study suggest that, under con- ditions of MAO-B induction related to aging and PD, parkin’s E3 ligase activity is inhibited. This is in agree- ment with previous studies demonstrating that applica- tion of exogenous oxidants such as H 2 O 2 result in reductions in parkin solubility, formation of insoluble aggregates and loss of E3 ligase activity [11,19]. Parkin is still, however, able to translocate to the mitochon- dria; indeed this ability appears to be heightened par- ticularly under conditions of mitochondrial damage and depolarization (MAO-B, FCCP), suggesting that mitochondrial recruitment of the protein is separate from its enzymatic function. However, losses in par- kin’s E3 ligase activity appears to have marked effects on its ability to participate in mitochondrial turnover. The homeostatic turnover of damaged mitochondria is essential to cellular maintenance, as these organelles are recognized as master regulators of cellular function via their involvement in both energy production and the promotion of cell death via apoptosis [7,22]. Accu- mulation of damaged mitochondria within affected cells can lead to cellular dysfunction; we observed in our studies that reductions in lysosomally-mediated mitochondrial clearance as a consequence of elevations in MAO-B correlated with decreased efﬁciency of ATP synthesis, spare respiratory capacity, and mitochon- drial complex I activity. The mTOR inhibitor rapamy- cin was able to counteract the effects of loss of parkin function via increased mitophagy, resulting in improved cellular metabolic function. An interesting ﬁnding in this study is the noted increase in parkin protein levels in response to MAO-B induction. We speculate that this may a compensatory response to the presence of associated elevations in mitochondrial damage, but likely abortive in the con- text of chronic on-going oxidative stress and continued inhibition of parkin E3 ligase activity. We also noted an increase in parkin protein levels in the context of rapamycin treatment, particularly in the presence of MAO-B or FCCP–mediated mitochondrial stress. Its up-regulation in this context could be a consequence of increased levels of the downstream mTOR target 4EBP1 [25]. 4EBP1 levels are increased following