Rapamycin reverses effects of parkin activity loss 1017 Figure 6. (A) Mitochondrial respiration measurements reported as percent oxygen consumption rate (OCR) versus MAO-B controls in the absence or presence of oligomycin and FCCP application (at time of arrow) / rapamycin pre-treatment. (B) Mitochondrial complex I activity reported as percent MAO-B controls. Values are expressed as mean SD ***p 0.01 & **p 0.001 compared to MAO-B. mTOR inhibition and have been demonstrated to sub- sequently increase the translation of several mitochon- drial proteins [25]. Whether parkin is a target of this action of 4EBP1 is currently under investigation in our laboratory. We hypothesize, however, that rapamycin’s ability to increase parkin levels in stressed cells is also likely to be an abortive compensatory event and that its neuroprotective affects are more likely due to its ability to prevent mTOR-mediated repression of lyso- somal autophagy independent of parkin. In the case of PD, mitochondrial dysfunction is believed to occur in response to accelerated rates of oxidative stress although the mechanisms involved have yet to be completely elucidated [7]. Our data suggests that losses in cellular mitochondrial function as a consequence of MAO-B induction may be at least in part due to effects on oxidative effects on parkin activity that impact on its ability to remove damaged mitochondria and preserve overall cellular metabolic function. Many recent therapeutic studies have indeed focused on drugs that can restore the proper turnover of damaged mitochondria including rapamycin. Rapamycin appears to promote the deg- radation of both aggregated proteins and damaged mitochondria [1] and has been shown to exhibit neu- roprotective properties in several disease models including rotenone treatment [1,14–16]. Rapamycin treatment has recently been shown to prevent mito- chondrial defects and neuronal death in cell lines derived from parkin-deﬁcient PD patients [21]. Our studies suggest that rapamycin may be an effective means of counteracting the effects of loss in parkin function by allowing cells to regain the ability to facil- itate the removal of damaged mitochondria. In conclusion, clarifying the mechanistic pathways through which oxidative stress including that induced by MAO-B induction alters parkin function and the process of parkin-mediated mitophagy will aid in the understanding hallmarks associated with autosomal recessive forms of PD. Elucidation of parkin dysfunc- tion induced by oxidative stress may in addition one day facilitate a better understanding of the more com- mon sporadic form of PD and provide mechanistic insights that will aid in the development of new ther- apeutic agents for the treatment of PD. Declaration of interest These studies were funded by R01 NS045615 (JKA) and a U54 grant. References [1] Berger Z, Ravikumar B, Menzies FM, Oroz LG, Underwood BR, Pangalos MN, et al. Rapamycin alleviates toxicity of different aggregate-prone proteins. Hum Mol Genet 2006;15:433–442. [2] Bradford MM. A rapid and sensitive method for the quanti- tation of microgram quantities of protein utilizing the princi- ple of protein-dye binding. Anal Biochem 1976;72:248–254. [3] Forno LS. Neuropathology of Parkinson’s disease. J Neu- ropathol Exp Neurol 1996;55:259–272. [4] Geisler S, Holmstrom KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ, et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol 2010;12:119–131. [5] Hardy J, Cookson MR, Singleton A. Genes and parkinson- ism. Lancet Neurol 2003;2:221–228. [6] Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, et al. Mutations in the parkin gene cause