PINK1 Deficiency subunits in young (D5) and aged (D43) human neurons lacking PINK1 compared to controls. Note the increase in complex subunit expression in aged neurons lacking PINK1. Replicate lanes of pooled samples from three independent cultures are shown. Expression of OXPHOS subunits C-I-20 (ND6), C-II-30 (FeS), C-III-Core-2 and C-V-a is shown. b-actin; loading control. E) Graph showing significantly increased mean number of mitochondria/cell in aged PINK1 kd human neurons compared to controls, as quantified by TEM analysis. Histogram shows mean number mitochondria per cell 6sem averaged from at least 50 cells. F) Increased frequency of abnormal mitochondria of aged human PINK1 kd neurons compared to controls, quantified using TEM. Histogram shows mean frequency of abnormal mitochondria (per total number mitochondria in each cell) 6sem, n = 50 cells. G) TEM images showing abnormal mitochondria (arrowheads) within aged (d47) human neurons lacking PINK1 (panel b, and at higher magnification in d) compared to control neurons (panel a). There are increased numbers of mitochondria within cells lacking PINK1 (panel b) compared to controls (panel A) and a higher proportion of mitochondria in PINK1 kd neurons appear swollen with disorganised christae (panels c, d). Scale bar in a,b; 10 mM, c = 500 nM. doi:10.1371/journal.pone.0002455.g005 the mitochondria and cytochrome c release to the cytoplasm occurs earlier than in controls, and results in elevated levels of caspase activation (caspase-3 and caspase-9). In turn, increased levels of PARP cleavage and nuclear fragmentation are observed in PINK1 kd cells. Our findings are in line with previous reports demonstrating that PINK1 over-expression protects cells from STS-induced apoptosis [35] and transient RNAi-mediated PINK1 kd sensitises cells to various stressors [37,38,42,43]. Our findings confirm the requirement of PINK1 within human neurons for the regulation of mitochondrial permeability transition, and preven- tion of caspase-3 mediated neuronal apoptosis. Our model shows some similarities to toxin based models of PD: the administration of MPTP to mice results in dopaminergic neuronal loss mediated by Bax translocation to the mitochondria, cytochrome c release, and activation of caspase 9 and caspase 3. In corroboration, over- expression of PINK1 in the SN of rats protects against the effects of MPTP [38]. In the MPTP model, the sensitivity to Bax- mediated apoptosis is determined by high levels of ROS (resulting from MPTP induced complex 1 deficiency) increasing the soluble pool of cytochrome c in the intermembrane space that may be released on stimulation by Bax. [44,45] In our model, PINK1 deficiency is also associated with high levels of mitochondrial ROS production and thus the increased sensitivity to mitochondrial apoptosis may operate by a similar mechanism. PINK1 deficiency causes loss of mitochondrial membrane potential (ym) Mitochondrial dysfunction is strongly implicated in the pathogenesis of PD, either as a cause or downstream hallmark of dopaminergic degeneration [46]. PINK1 is targeted to mitochondria where it is has been proposed to maintain mitochondrial membrane potential (ym), the driving force behind oxidative phosphorylation and ion transportation. Over-expres- sion of wild type PINK1 in cell lines was found to protect cells from membrane depolarisation in response to proteasomal stress [27,47]. Using live imaging techniques we show that PINK1 deficiency causes a reduction in the basal ym in human neurons, implicating a role for PINK1 in the maintenance of membrane potential, even in the absence of stress. This is a significant finding as mitochondrial potential is critical for the synthesis of ATP through the dissipation of the transmembrane proton gradient. ATP availability is critical for cellular anabolism and importantly for neurons, maintaining a resting plasma membrane potential for cell excitability. A reduced proton gradient would explain decreased ATP levels in PINK1 fly knockout models [32,34] and it would be interesting to investigate whether this is due to a reduced ym in vivo. PINK1, ROS generation and glutathione levels One important sequelae of mitochondrial dysfunction is increased generation of reactive oxygen species, which in turn can cause oxidative stress and damage to macromolecules within cells [11]. This damage extends to the mitochondrial respiratory components themselves, compromising function further. Evidence of oxidative stress and damage to proteins, lipids and nucleic acids has been found in the substantia nigra of sporadic PD patients and more recently, in fibroblasts from patients carrying PINK1 mutations [48]. We show here that in the absence of PINK1, ROS generation in both mitochondria and cytoplasm is increased, even in young human neurons. PINK1 knockout flies demonstrate increased sensitivity to oxidative stress [31,33] and increased ROS generation and oxidative stress is similarly found in animal models of parkin and DJ-1 loss-of-function [49–52]. Hence ROS generation and elevated oxidative stress appears to be a unifying cellular phenotype in recessive Parkinsonism. This is the first demonstration that increased ROS production occurs early in a mammalian model of PD prior to neuronal stress and apoptosis, and hence suggests that it has an important role in the pathogenesis of neuronal dysfunction and death. PINK1 may exert its protective effect on ym and the prevention of ROS generation via phosphorylation of Tumor-necrosis factor associated protein-1 (TRAP1). TRAP1 (Hsp75) is an ATP-binding molecular chaperone co-localises with PINK1 in mitochondria, and is phosphorylated by PINK1 in vitro [43]. Silencing of TRAP1 in tumor cells also causes mitochondrial swelling, loss of ym, rapid increase in intracellular ROS generation and release of cyto- chrome C, identical to the effects of PINK1 knockdown [43,53]. Rescue experiments placed TRAP1 downstream of PINK1, with phosphorylation of TRAP1 required for its protective activity, which indirectly prevents the release of cytochrome C and ROS generation in response to apoptogenic agents such as hydrogen peroxide. The role of a putative PINK1-TRAP1 interaction in dopaminergic neurons has yet to be investigated, but could provide a key to selective degeneration of these cells if heavily utilised as a protective pathway during stress. The primary mechanism for detoxification of ROS in the cytoplasm is the oxidation of reduced glutathione (GSH), which is regenerated harmlessly to the reduced species by the flavoenzyme glutathione reductase. We show that there is a significant decrease in steady state levels of total glutathione in primary human neurons lacking PINK1, which is likely to greatly impair their antioxidant defences. Evidence for decreased total GSH levels in the brains of patients with idiopathic PD has been widely reported and may occur as a result of decreased GSH synthesis, which is ATP dependent and thus compromised as a result of failing membrane potential [54]. Chronic inhibition of glutathione synthesis in dopaminergic neurons in vitro using buthionone-S- sulfoximime (BSO) reduced long term neuronal viability [55]. Numerous studies in rodents have demonstrated that glutathione deficiency by BSO administration results in mitochondrial swelling with vacuolization and rupture of the cristae and mitochondrial membranes seen by EM [56]. This may explain our observations of altered mitochondrial morphology in PINK1 deficient human neurons (see below). The importance of glutathione in recessive PD has already been explored: depleting parkin flies of PLoS ONE | www.plosone.org 11 June 2008 | Volume 3 | Issue 6 | e2455