glutathione-S-transferase enzyme exacerbated the neurodegener- ative phenotype [57]. Conversely, re-introduction of GST was found to rescue the mitochondrial swelling and neuronal loss in parkin knockouts [58]. Clearly, there exists a complex interrela- tionship between glutathione levels, respiratory chain impairment, mitochondrial morphology, and cell viability within neurons which both PINK1 and parkin co-operate [32]. Altered mitochondrial integrity and proliferation Interestingly a disrupted mitochondrial morphology was seen in aged human neurons lacking PINK1. Neuronal mitochondria appeared enlarged, with disrupted cristae and greatly reduced matrix volume. This finding corroborates the striking muscle phenotype in PINK1 and parkin knockout fly models, where myofibrils contain vacuolated, swollen and dysmorphic mitochon- dria, with disorganised christae [31,32,34]. Enlarged mitochondria were also seen in surviving dopaminergic neurons, the frequency of which increased with age [32]. Using genetic complementation experiments PINK1 was subsequently shown to function ‘up- stream’ of parkin to regulate mitochondrial integrity [32]. Recently, it was found that loss-of-function of the mitochondrial fission-promoting component Drp1, caused lethality in PINK1 or parkin knockout flies [59]. Conversely, over-expression of Drp1 or mutations in mitochondrial fusion-promoting proteins (Mitofusin2 and OPA1) suppressed the PINK1 and parkin phenotype. These experiments suggest that these PD genes co-operate to regulate mitochondrial dynamics and that mitochondrial fission (splitting) is stimulated in response to inactivation of the PINK1- parkin pathway. We provide interesting evidence for mitochondrial proliferation in PINK1 knockdown neurons, including increases in mean mitochondrial number per neuron, increased Mitotracker uptake, enhanced expression of respiratory chain complex subunits and elevated citrate synthase activity. Despite an increase in mass, no significant change in respiratory chain activity was detected in neurons lacking PINK1, implying that oxidative phosphorylation may be impaired in the increased numbers of mitochondria in these cells. It is established that increased levels of intracellular ROS may mediate changes in mitochondrial abundance and mtDNA copy number which occur during the ageing process [60]. The oxidative stress induced proliferation of mitochondria may initially be beneficial to compensate for dysfunctional mitochon- dria, and to supply ATP needed for cell survival. However increased numbers of mitochondria will also result in a further excess of ROS, which in the presence of insufficient antioxidant defense mechanisms, will produce more oxidative damage to cells. Increased oxidative damage will render neurons susceptible to death by apoptosis from mitochondrial membrane permeabilisa- tion and cytochrome c release, or by necrosis due to insufficient ATP production. An increase in mitochondrial proliferation has so far not been reported in any other PD model. PINK1 deficiency and lysosomes We also report here a novel phenotype in the surviving neurons of PINK1 deficient neurons which resembles the pathology observed in lysosomal storage diseases. Large intracellular bodies comprised of multivesicular ‘aggregates’ were detected in ageing PINK1 kd neurons, which were lysosomal in nature. PINK1 has not previously been implicated in lysosomal dysfunction but endogenous PINK1 expression has been detected in microsomes [35]. Lysosomes are present within autophagosomes, the multi- lamellar bodies responsible for proteolytic degradation of macro- molecules and thought to be crucial in the clearance of amyloidogenic proteins such as a-synuclein and huntingtin [61]. PINK1 Deficiency Autophagy involves the non-selective degradation of proteins and organelles by lysosomes. However selective autophagy for certain damaged organelles such as mitochondria may occur in response to ROS. Moreover it has been proposed that mitochondria may act as a major source of the ROS signal required to activate autophagy. In our model of PINK1 deficiency we see an increase in autophagolysosomes suggesting an upregulation of autophagy. This may be occurring as a response to remove oxidatively damaged proteins and/or structurally abnormal mitochondria. The concurrent increase in cytosolic and mitochondrial ROS production may act as a signal for this process [62]. The lysosomal pathology we find in neurons resembles that seen in SHSY5Y cells over-expressing mutant b-synuclein protein [63]. b-synuclein is thought to be neuroprotective by interacting with and preventing the formation of a-synuclein inclusions. Pharmacological preven- tion of autophagy exacerbated cell death and a-synuclein toxicity, suggesting that they may fulfil a protective function in neurons. Enhancing formation of lysosomal clearance mechanisms in PD may therefore be a viable therapeutic strategy. In summary, to study the role of PINK1 in PD, we have generated models of PINK1 loss of function in human and mouse neurons. PINK1 is up-regulated during neuronal differentiation and plays a major role in the neuroprotection of mature neurons. Neurons that lack PINK1 function are prone to apoptosis via the intrinsic mitochondrial apoptosis pathway. Moreover loss of PINK1 function results in increased levels of oxidative stress and reduced mitochondrial membrane potential, suggesting that mitochondrial dysfunction directly leads to the increased suscep- tibility to apoptosis. Further characterisation of the molecular events within mitochondria that lack PINK1 will enable a better understanding of the pathophysiologic mechanisms that cause the phenotype of PINK1 loss-of-function. Persistent mitochondrial dysfunction over time ultimately results in structural changes of the mitochondria which are reminiscent of PINK1 knockout fly models, but to date have not been detected in mammalian models. To our knowledge this is the first study to provide a functional role for PINK1 in human neurons, and specifically midbrain derived neurons. Modelling human neurodegenerative disease may only be successfully achieved through recapitulation of the ageing process. Under these circumstances we find an age-specific phenotype for loss of PINK1 function. This provides unique insights into the pathogenesis of sporadic PD in which the phenotype of oxidative stress, mitochondrial dysfunction and neuronal apoptosis has been well established. Further it suggests a convergence of the molecular pathways of PINK1-associated PD and sporadic PD. Supporting Information Methods S1 Supplementary Methods. Found at: doi:10.1371/journal.pone.0002455.s001 (0.04 DOC) Table S1 Found at: doi:10.1371/journal.pone.0002455.s002 (0.05 DOC) MB MB Figure S1 A) Graph showing the increase in mRNA of different markers of dopamine neurons following differentiation of NSCs to neurons, using the pre-D method. Expression of TH, DAT, lmx1a, and Nurr1 are significantly elevated upon differentiation. Levels are normalized to housekeeping gene ATPB5, and shown relative to fetal brain tissue expression. B) Graph showing the effects of four different shRNA constructs on PINK1 gene expression in SHSY5Y cells. Values are mean RQs of five PLoS ONE | www.plosone.org 14 June 2008 | Volume 3 | Issue 6 | e2455