both inherited and sporadic disease; namely protein aggregation and impairment of the ubiquitin proteasome system, mitochon- drial dysfunction, oxidative stress and protein phosphorylation [8]. The role of mitochondrial dysfunction in PD has been suggested since the original discovery that the complex I inhibitor 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused the develop- ment of PD in recreational drug users [9]. Other complex I inhibitors including rotenone and paraquat have similarly been found to cause PD–like symptoms in rodent models [10]. Inhibition of the mitochondrial respiratory chain is known to increase the generation of free radicals leading to cellular oxidative stress within cells [11]. Concordantly, evidence of impaired complex I activity has been reported in post-mortem PD brain tissue with an increase in markers of oxidative stress [12]. However the most convincing evidence to date has been the characterisation of genes mutated in familial PD with putative functional roles within mitochondria. Both PINK1 and Omi/HtrA2 have predicted mitochondrial targeting sequences and appear to exhibit protective functions within this organelle [5,6,13]. Parkin, DJ-1, a- synuclein and LRRK2 have also been shown to associate with the mitochondrion or impact upon its function, but the mechanisms involved remain unclear [14–17]. PINK1 is a highly conserved 581 amino acid protein with a catalytic serine/threonine kinase domain with close sequence homology to CAMK1 [5]. Several studies have demonstrated that recombinant PINK1 can undergo autophosphorylation as well as phosphorylate generic substrates in vitro [18–20]. It has a predicted N-terminal mitochondrial targeting motif and a significant proportion has been localised to both the inner and outer mitochondrial membranes [5,19,21]. Full length PINK1 pre- protein (,63 kDa) can also be cleaved to a ‘mature’ form (54 kDa) by an unknown protease [21,22]. PINK1 mRNA is ubiquitously expressed in human tissues, with highest expression in heart, muscle and testes [23]. It is uniformly expressed in mammalian brain, with highest expression levels found within the cell bodies of neurons and glia [21,24]. Homozygous and compound heterozygous mutations in the PINK1 gene locus are known to cause PARK6 familial Parkinsonism, which is indistinguishable from idiopathic PD apart from an earlier age of onset [5]. To date there are no neuropathological data from any individual affected with a homozygous mutation in the PINK1 gene. However, brains of patients with PINK1 heterozygote mutations display the typical pathological hallmarks of idiopathic PD [21]. The prevalence of PINK1 mutations in autosomal recessive early onset PD range from 0-15%, depending on the patient series analysed [25–27]. This suggests that PINK1 is the second most common causative gene in early onset PD after parkin [25]. The vast majority of pathogenic mutations in PINK1 gene are located within the kinase domain and include nonsense, missense and deletion mutations which are predicted to either reduce or obliterate kinase activity [20]. Accordingly, reduced kinase activity has been demonstrated in vitro for the pathogenic mutations G309D, L437P, G386A and G409V [18,20]. The most common mutation, a C1366T transition, reportedly triggers nonsense-mediated mRNA decay, resulting in a 80–90% reduction in transcript levels in tissues from homozygous patients [28]. Taken together, these findings suggest that PARK6 parkinsonism results from a loss-of-function of the PINK1 protein. Valid animal models of PINK1 parkinsonism should recapitu- late the motor symptoms seen in patients as well as the underlying nigrostriatal deficiencies and neuropathological findings. Howev- er, no motor impairments or other non-motor parkinsonian symptoms have been reported in PINK1 loss-of-function mouse PINK1 Deficiency models to date [29,30]. In contrast, Drosophila models of PINK1 deficiency have given us closer insights into the molecular mechanisms involved [31–34]. Motor deficits include abnormal wing posture, rigidity, flight impairment and reduced climbing ability [31,32,34]. Moreover, some of these motor phenotypes were found to be progressive with age [32,34] and showed partial but significant decreases (,10%) in the numbers of dopaminergic neurons in certain key clusters within the fly brain [32–34]. Ultrastructurally, muscle fibres contained vacuolated, swollen and dysmorphic mitochondria, with disorganised christae which was also seen in surviving dopaminergic neurons [32]. Both ATP production and resistance to oxidative stressors was compromised in PINK1 deficient flies. Although fly models of PINK1 loss-of- function have recapitulated mitochondrial dysfunction and age- related neuronal death, the actual mechanisms remain unclear. Cellular pathogenesis of PINK1 loss of function in PD The molecular mechanism by which PINK1 operates in the mitochondria is undefined, but the first in vitro cellular studies of PINK1 loss-of-function implicated a role for the kinase in maintenance of mitochondrial membrane potential and preven- tion of stress-induced apoptosis [5]. Over-expression of wild-type (but not pathogenic variants) in tumour cell lines was subsequently shown to prevent release of cytochrome c from mitochondria and caspase-3 activation during stress [5,35,36]. In contrast, depletion of PINK1 from cells using transient RNA interference (RNAi) was shown to increase the rate of basal and stress-induced apoptosis [37,38]. A novel pathway for PINK1 has recently been proposed following the identification of the serine protease HtrA2/Omi as a PINK1 interactor. Mutations in HtrA2 are a susceptibility factor for PD (PARK13 locus) and loss of function models in mice display a neurodegenerative phenotype resembling parkinsonism. Phos- phorylation of HtrA2 at Ser142 is dependent on PINK1 and the phosphorylated form demonstrates increased protease activity and enhanced protective effect in cells upon stress [13]. To date the majority of studies of PINK1 function come from non-mammalian models, or non-neuronal in vitro cell models cultured over relatively short periods of time. Whilst yielding important insights they do not recapitulate the human disease, that is, in ageing human dopaminergic neurons. To address this issue, we created a unique model of PINK1 loss-of-function, in a human neural stem cell (NSC) line that is able to differentiate into functional dopaminergic neurons [39]. This novel cellular system recapitulates the key features of PD namely dysfunction and death in ageing midbrain neurons. In addition, we utilised primary mouse neurons taken from mice and a widely used human dopaminergic neuroblastoma cell line (SHSY5Y), which allowed cross-validation in three complementary cellular systems. We report the spatio-temporal expression of PINK1 in human dopaminergic neurons, and the effects of PINK1 deficiency including chronic mitochondrial dysfunction, an increase in oxidative stress, and lysosomal pathology. These phenotypic effects may all contribute to the marked decrease in long term viability of PINK1 deficient neurons and enhanced basal and stress-induced caspase-3 activation leading to apoptosis. Our findings give new insights into the underlying dysfunction of human dopaminergic neurons in PINK1 parkinsonism and sporadic PD, by utilising a truly representative in vitro cellular model of the disease. Materials and Methods All chemicals and reagents were obtained from Sigma or Invitrogen unless otherwise stated. PLoS ONE | www.plosone.org 2 June 2008 | Volume 3 | Issue 6 | e2455