Figure 4. Apoptosis in PINK1 kd human neurons. Western blots showing increase in basal (2) and STS-induced (+) cleaved caspase 3, cleaved caspase-9 (two bands ,36 and ,32 kDa) and cleaved PARP without PINK1 in young (A) (dd5) and aged (B) (dd43) human control and PINK1 kd neuronal cultures. Each lane is a sample from three pooled independent culture lysates. The lowest bands are immunore- active degradation products which run at the foot of an SDS-PAGE gel. doi:10.1371/journal.pone.0002455.g004 plasmic vesicular aggregates only in PINK1 kd neurons, reminiscent of autophagosomes, at a frequency of approximately 30% (Figure 7A). To determine whether these structures were lysosomal in origin, we applied the acidophilic Lysotracker dye to aged cultures and found this dye was taken up into these distinctive aggregates, which were considerably larger and more numerous in PINK1 kd neurons compared to controls (Figure 7B). The phenotypes observed in the different cell models of PINK1 loss-of-function are summarised in Table 1. Discussion The aim of this study was to further elucidate the function of PINK1 in the prevention of DAergic degeneration seen in PINK1 Deficiency Parkinson’s disease. We produced stable knockdown models of PINK1 using small interfering RNAs in two complementary cell lines–human neuroblastoma SHSY5Y and a novel human NSC line capable of high levels of DAergic differentiation. In addition, we used primary neuronal cultures from PINK1 knockout mice to corroborate our findings and control for potential non-specific effects of RNAi. Human neurons are generated from immortalised human fetal ventral mesencephalic neural stem cells (NSCs) which maintain a stable karyotype in culture, stable growth rates and readily differentiate into function neurons–thus providing advan- tages over commonly used ‘‘cancer’’ cell lines [39]. Differentiated NSCs cultures contain a high percentage of DAergic neurons which express typical neuronal markers and display characteristic action potentials. The use of stable RNAi allowed us to study the molecular changes occurring in human DAergic neurons during the process of ageing in a PINK1 background. Our model therefore represents an in vitro system highly relevant to human PD, which is both progressive and age-dependent. PINK1 expression in dopaminergic neurons We found that PINK1 expression is up-regulated ,100 fold during differentiation of human NSCs into human neurons, suggesting that PINK1 has an important function in differentiated neurons. However, it is important to note that PINK1 did not affect differentiation into the DAergic phenotype. This suggests PINK1 is not necessary for development which is in-line with the progression of the PINK1 mutation onset of PD. Endogenous PINK1 was expressed within soma and neurites of post-mitotic dopaminergic neurons, although it was not expressed exclusively in these subtypes. This is consistent with previous reports which show endogenous PINK1 expression in both neurons and glia throughout brain [21,24]. PINK1 expression in neurons was highest within mature processes, where its expression may be most critical. PINK1 deficiency and long term survival A major finding in our study was the reduction in long term viability of human and mouse neurons deficient in PINK1. To our knowledge this is the first age-dependent phenotype reported for any in vitro cell model of PD. A progressive phenotype has been reported in PINK1 knockout Drosophila models, attributable to an increased level of apoptosis, but so far not in mammalian models [32,34]. We observed significant age-dependent increases in cell death and apoptosis in mammalian neurons which provide a mechanism for the progressive phenotype seen in both sporadic and genetic forms of PD which have up until now been difficult to recapitulate in vitro. PINK1deficiency sensitises neurons to mitochondrial apoptosis An increase in apoptosis has been previously reported in SHSY5Y cells following transient RNAi-mediated PINK1 kd, and in fibroblasts taken from patients with compound heterozygous PINK1 mutations [35,37]. Interestingly, no basal increase in cell death and apoptosis was observed with PINK1 deficient differentiated human neurons or primary mouse neurons at early time points. However, molecular differences do exist between control and PINK1 kd cells at this early stage, specifically in their ability to handle mitochondrial stress. We find that PINK1 deficient cells are acutely sensitive to the toxin STS, which triggers mitochondrial apoptosis via opening of the mitochondrial permeability transition pore (MPTP) and subsequent cytochrome c release. In SHSY5Y cells lacking PINK1, Bax translocation to PLoS ONE | www.plosone.org 9 June 2008 | Volume 3 | Issue 6 | e2455