with controls (Figure 1B). We similarly confirmed absence of PINK1 expression in cultured cortical neurons from the PINK1 knockout mouse model by Western blot (Figure S1D). PINK1 is not necessary for differentiation of human neural stem cells to dopaminergic neurons Human NSC lines, carrying the stable #1029 PINK1 kd shRNA, does not prevent differentation into human DAergic neurons as demonstrated by tyrosine hydroxylase (TH) immuno- staining, following the PreD differentiation protocol (Figure 1E). No difference in TH expression was found between PINK1 kd neurons and controls using Western blot (data not shown). The PreD protocol generates the highest proportion of DAergic neurons in ReNcell cultures [39]. However, due to the formation of ‘neuronal aggregates’ in these cultures, accurate quantification of percentage TH+ neurons is extremely difficult using microsco- py. In contrast, the StdD protocol gives rise to a monolayer of neurons, which permits the use of the Cellomics microscope and BioApplication software to robotically count (cytoplasmic) TH+ neurons. Control or PINK1 kd human neuronal cultures contained 1–5% TH+ neurons using the StdD protocol. There was no significant difference in the proportion of TH+ cells in control or PINK1 kd cells, either as a percentage of total cells or percentage of total neurons (data not shown). PINK1 deficiency results in age-related reduction in basal viability of human and mouse neurons We then investigated if PINK1 was necessary for the survival of neurons. Due to the characteristics of the different cell models, we employed different methods to assess viability and apoptosis. SHSY5Y cells are proliferative neuroblastoma cells that may be assessed using flow cytometry for both viability and apoptosis. Annexin V/PI flow cytometry of PINK1 kd SHSY5Y cells showed a significant reduction in the number of live cells after 96 h in culture when compared to control cells (p = 0.0263). Furthermore we observed a concomitant increase in cells undergoing early apoptosis, as shown by cells expressing annexin V only (p = 0.0023), and cells staining for both PI and annexin V (p = 0.0218) (Figure S2B). To assess the viability of differentiated human neurons, we employed a cytotoxicity assay for neurons at days 15, 30, 43 and 52 in culture. Cytotoxicity Indices (CI) for control and PINK kd cultures were calculated at each time point. There was no significant difference in basal viability at day 15, but from days 30 to 52, there was a significant and increasing difference in the mean CI of PINK1 kd neurons compared to controls, demonstrating an age-dependent effect of loss-of-PINK1 function (Figure 2A). By 52 days differentiation, the mean CI of PINK1 kd neurons was ,30% above that of controls (p,0.001). Using the Cellomics software we examined differences in the various parameters quantified using the Multiparameter Cytotox- icity 1 BioApplication Software which are collated into the final CI readout. These revealed a striking reduction in mean nuclear size and fluorescence intensity, indicative of apoptosis (Figure 2C,D). Indeed we found a significant increase in the percentage apoptotic neurons in aged PINK1 kd cultures compared to controls by manual counting of pyknotic nuclei (Figure 2F). In addition there was a significant increase in mean lysosomal mass/pH in aged PINK1 kd neurons (Figure 2E). We then sought to confirm these findings using primary embryonic cortical cultures from PINK1 KO mice (Figure S2C). Again we found the basal viability of neuronal cultures lacking PINK1 declined with age with mouse cortical KO neurons having significantly higher CI than controls by later time points PINK1 Deficiency (Figure 2B). Assessment of individual Multiparameter Cytotox- icity assay parameters at later time points (d30) revealed a decrease in nuclear intensity, and an increase in lysosomal mass/pH (Figure S2Di,ii) indicative of early cellular apoptosis. Lack of PINK1 sensitises neurons to mitochondrial- mediated apoptosis Using annexin V based flow cytometry we demonstrated that SHSY5Y cells exposed to 60nM staurosporine (STS) for 24hrs exhibited a significant reduction in the number of live cells in PINK1 kd clones compared to controls (p = 0.0002), as well as higher levels of early apoptosis in PINK1 kd cells (p,0.0001), and higher levels of total apoptosis in PINK1 kd cells (p = 0.0002) (Figure 3A). Preincubation of PINK1 kd cultures with 100 mM of the pan-caspase inhibitor Z-VAD-FMK 1hr prior to exposure with STS abolished cell death and markers of early and late apoptosis (data not shown). To assess the pathway of STS-induced apoptosis, and how it is sensitized in PINK1 kd cells, we used 0.75 mM STS for 4hrs, fixed cells at different intervals and performed double immunofluores- cence to assess the localisation of Bax and cytochrome c. Under basal conditions Bax was observed in the cytosol and nucleus of cells, cells but not in the mitochondria (demonstrated by the failure of Bax to co-localise with the mitochondrial protein Cox V) whereas cytochrome c was localised to the mitochondria and showed overlap with Cox V staining. At 120 mins after addition of STS, some cells in the PINK1 kd cultures appeared apoptotic with nuclear fragmentation and condensation. In these cells, Bax staining co- localised with Cox V, suggesting translocation of Bax to the mitochondria from the cytosol in PINK1 kd cells undergoing apoptotic stress (Figure 3B,a–h). We further confirmed this finding using subcellular fractionation into mitochondrial and cytosolic fractions and immunoblotting for Bax (data not shown). After 180 mins of exposure to STS, we observed several cells per field in PINK1 kd cultures undergoing apoptosis as evidenced by nuclear fragmentation and condensation. In these cells Cox V staining demonstrated a loss of the tubular network of mitochondria, mitochondrial fragmentation and abnormal clustering. In addition to these nuclear and mitochondrial abnormalities, cytochrome c was distributed diffusely in the cytoplasm of these cells, confirming that it had been released from the mitochondria during apoptosis (Figure 3B, i–p). To further confirm the induction of apoptosis in PINK1 kd cells, we assessed PINK1 kd cell lysates for active caspase-3 and PARP cleavage at 90 minute intervals following exposure to 0.75 mM STS. We demonstrate an increase in cleaved caspase 3, caspase 9, and an increase in cleaved PARP in PINK1 kd cultures compared to controls (Figure 3C). All these molecular events were activated at earlier time points in the PINK1 kd cells compared to controls suggesting an enhanced susceptibility to apoptosis via the mitochondrial pathway. We investigated whether a similar sensitivity to mitochondrial apoptosis was evident in the human neurons as well as the SHSY5Y cells. Both young and aged human PINK1 kd neurons demonstrated enhanced sensitivity to STS and a higher induction of mitochondrial apoptosis with increased expression of cleaved caspase 3, caspase 9, and cleaved PARP (Figure 4 A, B). The increased expression was confirmed using band densitometry. Of note, the immunoreactive bands obtained using lysates from d43 neurons are less intense than those of young neurons due to the reduced amount of material obtained from aged cultures. Interestingly, activated caspase-3 was also detected in lysate from PINK1 kd neurons treated with vehicle alone at d43 (Figure 4 B) PLoS ONE | www.plosone.org 6 June 2008 | Volume 3 | Issue 6 | e2455