PINK1 Deficiency Figure 3. Apoptosis pathway in PINK1 kd SHSY5Y cells. A) Histogram showing a significant increase in apoptosis in PINK1 kd SHSY5Y cells in response to STS compared to controls. Cells were incubated with 60nM STS or vehicle overnight prior to analysis of annexinV/PI binding by FACS. Values represent mean of 20,000cells/well, of duplicate wells of 3 clones across 5 independent experiments6s.e.m. B) Panel a-h shows double immunofluorescence using Bax and Cox V antibodies in SHSY5Y cells treated with 0.75 mM STS. At 120 minutes, Bax (green) is seen in the cytoplasm and nucleus of control cells, with no overlap with a mitochondrial marker Cox V (red). In PINK1 kd cultures, arrows highlight two cells with evidence of nuclear condensation suggestive of apoptosis. In these cells, Bax immunofluorescence overlaps with Cox V immunofluorescence indicating Bax localisation in the mitochondria. Panel l-p shows double immunofluorescence using cytochrome c and Cox V antibodies. At 180 minutes, there is overlap of cytochrome c (green) and Cox V (red) in control cells indicating cytochrome c localisation in the mitochondria. However in PINK1 kd cells arrows indicate several apoptotic cells with diffuse cytosolic cytochrome c, indicating translocation from mitochondria to cytoplasm. Images are representative of two independent experiments with 3 clones. Images were acquired by obtaining z-stacks through cells at x63 oil objective. C) Western blot showing increased apoptosis on exposure to STS in PINK1 kd SHSY5Y cells. Cells were incubated with 0.75 mM mM STS for 6hrs. Cleaved caspase 9, cleaved caspase 3, and PARP expression is increased at earlier time points in PINK1 kd cells than in controls. doi:10.1371/journal.pone.0002455.g003 increase in respiratory complex activity was detected using biochemical techniques in neuronal models (data not shown). Together these data suggest a compensatory increase in mito- chondrial density in neurons lacking PINK-1. TEM analysis of young and aged human neurons lacking PINK1 also revealed an increase in the proportion of abnormal swollen mitochondria within cells (Figure 5F,G) further supporting the hypothesis that PINK1 functions to maintain mitochondrial integrity in neurons. PINK1 deficiency in neurons is associated with an increase in basal free radical production and decreased steady state levels of glutathione We utilised live cell imaging techniques using the redox-sensitive dye dihydroethidium (HEt) which measures cytosolic ROS production and the mitochondrial targeted variant of this dye, Mitosox, to measure mitochondrial ROS production. (Figure 6A,B) The basal rate of ROS generation was significantly increased in the cytoplasm of PINK1 kd human neurons, showing a 2.79-fold increase in basal rate of fluorescence increase (mean = 279611.3%, n = 189 cells, p,0.001). Stimulation of neurons with 50 mM KCl to transiently raise [Ca2+]c, increased the rate of ROS production in control cells, which showed a 3.2 fold increase in the rate of HEt fluorescence (from basal 100% to 321619.3% while the response in PINK1 kd neurons was much smaller (from 279611.3% to 300.1614.7%, where 100%- basal rate of ROS in control neurons) (Figure 6 C,D). We demonstrated a higher basal rate of mitochondrial superoxide production in PINK1 kd neurons compared to controls (the rate of increase of MitoSOX fluorescence was 2.1-fold higher in PINK1 kd neurons (mean = 212614.1%, p,0.001). The complex I inhibitor, rotenone caused a smaller proportional increase in superoxide production in PINK1 kd (210.2% to 255.469.9%) compared to controls (100% to 212.1611.6%) due to the higher basal levels of ROS generation, although the absolute rate of ROS generation in response to rotenone was higher in PINK1 kd neurons. (Figure 6A,B) In addition to measuring ROS production we assayed the antioxidant defense mechanisms by analysing glutathione levels in young (dd5) and aged (dd43) human neurons. (Figure 6Ei, ii). We demonstrate a significant reduction in the total levels of glutathione (reduced+oxidised) in PINK1 kd cells, implying an impairment of glutathione synthesis. A decrease in the ratio of reduced:oxidised glutathione was not found. Aged midbrain derived neurons deficient in PINK1 contain lysosomal aggregates In addition to morphological mitochondrial abnormalities in aged human PINK1 kd neurons, we also found large intracyto- PLoS ONE | www.plosone.org 8 June 2008 | Volume 3 | Issue 6 | e2455