Western blots showing increase in basal (−) 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 immunoreactive degradation products which run at the foot of an SDS-PAGE gel.

A) TEM images of vesicular aggregates within surviving aged human neurons lacking PINK1 at dd47. A control neuron is shown in panel A. Panels b-d show aggregates in PINK1 kd neurons, panel c is an aggregate enlarged from b. Scale bars; a, b; 20 µM, d; 2 µM. B) Immunofluorescence images of aged (dd47) control human neurons (panels a-c) and PINK1 kd neurons (d–f) showing lysosomal morphology (red staining; panels b and e). Nuclei are shown in blue (panels a and d), merged images presented in panels c and f. Large and multiple aggregates seen only in PINK1 kd neurons are positive for the dye Lysotracker (panel d). ,Scale bar  =  20 µM

A) Graph showing an increase in mean CI over time of human PINK1 kd neurons compared to controls. Values shown are means±s.e.m of 3 control and 4 kd cultures, each measured in triplicate. B) Graph showing an increase in mean CI over time of mouse embryonic cortical neuronal cultures taken from wild type and PINK1 knockout mice. Values shown are means±s.e.m of wild type (PINK1+/+, n = 2), and homozygote (PINK1−/−, n = 3) cultures each measured in triplicate. C,D,E) Comparison of individual cell parameters assayed by the Cytotoxicity algorithm for aged human neurons (dd 52) with PINK1 kd compared to controls, including mean nuclear size, mean nuclear intensity, plasma membrane permeability and lysosomal mass/pH. Values shown are means±s.e.m of 3 control and 4 kd cultures, each measured in triplicate. F) Histogram showing percentage apoptotic neuronal nuclei in aged control and PINK1 kd human neurons under basal conditions. Values shown are mean percentages of neuronal nuclei that are pyknotic from 10 fields of view, ±sem from 3 independent cultures.

A) and (B) demonstrate a higher basal production of ROS production in mitochondria of PINK1 kd neurons compared to controls. Image in Figure 6A confirms mitochondrial localisation of Mitosox probe in neurons. Inhibition of complex 1 with rotenone induced a significant increase of ROS production in control cells. Inhibition of complex 1 in PINK1 kd cells slightly increased ROS production which is still higher than rotenone induced ROS production in control mitochondria. The basal rate of Mitosox fluorescence in control cells was taken as 100%. C) Shows an increase in the rate of HEt fluorescence in response to 50 mM KCL in control neurons. D) PINK1 kd neurons have higher basal rates of cytosolic ROS production compared to control neurons. Stimulation using KCl results in an increase in ROS production which is more marked in control than PINK1 kd neurons. E) Histogram showing decreased reduced and total glutathione concentrations in young (dd5 i) and in aged (dd43 ii) and human PINK1 kd neurons compared to controls. Values represent means ±sem from triplicates of three independent samples.

A) PINK1 expression in human dopaminergic neurons. Red; PINK1, green; TH, blue; hoescht. B) Western blot showing endogenous PINK1 protein expression in control (c) and PINK1 kd (with 90% mRNA knockdown) human neuronal lysates after 5 days differentiation. Anti-PINK1 (Novus) was used to detect PINK expression; β-actin levels are shown as loading control. C) Histogram showing relative fold change in PINK1 mRNA expression levels between NSCs, 5 day differentiation (dd5), 30 day differentiation (dd30). PINK1 expression levels from pre-immortalised neural stem cells from 8 week old ventral mesencephalon (Midbrain P5) and adult human brain (Human Cb) are shown for comparison. Values represent mean of three replicate samples±s.e.m. D) Histogram showing expression of PINK1 mRNA in human neurons in four control (c1-4) and four PINK1 kd (kd1-4) selected clones relative to an endogenous control gene. RQs were normalised to that of c1; values represent mean of 3 replicate samples±standard error (s.e.m). E) Immunofluorescence images showing dopaminergic cells in control (i) and PINK1 kd (ii) human neuronal cultures after 5 days differentiation. Red; TH, green; βIII tubulin, blue; Hoecsht.

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 experiments±s.e.m. B) Panel a-h shows double immunofluorescence using Bax and Cox V antibodies in SHSY5Y cells treated with 0.75 µM 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 µM µM 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.

A) PINK deficiency leads to a reduced basal mitochondrial membrane potential (ψm) in young PINK1 kd neurons compared to controls, analysed using TMRM fluorescence. Values are mean percentages of the control cultures (normalised to 100%) from three independent experiments. B) Histogram showing increased uptake of the redox sensitive dye MitotrackerRed in human PINK1 kd neuronal cultures compared to controls at early and later time points. Values represent means±sem of 3 control or three PINK1 kd cultures (measured in triplicate). C) Histogram showing increased citrate synthase (CS) activity in aged (d30) PINK1 kd human neurons compared to controls. Values represent means±sem from three independent cultures. D) Western blot showing expression of mitochondrial subunits in young (D5) and aged (D43) human neurons lacking PINK1 compared to controls. Note the increase in complex subunit expression in aged neurons lacking PINK1. Replicate lanes of pooled samples from three independent cultures are shown. Expression of OXPHOS subunits C-I-20 (ND6), C-II-30 (FeS), C-III-Core-2 and C-V-α is shown. β-actin; loading control. E) Graph showing significantly increased mean number of mitochondria/cell in aged PINK1 kd human neurons compared to controls, as quantified by TEM analysis. Histogram shows mean number mitochondria per cell ±sem averaged from at least 50 cells. F) Increased frequency of abnormal mitochondria of aged human PINK1 kd neurons compared to controls, quantified using TEM. Histogram shows mean frequency of abnormal mitochondria (per total number mitochondria in each cell) ±sem, n = 50 cells. G) TEM images showing abnormal mitochondria (arrowheads) within aged (d47) human neurons lacking PINK1 (panel b, and at higher magnification in d) compared to control neurons (panel a). There are increased numbers of mitochondria within cells lacking PINK1 (panel b) compared to controls (panel A) and a higher proportion of mitochondria in PINK1 kd neurons appear swollen with disorganised christae (panels c, d). Scale bar in a,b; 10 µM, c  =  500 nM.