PINK1 Deficiency Figure 2. Long term viability is reduced in PINK1 deficient neurons. A) Graph showing an increase in mean CI over time of human PINK1 kd neurons compared to controls. Values shown are means6s.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 means6s.e.m of wild type (PINK1+/+, n = 2), and homozygote (PINK12/2, 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 means6s.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, 6sem from 3 independent cultures. doi:10.1371/journal.pone.0002455.g002 suggesting an enhanced basal level of caspase-3-mediated apoptosis in the absence of PINK1 in aged neurons. PINK1 deficiency in neurons leads to mitochondrial morphometric abnormalities We investigated mitochondrial dysfunction in PINK1 kd cells using live cell imaging of TMRM fluorescence. We found that PINK1 deficiency leads to a reduction in mitochondrial membrane potential (ym) in both SHSY5Y cells (data not shown) and human neurons (Figure 5A). There was a mean reduction of 22% in the TMRM signal in PINK1 kd compared to controls (p = 0.046)(n = 60 neurons/culture taken from 3 individual clones). In addition, we found several lines of evidence suggesting increased mitochondrial proliferation in both young and aged human PINK1 kd neurons and in cortical PINK1 ko mouse neurons. First, with human PINK1 kd neurons, we found an increased uptake of the redox-sensitive dye, Mitotracker CMXROS, suggesting an increase in mitochondrial mass compared to controls (Figure 5B). Second, in PINK1 deficient neurons we found a marked up-regulation of mitochondrial OXPHOS complex subunits in aged human neurons as analysed by Western blot (Figure 5D). There was no significant percentage change in subunit expression between PINK1 kd and control neurons at dd5. However, by dd43, expression of complex I, III and V subunits had significantly increased in PINK1 kd neurons by 107.8622.5 %, 125.165.4% and 233.8617.9% respectively, as determined by band densitometry (p,0.01). Third, in vitro assays showed an increase in the mitochondrial citrate synthase activity within these cells (Figure 5C). We also found a significant increase in mitochondrial mass using direct quantification of mitochondria within individual neurons using transmission electron microscopy (TEM) (Figure 5E). However, no parallel PLoS ONE | www.plosone.org 7 June 2008 | Volume 3 | Issue 6 | e2455