we have described for the first time using HESC how over-expression of Pax4, in combination with simple changes to the cell culture environment

ss compared to controls. Second, in PINK1 deficient neurons we found a marked up-regulation of mitochondrial OXPHOS XL-518 site complex subunits in aged human neurons as analysed by Western blot. 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. Third, in vitro assays showed an increase in the mitochondrial citrate synthase activity within these cells. We also found a significant increase in mitochondrial mass using direct quantification of mitochondria within individual neurons using transmission electron microscopy . However, no parallel PINK1 Deficiency increase in respiratory complex activity was detected using biochemical techniques in neuronal models. Together these data suggest 11906293 a compensatory increase in mitochondrial 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 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 which measures cytosolic ROS production and the mitochondrial targeted variant of this dye, Mitosox, to 17804601 measure mitochondrial ROS production. 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. Stimulation of neurons with 50 mM KCl to transiently raise c, increased the rate of ROS production in control cells, which showed a 3.2 fold increase in the rate of HEt fluorescence . We demonstrated a higher basal rate of mitochondrial superoxide production in PINK1 kd neurons compared to controls. The complex I inhibitor, rotenone caused a smaller proportional increase in superoxide production in PINK1 kd compared to controls 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. In addition to measuring ROS production we assayed the antioxidant defense mechanisms by analysing glutathione levels in young and aged human neurons.. We demonstrate a significant reduction in the total levels of glutathione 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 PINK1 Deficiency Parkinson’s disease. We produced stable knockdown models of PINK1 using small interfering RNAs in two complementary cell lineshuman 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 which maintain a stable karyotype in culture, stable growth rates and readily differentiate into function neuronsthus providing advantages over commonly u