? Glucocerebrosidase gene mutations are a risk factor for Parkinson’s disease. line WYE-354 to identify the biochemical abnormalities that follow GCase inhibition. We show that GCase inhibition leads to decreased ADP phosphorylation reduced mitochondrial membrane potential and increased free radical formation and damage together with accumulation of alpha-synuclein. Taken together inhibition of GCase by CβE induces abnormalities in mitochondrial function and oxidative stress in our cell culture model. We suggest that mutations and reduced GCase activity may increase the risk for PD by inducing these same abnormalities in PD brain. 1 Glucocerebrosidase 1 (GCase) is usually a ubiquitous lysosomal enzyme responsible for the breakdown of glucocerebroside to glucose and ceramide. Diverse mutations within the gene (mutations cause a reduction in enzyme activity this may not necessarily be the mechanism that mediates the pathogenesis of GD and alternative models include mis-trafficking of GCase and endoplasmic reticulum stress WYE-354 (Kov-Bar et al. 2011 Alpha-synuclein positive Lewy bodies have been identified in the brains of GD patients and carriers who died with PD (Neumann et al. 2009 Wong et al. 2004 There are now persuasive data that mutations are a major risk factor for PD and result in a clinical and pathological phenotype that is virtually indistinguishable from sporadic PD (Sidransky et al. 2009 The mechanism(s) whereby mutations increase the risk for PD remain unidentified. PD pathogenesis is usually thought to involve a number of WYE-354 pathways Kcnj8 including mitochondrial dysfunction and oxidative stress (Schapira 2006 Given the similar clinical and pathological phenotypes of knockdown SHSY-5Y stable cell lines SHSY-5Y cells were transfected with a ‘Hush’ GBA knockdown plasmid (Origene USA) empty plasmid and scrambled control (The sequence chosen for the knockdown was: GTGTGTGTCTGCAATGCCACATACTGTGA). Stable clones were isolated following selection with puromycin (Sigma UK) at 4?μg/ml and characterised by analysis of GCase activity actin-normalised mRNA by a ‘StepOne’ QPCR machine (Applied Biosystems UK) using SyBr Green (Life Technologies UK) and appropriate primers for and β-actin (Eurofins Germany) and GCase protein levels (by Western blotting). Clones were assessed after several passages (in the presence of a maintenance dose of 2?μg/ml puromycin) to check for the continuation of any knockdown effect. 2.7 Statistical analysis Where multiple comparisons were made one-way ANOVA tests were performed followed by Dunnett post test analysis in order to determine WYE-354 statistical significance. Student’s value of?0.05 was considered as significantly different. 3 3.1 CβE CβE has been reported to be a selective inhibitor of GCase activity (Prence et al. 1996 Newburg et al. 1986 and we have confirmed in SHSY-5Y cells that 50?μM CβE decreased GCase activity to ?5% of untreated cells and maintained the inhibition of GCase activity over 30?days (Suppl. Fig. 1). This concentration of CβE has also been previously reported to result in a WYE-354 greater than 2-fold increase of glucocerebroside over 24?days (Prence et al. 1996 In our experiments 30 CβE treatment had no effect on cell viability as judged by LDH release (Suppl. Fig. 2). 3.2 Mitochondrial studies 3.2 ATP synthesis (ADP phosphorylation) Fig. 1 shows the ADP phosphorylation capacity of digitonin-permeabilised cells following incubation with CβE. There was no measurable effect before 10?days but complex I-linked ADP phosphorylation with glutamate/malate as substrate was significantly decreased by 47% at 20?days (knockdown To confirm the effects of GCase inhibition by CβE we generated a stable shRNA-mediated knockdown model of in SH-SY5Y cells. Suppl. Fig. 4A shows that the enzyme activity was reduced by 62% and Western blot band densities indicated that the level of protein was decreased by 59% (Suppl. Fig. 4B and C) compared to the scrambled control levels. Quantitative WYE-354 PCR data also showed a significant decrease of 60% in the mRNA for relative to the scrambled control (data not shown). As shown in Suppl. Fig. 4D knockdown of caused a significant fall in TMRM fluorescence (mutations have now been reproducibly.