Mitochondrial dysfunction and perturbed degradation of proteins have been implicated in Parkinson's disease (PD) pathogenesis. Mutations in the Parkin and PINK1 genes are a cause of familial PD. PINK1 is a putative kinase associated with mitochondria, and loss of PINK1 expression leads to mitochondrial dysfunction, which increases with time. Parkin is suggested to be downstream of PINK1 and also mediates the removal of damaged mitochondria by macroautophagy (mitophagy). We investigated whether mitochondrial dysfunction in dopaminergic SH-SY5Y cells following decreased PINK1 expression by RNAi may in part be due to the inhibition of mitophagy. Reduced flux through the macroautophagy pathway was found to be coincident with the inhibition of ATP synthesis following 12 days of PINK1 silencing. Overexpression of parkin in these cells restored both autophagic flux and ATP synthesis. Overexpression and RNAi studies also indicated that PINK1 and parkin were required for mitophagy following CCCP-induced mitochondrial damage. The ubiquitination of several mitochondrial proteins, including mitofusin 1 and mitofusin 2, were detected within 3 h of CCCP treatment. These post-translational modifications were reduced following the silencing of parkin or PINK1. The ubiquitination of mitochondrial proteins appears to identify mitochondria for degradation and facilitate mitophagy. PINK1 and parkin are thus required for the removal of damaged mitochondria in dopaminergic cells, and inhibition of this pathway may lead to the accumulation of defective mitochondria which may contribute to PD pathogenesis.

Objective: Mutations in the glucocerebrosidase gene (GBA) represent a significant risk factor for developing Parkinson disease (PD). We investigated the enzymatic activity of glucocerebrosidase (GCase) in PD brains carrying heterozygote GBA mutations (PD+GBA) and sporadic PD brains.Methods: GCase activity was measured using a fluorescent assay in cerebellum, frontal cortex, putamen, amygdala, and substantia nigra of PD+GBA (n = 9–14) and sporadic PD brains (n = 12–14). Protein expression of GCase and other lysosomal proteins was determined by western blotting. The relation between GCase, α‐synuclein, and mitochondria function was also investigated in vitro.Results: A significant decrease in GCase activity was observed in all PD+GBA brain areas except the frontal cortex. The greatest deficiency was in the substantia nigra (58% decrease; p < 0.01). GCase activity was also significantly decreased in the substantia nigra (33% decrease; p < 0.05) and cerebellum (24% decrease; p < 0.05) of sporadic PD brains. GCase protein expression was lower in PD+GBA and PD brains, whereas increased C/EBP homologous protein and binding immunoglobulin protein levels indicated that the unfolded protein response was activated. Elevated α‐synuclein levels or PTEN‐induced putative kinase 1 deficiency in cultured cells had a significant effect on GCase protein levels.Interpretation: GCase deficiency in PD brains with GBA mutations is a combination of decreased catalytic activity and reduced protein levels. This is most pronounced in the substantia nigra. Biochemical changes involved in PD pathogenesis affect wild‐type GCase protein expression in vitro, and these could be contributing factors to the GCase deficiency observed in sporadic PD brains. ANN NEUROL 2012;72:455–463.

Mutations in the glucocerebrosidase (gba) gene cause Gaucher disease (GD), the most common lysosomal storage disorder, and increase susceptibility to Parkinson's disease (PD). While the clinical and pathological features of idiopathic PD and PD related to gba (PD-GBA) mutations are very similar, cellular mechanisms underlying neurodegeneration in each are unclear. Using a mouse model of neuronopathic GD, we show that autophagic machinery and proteasomal machinery are defective in neurons and astrocytes lacking gba. Markers of neurodegeneration--p62/SQSTM1, ubiquitinated proteins, and insoluble α-synuclein--accumulate. Mitochondria were dysfunctional and fragmented, with impaired respiration, reduced respiratory chain complex activities, and a decreased potential maintained by reversal of the ATP synthase. Thus a primary lysosomal defect causes accumulation of dysfunctional mitochondria as a result of impaired autophagy and dysfunctional proteasomal pathways. These data provide conclusive evidence for mitochondrial dysfunction in GD and provide insight into the pathogenesis of PD and PD-GBA.

Gaucher disease is caused by mutations in the glucocerebrosidase gene, which encodes the lysosomal hydrolase glucosylceramidase. Patients with Gaucher disease and heterozygous glucocerebrosidase mutation carriers are at increased risk of developing Parkinson’s disease. Indeed, glucocerebrosidase mutations are the most frequent risk factor for Parkinson’s disease in the general population. Therefore there is an urgent need to understand the mechanisms by which glucocerebrosidase mutations predispose to neurodegeneration to facilitate development of novel treatments. To study this we generated fibroblast lines from skin biopsies of five patients with Gaucher disease and six heterozygous glucocerebrosidase mutation carriers with and without Parkinson’s disease. Glucosylceramidase protein and enzyme activity levels were assayed. Oxidative stress was assayed by single cell imaging of dihydroethidium. Glucosylceramidase enzyme activity was significantly reduced in fibroblasts from patients with Gaucher disease (median 5% of controls, P = 0.0001) and heterozygous mutation carriers with (median 59% of controls, P = 0.001) and without (56% of controls, P = 0.001) Parkinson’s disease compared with controls. Glucosylceramidase protein levels, assessed by western blot, were significantly reduced in fibroblasts from Gaucher disease (median glucosylceramidase levels 42% of control, P < 0.001) and heterozygous mutation carriers with (median 59% of control, P < 0.001) and without (median 68% of control, P < 0.001) Parkinson’s disease. Single cell imaging of dihydroethidium demonstrated increased production of cytosolic reactive oxygen species in fibroblasts from patients with Gaucher disease (dihydroethidium oxidation rate increased by a median of 62% compared to controls, P < 0.001) and heterozygous mutation carriers with (dihydroethidium oxidation rate increased by a median of 68% compared with controls, P < 0.001) and without (dihydroethidium oxidation rate increased by a median of 70% compared with controls, P < 0.001) Parkinson’s disease. We hypothesized that treatment with the molecular chaperone ambroxol hydrochloride would improve these biochemical abnormalities. Treatment with ambroxol hydrochloride increased glucosylceramidase activity in fibroblasts from healthy controls, Gaucher disease and heterozygous glucocerebrosidase mutation carriers with and without Parkinson’s disease. This was associated with a significant reduction in dihydroethidium oxidation rate of ∼50% (P < 0.05) in fibroblasts from controls, Gaucher disease and heterozygous mutation carriers with and without Parkinson’s disease. In conclusion, glucocerebrosidase mutations are associated with reductions in glucosylceramidase activity and evidence of oxidative stress. Ambroxol treatment significantly increases glucosylceramidase activity and reduces markers of oxidative stress in cells bearing glucocerebrosidase mutations. We propose that ambroxol hydrochloride should be further investigated as a potential treatment for Parkinson’s disease.

Impairment of the autophagy–lysosome pathway is implicated with the changes in α‐synuclein and mitochondrial dysfunction observed in Parkinson's disease (PD). Damaged mitochondria accumulate PINK1, which then recruits parkin, resulting in ubiquitination of mitochondrial proteins. These can then be bound by the autophagic proteins p62/SQSTM1 and LC3, resulting in degradation of mitochondria by mitophagy. Mutations in PINK1 and parkin genes are a cause of familial PD. We found a significant increase in the expression of p62/SQSTM1 mRNA and protein following mitophagy induction in human neuroblastoma SH‐SY5Y cells. p62 protein not only accumulated on mitochondria, but was also greatly increased in the cytosol. Increased p62/SQSMT1 expression was prevented in PINK1 knock‐down cells, suggesting increased p62 expression was a consequence of mitophagy induction. The transcription factors Nrf2 and TFEB, which play roles in mitochondrial and lysosomal biogenesis, respectively, can regulate p62/SQSMT1. We report that both Nrf2 and TFEB translocate to the nucleus following mitophagy induction and that the increase in p62 mRNA levels was significantly impaired in cells with Nrf2 or TFEB knockdown. TFEB translocation also increased expression of itself and lysosomal proteins such as glucocerebrosidase and cathepsin D following mitophagy induction. We also report that cells with increased TFEB protein have significantly higher PGC‐1α mRNA levels, a regulator of mitochondrial biogenesis, resulting in increased mitochondrial content. Our data suggests that TFEB is activated following mitophagy to maintain autophagy–lysosome pathway and mitochondrial biogenesis. Therefore, strategies to increase TFEB may improve both the clearance of α‐synuclein and mitochondrial dysfunction in PD. image Damaged mitochondria are degraded by the autophagy–lysosome pathway and is termed mitophagy. Following mitophagy induction, the transcription factors Nrf2 and TFEB translocate to the nucleus, inducing the transcription of genes encoding for autophagic proteins such as p62, as well as lysosomal and mitochondrial proteins. We propose that these events maintain autophagic flux, replenish lysosomes and replace mitochondria.

Glucocerebrosidase (GBA1) gene mutations increase the risk of Parkinson disease (PD). While the cellular mechanisms associating GBA1 mutations and PD are unknown, loss of the glucocerebrosidase enzyme (GCase) activity, inhibition of autophagy and increased α-synuclein levels have been implicated. Here we show that autophagy lysosomal reformation (ALR) is compromised in cells lacking functional GCase. ALR is a cellular process controlled by mTOR which regenerates functional lysosomes from autolysosomes formed during macroautophagy. A decrease in phopho-S6K levels, a marker of mTOR activity, was observed in models of GCase deficiency, including primary mouse neurons and the PD patient derived fibroblasts with GBA1 mutations, suggesting that ALR is compromised. Importantly Rab7, a GTPase crucial for endosome-lysosome trafficking and ALR, accumulated in GCase deficient cells, supporting the notion that lysosomal recycling is impaired. Recombinant GCase treatment reversed ALR inhibition and lysosomal dysfunction. Moreover, ALR dysfunction was accompanied by impairment of macroautophagy and chaperone-mediated autophagy, increased levels of total and phosphorylated (S129) monomeric α-synuclein, evidence of amyloid oligomers and increased α-synuclein release. Concurrently, we found increased cholesterol and altered glucosylceramide homeostasis which could compromise ALR. We propose that GCase deficiency in PD inhibits lysosomal recycling. Consequently neurons are unable to maintain the pool of mature and functional lysosomes required for the autophagic clearance of α-synuclein, leading to the accumulation and spread of pathogenic α-synuclein species in the brain. Since GCase deficiency and lysosomal dysfunction occur with ageing and sporadic PD pathology, the decrease in lysosomal reformation may be a common feature in PD.