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.

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.

Mutations of the gene for glucocerebrosidase 1 (GBA) cause Gaucher disease (GD), an autosomal recessive lysosomal storage disorder. Individuals with homozygous or heterozygous (carrier) mutations of GBA have a significantly increased risk for the development of Parkinson's disease (PD), with clinical and pathological features that mirror the sporadic disease. The mechanisms whereby GBA mutations induce dopaminergic cell death and Lewy body formation are unknown. There is evidence of mitochondrial dysfunction and oxidative stress in PD and so we have investigated the impact of glucocerebrosidase (GCase) inhibition on these parameters to determine if there may be a relationship of GBA loss-of-function mutations to the known pathogenetic pathways in PD. We have used exposure to a specific inhibitor (conduritol-β-epoxide, CβE) of GCase activity in a human dopaminergic cell line 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 GBA mutations and reduced GCase activity may increase the risk for PD by inducing these same abnormalities in PD brain.

Mitochondrial fission is essential for the degradation of damaged mitochondria. It is currently unknown how the dynamin-related protein 1 (DRP1)–associated fission machinery is selectively targeted to segregate damaged mitochondria. We show that PTEN-induced putative kinase (PINK1) serves as a pro-fission signal, independently of Parkin. Normally, the scaffold protein AKAP1 recruits protein kinase A (PKA) to the outer mitochondrial membrane to phospho-inhibit DRP1. We reveal that after damage, PINK1 triggers PKA displacement from A-kinase anchoring protein 1. By ejecting PKA, PINK1 ensures the requisite fission of damaged mitochondria for organelle degradation. We propose that PINK1 functions as a master mitophagy regulator by activating Parkin and DRP1 in response to damage. We confirm that PINK1 mutations causing Parkinson disease interfere with the orchestration of selective fission and mitophagy by PINK1.

GBA gene mutations are the greatest cause of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase) but the mechanisms by which loss of GCase contributes to PD remain unclear. Inhibition of autophagy and the generation of endoplasmic reticulum (ER) stress are both implicated. Mutant GCase can unfold in the ER and be degraded via the unfolded protein response, activating ER stress and reducing lysosomal GCase. Small molecule chaperones that cross the blood brain barrier help mutant GCase refold and traffic correctly to lysosomes are putative treatments for PD. We treated fibroblast cells from PD patients with heterozygous GBA mutations and Drosophila expressing human wild-type, N370S and L444P GBA with the molecular chaperones ambroxol and isofagomine. Both chaperones increased GCase levels and activity, but also GBA mRNA, in control and mutant GBA fibroblasts. Expression of mutated GBA in Drosophila resulted in dopaminergic neuronal loss, a progressive locomotor defect, abnormal aggregates in the ER and increased levels of the ER stress reporter Xbp1-EGFP. Treatment with both chaperones lowered ER stress and prevented the loss of motor function, providing proof of principle that small molecule chaperones can reverse mutant GBA-mediated ER stress in vivo and might prove effective for treating PD.