Matthew E. Gegg

Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy

Mitochondrial dysfunction and perturbed degradation of proteins have been implicated in Parkinsons 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.

Glucocerebrosidase deficiency in substantia nigra of parkinson disease brains

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.

Silencing of PINK1 Expression Affects Mitochondrial DNA and Oxidative Phosphorylation in DOPAMINERGIC Cells

Background Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinsons disease (PD). Impairment of the mitochondrial electron transport chain (ETC) and an increased frequency in deletions of mitochondrial DNA (mtDNA), which encodes some of the subunits of the ETC, have been reported in the substantia nigra of PD brains. The identification of mutations in the PINK1 gene, which cause an autosomal recessive form of PD, has supported mitochondrial involvement in PD. The PINK1 protein is a serine/threonine kinase localized in mitochondria and the cytosol. Its precise function is unknown, but it is involved in neuroprotection against a variety of stress signalling pathways. Methodology/Principal Findings In this report we have investigated the effect of silencing PINK1 expression in human dopaminergic SH-SY5Y cells by siRNA on mtDNA synthesis and ETC function. Loss of PINK1 expression resulted in a decrease in mtDNA levels and mtDNA synthesis. We also report a concomitant loss of mitochondrial membrane potential and decreased mitochondrial ATP synthesis, with the activity of complex IV of the ETC most affected. This mitochondrial dysfunction resulted in increased markers of oxidative stress under basal conditions and increased cell death following treatment with the free radical generator paraquat. Conclusions This report highlights a novel function of PINK1 in mitochondrial biogenesis and a role in maintaining mitochondrial ETC activity. Dysfunction of both has been implicated in sporadic forms of PD suggesting that these may be key pathways in the development of the disease.

Differential effect of nitric oxide on glutathione metabolism and mitochondrial function in astrocytes and neurones: implications for neuroprotection/neurodegeneration?

Primary culture rat astrocytes exposed to the long acting nitric oxide donor (Z)‐1‐[2‐aminoethyl)‐N‐(2‐ammonioethyl)amino]diazen‐1‐ium‐1,2‐diolate (DETA‐NO) for 24 h approximately double their concentration of glutathione (GSH) and show no sign of cell death. In contrast, GSH was depleted by 48%, and significant loss of mitochondrial respiratory chain complex activity and cell death were observed in primary culture rat neurones subjected to DETA‐NO for 18 h. Northern blot analysis suggested that mRNA amounts of both subunits of glutamate‐cysteine ligase (GCL), the rate‐limiting enzyme in GSH synthesis, were elevated in astrocytes following nitric oxide (NO) exposure. This correlated with an increase in astrocytic GCL activity. Neurones on the other hand did not exhibit increased GCL activity when exposed to NO. In addition, the rate of GSH efflux was doubled and γ‐glutamyltranspeptidase (γ‐GT) activity was increased by 42% in astrocytes treated with NO for 24 h. These results suggest that astrocytes, but not neurones, up‐regulate GSH synthesis as a defence mechanism against excess NO. It is possible that the increased rate of GSH release and activity of γ‐GT in astrocytes may have important implications for neuroprotection in vivo by optimizing the supply of GSH precursors to neurones in close proximity.

Mitochondria and Quality Control Defects in a Mouse Model of Gaucher Disease—Links to Parkinson’s Disease

Summary 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.

Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells

Heterozygous GBA gene mutations are the most frequent Parkinson’s disease risk factor. Using Parkinson’s disease patient derived fibroblasts McNeill et al. show that heterozygous GBA mutations reduce glucosylceramidase activity, and are associated with endoplasmic reticulum and oxidative stress. Ambroxol treatment improved glucosylceramidase activity and reduced oxidative stress in these cells.

Mitochondrial Contribution to Parkinson's Disease Pathogenesis

The identification of the etiologies and pathogenesis of Parkinsons disease (PD) should play an important role in enabling the development of novel treatment strategies to prevent or slow the progression of the disease. The last few years have seen enormous progress in this respect. Abnormalities of mitochondrial function and increased free radical mediated damage were described in post mortem PD brain before the first gene mutations causing familial PD were published. Several genetic causes are now known to induce loss of dopaminergic cells and parkinsonism, and study of the mechanisms by which these mutations produce this effect has provided important insights into the pathogenesis of PD and confirmed mitochondrial dysfunction and oxidative stress pathways as central to PD pathogenesis. Abnormalities of protein metabolism including protein mis-folding and aggregation are also crucial to the pathology of PD. Genetic causes of PD have specifically highlighted the importance of mitochondrial dysfunction to PD: PINK1, parkin, DJ-1 and most recently alpha-synuclein proteins have been shown to localise to mitochondria and influence function. The turnover of mitochondria by autophagy (mitophagy) has also become a focus of attention. This review summarises recent discoveries in the contribution of mitochondrial abnormalities to PD etiology and pathogenesis.

Differential effects of PINK1 nonsense and missense mutations on mitochondrial function and morphology

Mutations of the PINK1 gene are a cause of autosomal recessive Parkinsons disease (PD). PINK1 encodes a mitochondrial kinase of unknown function which is widely expressed in both neuronal and non-neuronal cells. We have studied fibroblast cultures from four family members harbouring the homozygous p.Q456X mutation in PINK1, three of their wild-type relatives, one individual with the homozygous p.V170G mutation and five independent controls. Results showed bioenergetic abnormalities involving decreased activities of complexes I and IV along with increased activities of complexes II and III in the missense p.V170G mutant. There were increased basal levels of mitochondrial superoxide dismutase in these cells and an exaggerated increase of reduced glutathione in response to paraquat-induced free radical formation. Furthermore, swollen and enlarged mitochondria were observed in this sample. In the p.Q456X nonsense mutants, the respiratory chain enzymes were unaffected, but ATP levels were significantly decreased. These results confirm that mutations of PINK1 cause abnormal mitochondrial morphology, bioenergetic function and oxidative metabolism in human tissues but suggest that the biochemical consequences may vary between mutations.

Suppression of Autoimmune Retinal Disease by Lovastatin Does Not Require Th2 Cytokine Induction

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In an acute mouse model of autoimmune retinal disease, we demonstrate that treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, lovastatin, suppresses clinical ocular pathology, retinal vascular leakage, and leukocytic infiltration into the retina. Efficacy was reversed by coadministration of mevalonolactone, the downstream product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not by squalene, which is distal to isoprenoid pyrophosphate metabolites within the cholesterol biosynthetic pathway. Lovastatin treatment (20 mg/kg/day i.p.) over 7 days, which resulted in plasma lovastatin hydroxyacid concentrations of 0.098 ± 0.03 μM, did not induce splenocyte Th2 cytokine production but did cause a small reduction in Ag-induced T cell proliferation and a decrease in the production of IFN-γ and IL-10. Thus, it is possible to dissociate the therapeutic effect of statins in experimental autoimmune uveitic mice from their activity on the Th1/Th2 balance. Statins inhibit isoprenoid pyrophosphate synthesis, precursors required for the prenylation and posttranslational activation of Rho GTPase, a key molecule in the endothelial ICAM-1-mediated pathway that facilitates lymphocyte migration. Consistent with inhibition of leukocyte infiltration in vivo, lovastatin treatment of retinal endothelial cell monolayers in vitro leads to inhibition of lymphocyte transmigration, which may, in part, account for drug efficacy. Unlike lovastatin, atorvastatin treatment showed little efficacy in retinal inflammatory disease despite showing significant clinical benefit in experimental autoimmune encephalomyelitis. These data highlight the potential differential activity of statins in different inflammatory conditions and their possible therapeutic use for the treatment of human posterior uveitis.

Bioenergetic Consequences of PINK1 Mutations in Parkinson Disease

Background Mutations of the gene for PTEN-induced kinase 1 (PINK1) are a cause of familial Parkinsons disease (PD). PINK1 protein has been localised to mitochondria and PINK1 gene knockout models exhibit abnormal mitochondrial function. The purpose of this study was to determine whether cells derived from PD patients with a range of PINK1 mutations demonstrate similar defects of mitochondrial function, whether the nature and severity of the abnormalities vary between mutations and correlate with clinical features. Methodology We investigated mitochondrial bioenergetics in live fibroblasts from PINK1 mutation patients using single cell techniques. We found that fibroblasts from PINK1 mutation patients had significant defects of bioenergetics including reduced mitochondrial membrane potential, altered redox state, a respiratory deficiency that was determined by substrate availability, and enhanced sensitivity to calcium stimulation and associated mitochondrial permeability pore opening. There was an increase in the basal rate of free radical production in the mutant cells. The pattern and severity of abnormality varied between different mutations, and the less severe defects in these cells were associated with later age of onset of PD. Conclusions The results provide insight into the molecular pathology of PINK1 mutations in PD and also confirm the critical role of substrate availability in determining the biochemical phenotype – thereby offering the potential for novel therapeutic strategies to circumvent these abnormalities.