Loss of DJ-1 Does Not Affect Mitochondrial Respiration but Increases ROS Production and Mitochondrial Permeability Transition Pore Opening

Background: Loss of function mutations in the DJ-1 gene have been linked to recessively inherited forms of Parkinsonism. Mitochondrial dysfunction and increased oxidative stress are thought to be key events in the pathogenesis of Parkinson’s disease. Although it has been reported that DJ-1 serves as scavenger for reactive oxidative species (ROS) by oxidation on its cysteine residues, how loss of DJ-1 affects mitochondrial function is less clear. Methodology/Principal Findings: Using primary mouse embryonic fibroblasts (MEFs) or brains from DJ-1−/− mice, we found that loss of DJ-1 does not affect mitochondrial respiration. Specifically, endogenous respiratory activity as well as basal and maximal respiration are normal in intact DJ-1−/− MEFs, and substrate-specific state 3 and state 4 mitochondrial respiration are also unaffected in permeabilized DJ-1−/− MEFs and in isolated mitochondria from the cerebral cortex of DJ-1−/− mice at 3 months or 2 years of age. Expression levels and activities of all individual complexes composing the electron transport system are unchanged, but ATP production is reduced in DJ-1−/− MEFs. Mitochondrial transmembrane potential is decreased in the absence of DJ-1. Furthermore, mitochondrial permeability transition pore opening is increased, whereas mitochondrial calcium levels are unchanged in DJ-1−/− cells. Consistent with earlier reports, production of reactive oxygen species (ROS) is increased, though levels of antioxidative enzymes are unaltered. Interestingly, the decreased mitochondrial transmembrane potential and the increased mitochondrial permeability transition pore opening in DJ-1−/− MEFs can be restored by antioxidant treatment, whereas oxidative stress inducers have the opposite effects on mitochondrial transmembrane potential and mitochondrial permeability transition pore opening. Conclusions/Significance: Our study shows that loss of DJ-1 does not affect mitochondrial respiration or mitochondrial calcium levels but increases ROS production, leading to elevated mitochondrial permeability transition pore opening and reduced mitochondrial transmembrane potential

Brain region specific mitophagy capacity could contribute to selective neuronal vulnerability in Parkinson's disease

Parkinson's disease (PD) is histologically well defined by its characteristic degeneration of dopaminergic neurons in the substantia nigra pars compacta. Remarkably, divergent PD-related mutations can generate comparable brain region specific pathologies. This indicates that some intrinsic region-specificity respecting differential neuron vulnerability exists, which codetermines the disease progression. To gain insight into the pathomechanism of PD, we investigated protein expression and protein oxidation patterns of three different brain regions in a PD mouse model, the PINK1 knockout mice (PINK1-KO), in comparison to wild type control mice. The dysfunction of PINK1 presumably affects mitochondrial turnover by disturbing mitochondrial autophagic pathways. The three brain regions investigated are the midbrain, which is the location of substantia nigra; striatum, the major efferent region of substantia nigra; and cerebral cortex, which is more distal to PD pathology. In all three regions, mitochondrial proteins responsible for energy metabolism and membrane potential were significantly altered in the PINK1-KO mice, but with very different region specific accents in terms of up/down-regulations. This suggests that disturbed mitophagy presumably induced by PINK1 knockout has heterogeneous impacts on different brain regions. Specifically, the midbrain tissue seems to be most severely hit by defective mitochondrial turnover, whereas cortex and striatum could compensate for mitophagy nonfunction by feedback stimulation of other catabolic programs. In addition, cerebral cortex tissues showed the mildest level of protein oxidation in both PINK1-KO and wild type mice, indicating either a better oxidative protection or less reactive oxygen species (ROS) pressure in this brain region. Ultra-structural histological examination in normal mouse brain revealed higher incidences of mitophagy vacuoles in cerebral cortex than in striatum and substantia nigra. Taken together, the delicate balance between oxidative protection and mitophagy capacity in different brain regions could contribute to brain region-specific pathological patterns in PD

Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis

Ellagic Acid Prevents Dopamine Neuron Degeneration from Oxidative Stress and Neuroinflammation in MPTP Model of Parkinson’s Disease

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases and is characterized by progressive dopaminergic neurodegeneration in the substantia nigra pars compacta area. In the present study, treatment of EA for 1 week at a dose of 10 mg/kg body weight prior to MPTP (25 mg/kg body weight) was carried out. MPTP administration caused oxidative stress, as evidenced by decreased activities of superoxide dismutase and catalase, and the depletion of reduced glutathione with a concomitant rise in the lipid peroxidation product, malondialdehyde. It also significantly increased the pro-inflammatory cytokines and elevated the inflammatory mediators like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the striatum. Immunohistochemical analysis revealed a loss of dopamine neurons in the SNc area and a decrease in dopamine transporter in the striatum following MPTP administration. However, treatment with EA prior to MPTP injection significantly rescued the dopaminergic neurons and dopamine transporter. EA treatment further restored antioxidant enzymes, prevented the depletion of glutathione and inhibited lipid peroxidation, in addition to the attenuation of pro-inflammatory cytokines. EA also reduced the levels of COX-2 and iNOS. The findings of the present study demonstrate that EA protects against MPTP-induced PD and the observed neuroprotective effects can be attributed to its potent antioxidant and anti-inflammatory properties

Increases of oxidative stress induce reduction of ΔΨ_m in <i>DJ-1</i>+/+ MEFs.

<p>(<b>A, B</b>) Confocal microscopic analysis. (<b>A</b>) Representative confocal live cell images of <i>DJ-1</i>−/− and +/+ MEFs stained with TMRM (50 nM, red) and Mitotracker Green (200 nM) after incubation with or without H₂O₂ (500 µM, 3 hr) or pyocyanin (100 µM, 24 hr). Insets show higher power views of the boxed area in the panel. Scale bar: 10 µm. (<b>B</b>) The bar graph shows quantification of TMRM signal in <i>DJ-1</i>−/− and +/+ MEFs after incubation with or without H₂O₂ (500 µM, 3 hr) or pyocyanin (100 µM, 24 hr). The TMRM signal is markedly reduced in <i>DJ-1</i>+/+ cells after induction of oxidative stress. The number shown in the panel indicates the number of cells quantified per genotype in the graph. (<b>C, D</b>) FACS analysis. (<b>C</b>) Representative flow cytometry dot plots show the intensity of TMRM signal in <i>DJ-1</i>−/− and +/+ MEFs following incubation with TMRM (50 nM) after incubation with or without H₂O₂ (500 µM, 3 hr) or pyocyanin (100 µM, 24 hr). (<b>D</b>) The bar graph shows quantification of TMRM signal in <i>DJ-1</i>−/− and +/+ MEFs. The TMRM fluorescence in <i>DJ-1</i>+/+ cells is decreased after incubation with oxidative stress inducers. The number shown in the panel indicates the number of embryos used to derive primary MEFs per genotype, and the data were obtained from three independent experiments. All data are expressed as mean ± SEM. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p