The Investigation Of The Potential Contribution Of Dopamine Quinone To Parkinson's Disease

Parkinson's disease (PD) is an age-dependent neurodegenerative disorder characterized by dopaminergic neuronal loss in substantia nigra. Previous studies have implicated a role of dopamine oxidation in PD. Dopamine oxidation leads to the formation of dopamine quinone and reactive oxygen species through redox cycling. Dopamine quinone can covalently modify cysteinyl proteins to form quinoprotein adducts, and reactive oxygen species can cause lipid peroxidation or protein peroxidatin in cells. We compared quinoprotein adduct formation and lipid peroxidation in different brain regions of young and old rats. To determine whether quinoprotein adduct formation correlates with dopamine-induced cytotoxicity, we analyzed dopamine and rotenone treated SH-SY5Y cells. We detected the correlationship between cell viability and quinoprotein adduct formation/lipid peroxidation levels. Together, our results support that quinoprotein adduct formation plays a role in the selective dopaminergic neurodegeneration in PD. The specific results are listed here:1. The establishment of specific detection method for quinoprotein and determination of dopamine auto-oxidation.Spectrophotometer method and western blot/NBT staining method were used to detect dopamine auto-oxidation and ability of NBT/Glycine method for quinoprotein specific detection. Results showed free dopamine can auto-oxidized,and oxidized products could covalently combine with specific proteins. NBT/Glycine could detect dopamine quinone and quinoprotein adduct formation with high specificity and stability.2. Detection of quinoprotein adduct formation and lipid peroxidation levels in different rat brain regions during aging.Four different brain regions (prefrontal cortex, hippocampus, caudate nucleus,substantia nigra) were dissected from 2-,6-,10-,and 15-month old rat brains. Spectrophotometer method and western blot/NBT staining method were used to detect quinoprotein adduct formation during aging in each sample. MDA assay was used to determine lipid peroxidation levels during aging in each sample. Two dimensional electrophoresis and western blot/NBT staining method were used to separate target proteins for dopamine quinone modification. Results showed that no age-dependent alteration in quinoprotein adduct formation was detected in the hippocampus. A small but statistically significant increase in quinoprotein adducts was detected in the prefrontal cortex and caudate necleus. Notably, the most prominent age-dependent increase was in substantia nigra. Compared to 2-months old rats, the quinoprotein adducts in substantia nigra of 15-months old rats increased almost 50%. Two dimensional electrophoresis and western blot/NBT staining method revealed that more quinoprotein spots were detected in substantia nigra of 15-months old rats. The same brain tissues were also subjected to the MDA assay to determine the levels of lipid peroxidation. We did not detect a significant age-dependent changes in any of the four brain regions. Collectively, our results revealed that there is a prominent age-dependent accumulation of quinoprotein adduct in substantia nigra region. Quinoprotein adduct formation may play an important role in the selective dopaminergic neurodegeneration in PD.3. Investigating dopamine and rotenone caused cytotoxicity.To determine whether quinoprotein adduct formation correlates with dopamine-induced cytotoxicity, we analyzed dopamine and rotenone treated SH-SY5Y cells. MTT assay and live cell counting were used to detect the effects of cytotoxicity induced by dopamine and rotenone. MTT assay was also used to detect the cytoprotective effects of SF against dopamine induced cytotoxicity. NBT/Glycine and MDA assay were used to investigate the mechanism of cytotoxicity induced by dopamine and rotenone. Results showed dopamine could decrease cell viability in a dosage-dependent manner. Levels of quinoprotein adducts in both supernatant and pellet fractions exhibited a dosage-dependent increase, while higher levels of quinoprotein adducts were detected in the pellet fractions. A 24-hour pre-incubation of 1μM SF resulted in a small but statistically significant improvement in cell viability, accompanied with reductions of quinoprotein adducts in both supernatant and pellet fractions. No significant changes of lipid peroxidatin were detected in cells incubated with concentrations of DA from 0 to 400μmol/L. Cell viability were siginificantly reduced after an incubation with 1000 nmol/L rotenone. Levels of quinoprotein adducts exhibited a dosage-dependent decrease in supernatant fractions, but a dosage-dependent increase in pellet fractions. A significant increase of lipid peroxidation was detected in cells incubated with rotenone in a dosage-dependent manner.PD is an age-dependent neurodegenerative disorder. Studies have implicated a role of dopamine oxidation in PD. Dopamine oxidation leads to the formation of dopamine quinone and reactive oxygen species. In the study, we analyzed the two oxidized products respectively. We found quinoprotein adduct formation had a much stronger correlation with cytotoxicity than lipid peroxidation. Our results support that quinoprotein adduct formation plays a role in the selective dopaminergic neurodegeneration in PD.