Abstract
Mutations of parkin are linked to early onset Parkinson disease. Here we show that stable transfection of parkin in the human dopaminergic neuroblastoma cell line SH-SY5Y markedly reduced the activities of both monoamine oxidase (MAO) A and B. The amount of 3,4-dihydroxyphenylacetic acid, which is produced during dopamine oxidation by MAO, was greatly reduced by parkin overexpression. Radioligand binding assays showed that MAO binding sites were decreased accordingly. Consistent with these, MAO-B protein level was much lower, whereas the amount of MAO-A protein was not determined due to the lack of a suitable antibody. Co-transfection of either MAO with parkin in HEK293 cells did not significantly alter ubiquitination and degradation of each MAO. When we measured MAO expression by real-time quantitative reverse transcription-PCR, marked reductions were seen in SH-SY5Y cells stably expressing parkin compared with the parental cells or a control line stably transfected with luciferase. In addition, parkin mutants defective in E3 ligase activity exhibited different effects on MAO expression. We found that parkin also significantly decreased mRNA levels of both MAOs in the mouse fibroblast cell line NIH3T3. Furthermore, MAO expression was significantly increased in human B lymphocyte cell lines derived from Parkinson disease patients with homozygous but not heterozygous deletion of exon 4 of parkin. Together these results suggest that parkin suppresses MAO expression. This function may limit the production of reactive oxygen species generated by MAO in dopamine oxidation and would, thus, be beneficial to the survival of dopaminergic neurons.
Highlights
Parkin has a protein-ubiquitin E3 ligase activity [7] that targets a variety of substrates for ubiquitin-dependent proteolysis by the 26 S proteasome [8]
To study the mechanism further, we measured the amounts of dopamine and its metabolites in SH-SY5Y cells (SH) and SH-SY5Y cells stably transfected with wild-type human parkin (SH/PKN)
Our previous study has shown that parkin attenuates dopamine toxicity on human dopaminergic neuroblastoma cell line SH-SY5Y by suppressing the production of reactive oxygen species [23]
Summary
MAO expression was significantly increased in human B lymphocyte cell lines derived from Parkinson disease patients with homozygous but not heterozygous deletion of exon 4 of parkin. Oxidation of dopamine by monoamine oxidase (MAO) produces large amounts of reactive oxygen species (ROS) that are toxic to the cell [15]. Endogenous dopamine in DA neurons represents a significant toxicity to the cell This notion is consistent with the observations that basal protein oxidation is 2-fold higher in substantia nigra pars compacta than other regions in the normal human brain [21] and is significantly increased in brain tissues from PD compared with incidental Lewy body disease [22]. Our previous studies have shown that overexpression of parkin in the human dopaminergic neuroblastoma cell line SH-SY5Y reduces dopamine-induced apoptosis by decreasing ROS production [23]. Such a function would protect DA neurons from their own toxin, dopamine
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