Abstract
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies. Many recent studies focused on the interaction between α-synuclein (α-syn) and dopamine in the pathogenesis of PD, and fluorescent anisotropy suggested that the C-terminal region of α-syn may be a target for modification by dopamine. However, it is not well understood why PD-related pathogenesis occurs selectively in dopaminergic neurons. We investigated the interaction between dopamine and α-syn with regard to cytotoxicity. A soluble oligomer was formed by co-incubating α-syn and dopamine in vitro. To clarify the effect of dopamine on α-syn in cells, we generated PC12 cells expressing human α-syn, as well as the α-syn mutants, M116A, Y125D, M127A, S129A, and M116A/M127A, in a tetracycline-inducible manner (PC12-TetOFF-α-syn). Overexpression of wildtype α-syn in catecholaminergic PC12 cells decreased cell viability in long-term cultures, while a competitive inhibitor of tyrosine hydroxylase blocked this vulnerability, suggesting that α-syn-related cytotoxicity is associated with dopamine metabolism. The vulnerabilities of all mutant cell lines were lower than that of wildtype α-syn-expressing cells. Moreover, α-syn containing dopamine-mediated oxidized methionine (Met(O)) was detected in PC12-TetOFF-α-syn. Met(O) was lower in methionine mutant cells, especially in the M127A or M116A/M127A mutants, but also in the Y125D and S129A mutants. Co-incubation of dopamine and the 125YEMPS129 peptide enhanced the production of H2O2, which may oxidize methionine residues and convert them to Met(O). Y125- or S129-lacking peptides did not enhance the dopamine-related production of H2O2. Our results suggest that M127 is the major target for oxidative modification by dopamine, and that Y125 and S129 may act as enhancers of this modification. These results may describe a mechanism of dopaminergic neuron-specific toxicity of α-syn in the pathogenesis of PD.
Highlights
Parkinson’s disease (PD) is one of the major neurodegenerative diseases, and is characterized by a selective degeneration of dopamine (DA) neurons and the formation of a-synuclein (a-syn)containing Lewy bodies in the substantia nigra, with the subsequent loss of their terminals in the striatum [1,2,3]
Numerous genetic risk factors are reported to induce and/or enhance PD onset [3,7,8,9,10], and a-syn is one of the most important molecules associated with the pathogenesis of familial and sporadic PD [9]. a-syn is a 140-amino acid protein that is a major component of Lewy bodies in the brains of PD patients as well as those with dementia with Lewy bodies [1,11]
Because phosphorylations of the serine residue at position 129 (S129) and the tyrosine residue at the position 125 (Y125) were identified as pathogenic modifications in the pathogenesis of synucleinopathy, we examined the phosphorylation of S129 and Y125 using phospho-S129- or phospho-Y125- specific antibodies
Summary
Parkinson’s disease (PD) is one of the major neurodegenerative diseases, and is characterized by a selective degeneration of dopamine (DA) neurons and the formation of a-synuclein (a-syn)containing Lewy bodies in the substantia nigra, with the subsequent loss of their terminals in the striatum [1,2,3]. To investigate DA-related modifications of a-syn and the DA neuron-specific pathogenesis of PD, we established several cell lines expressing mutant forms of a-syn, and examined the role of Met(O) in a-syn oligomerization and cytotoxicity.
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