DA, or with aMT-treatment. The Methionine residue at position 127 is important for DA-related cytotoxicity of a-syn in PC12 cells It 15060526 was reported that methionine residues in a-syn are oxidized in the presence of DA; thus, we examined the effects of mutating 5 DA-Related Oxidative Modification of a-Synuclein methionine residues 116 and 127 on a-syncytotoxicity in M116A, M127A, and M116A/M127A mutant asyn-expressing PC12 cells. All three a-syn mutants were induced 37 days after the withdrawal of Dox from the medium. Induced expression of M116A mutant a-syn was less toxic than the wildtype a-syn, while the M127A and M116A/M127A mutant cell lines showed no vulnerability to increased expression of respective a-syn mutants. Methionine sulfoxide is detected in a-syn in PC12 cells In the presence of DA, Met was reported to be an important modification of a-syn; thus, we used anti-Met antibodies to investigate whether a-syn Met was present in our cell lines. Following immunoprecipitation of total a-syn from PC12 cells, we observed Met-labeled a-syn that was decreased by the inhibition of catecholamine metabolism with aMT. On the other hand, reserpine, which increases Debio-1347 intracellular free DA, increased the amount of Met-labeled a-syn. To identify which a-syn methionine residue is predominantly oxidized in the presence of DA, we compared the degree of a-syn methionine oxidation in wildtype a-syn-, M116A-, M127A-, and M116A/M127A-expressing PC12 cells. Each of the a-syn mutants, but particularly those with M127A mutations, exhibited less Met than that in the wildtype a-syn, suggesting that M127 11478874 was the major target for 
methionine oxidation in a-syn. Despite the undetectable levels of phosphorylation of the wildtype a-syn Y125 and S129 residues, mutating the tyrosine and serine residues in the mutant a-synexpressing PC12 cells still reduced DA-associated cell vulnerability. In an attempt to explain this, we also checked for the presence of Met in the Y125D and S129A mutants of a-syn. Surprisingly, Met levels were lower in both the Y125D and S129A mutants of a-syn compared with wildtype, suggesting that the Y125 and/or S129 may also play an important role in the oxidation of M127 by DA. 125YEMPS129 and 125YEAPS129 peptides enhance DAQ-related H2O2 formation in vitro Because the sulfide moiety in the methionine residue is usually oxidized by H2O2 when Met is formed, we measured the levels of H2O2 production during the incubation of DA alone or with peptides designed from the amino acid sequence in the C-terminal of a-syn. H2O2 was produced in vitro during the incubation of the reaction mixture with DA alone, and the coincubation of DA with the 125YEMPS129 peptide increased the production of H2O2, while the peptides lacking the Y125 or S129 residues decreased the production of H2O2 in the present DA-Related Oxidative Modification of a-Synuclein experiments. Interestingly, co-incubation of DA with the mutant peptide 125YEAPS129 produced the highest levels of H2O2. Discussion a-syn is a key molecule in PD pathogenesis, and is associates not only with familial PD, but also with Lewy body formation in the brains of sporadic PD patients. Overexpression of a-syn in the PARK4 family of PD results in a typical parkinsonian phenotype and loss of dopaminergic neurons as well as common sporadic PD. Some reports suggest that a-syn exhibits its cytotoxic character only in the dopaminergic cells, and not in the non-dopaminergic cells. However, it is not well understo
