Dopamine oxidation has been previously demonstrated to cause dysfunction in mitochondrial respiration and membrane permeability, possibly related to covalent modification of critical proteins by the reactive dopamine quinone. However, specific mitochondrial protein targets have not been identified. In this study, we utilized proteomic techniques to identify proteins directly conjugated with 14 C-dopamine from isolated rat brain mitochondria exposed to radiolabeled dopamine quinone (150 μM) and differentiated SH-SY5Y cells treated with 14 C-dopamine (150 μM). We observed a subset of rat brain mitochondrial proteins that were covalently modified by 14 C-dopamine, including chaperonin, ubiquinol-cytochrome c reductase core protein 1, glucose regulated protein 75/ mitochondrial HSP70/mortalin, mitofilin, and mitochondrial creatine kinase. We also found the Parkinson's disease associated proteins ubiquitin carboxy-terminal hydrolase L1 and DJ-1 to be covalently modified by dopamine in both brain mitochondrial preparations and SH-SY5Y cells. The susceptibility of the identified proteins to covalent modification by dopamine may carry implications for their role in the vulnerability of dopaminergic neurons in Parkinson's disease pathogenesis.

Aberrant aggregation of ␣-synuclein (␣-syn) to form fibrils and insoluble aggregates has been implicated in the pathogenic processes of many neurodegenerative diseases. Despite the dramatic effects of dopamine in inhibiting the formation of ␣-syn fibrils by stabilization of oligomeric intermediates in cell-free systems, no studies have examined the effects of intracellular dopamine on ␣-syn aggregation. To study this process and its association with neurodegeneration, intracellular catechol levels were increased to various levels by expressing different forms of tyrosine hydroxylase, in cells induced to form ␣-syn aggregates. The increase in the steady-state dopamine levels inhibited the formation of ␣-syn aggregates and induced the formation of innocuous oligomeric intermediates. Analysis of transgenic mice expressing the disease-associated A53T mutant ␣-syn revealed the presence of oligomeric ␣-syn in nondegenerating dopaminergic neurons that do contain insoluble ␣-syn. These data indicate that intraneuronal dopamine levels can be a major modulator of ␣-syn aggregation and inclusion formation, with important implications on the selective degeneration of these neurons in Parkinson's disease.