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Parkinson's disease (PD) is a neurodegenerative disorder of the elderly characterized by resting tremor, rigidity, bradykinesia and postural instability. Loss of function mutations in DJ-1 lead to an early-onset form of inherited parkinsonism. To provide new insight into mechanisms of PD, we modeled loss of DJ-1 function in Drosophila by generating microdeletions in regions of the genome containing the fly DJ-1 homologs: DJ-1a and DJ-1b. Flies lacking DJ-1 function displayed selective sensitivity to toxins acting through oxidative stress, including pesticides and herbicides that are associated with increased risk of sporadic PD (paraquat, rotenone). Like the human protein, fly DJ-1b protein responded biochemically to oxidative stress by shifting to more acidic isoforms by 2-dimensional gel analysis, and demonstrated a novel property of shifting to a slower migrating species by 1-dimensional SDS-PAGE. To reveal insight into functional aspects of the DJ-1 protein, we focused on how modification of DJ-1b in response to paraquat exposure may modulate activity of the protein. In our studies, we found that the cysteine residues likely become oxidized, and oxidation of cysteine 104 results in the observed 1-dimensional SDS-PAGE shift. Functional analysis revealed that cysteine 104 in fly DJ-1b was critical for the protective function of the protein upon oxidative stress. Mutation of this cysteine residue to an alanine or aspartic acid led to a functionally inactive DJ-1b protein, despite the fact that the aspartic acid mutant form biochemically resembled the form of DJ-1b that normally occurs upon oxidative stress. Taken together, our biochemical and functional studies of the DJ-1b protein suggest that modification at cysteine 104 upon oxidative stress may lead to the functional inactivation of the DJ-1b protein. To further examine the significance of DJ-1b modification, we analyzed modification in older animals and found that it increases with age in flies, mice and human brain tissue. Furthermore, DJ-1b modification upon oxidative insult was greatly enhanced with age (to 50% in aged animals), and older flies were strikingly more susceptible to death upon paraquat exposure. These studies suggest a model for sporadic PD whereby modification of DJ-1 in response to oxidative stress and/or aging inhibits the protective activity of DJ-1 protein, leading to increased vulnerability to oxidative insult. As age and oxidative stress are two critical factors involved in several neurodegenerative diseases beyond PD, these findings suggest a potentially broad role for DJ-1 modification in age-associated neurodegenerative diseases. Manipulation of the DJ-1 pathway could serve as an attractive therapeutic target for age-associated diseases including PD.