Background: Exposure to the organophosphorus pesticide, chlorpyrifos, can have chronic adverse effects that are not explained by inhibition of acetylcholinesterase. Goal: The long-term objective is to establish a mechanism to explain neurotoxicity from chronic low dose exposure to organophosphorus pesticides. Hypothesis: The hypothesis developed in this proposal is based on our mass spectrometry observations which show that proteins treated with chlorpyrifos oxon make covalent adducts on lysine and some of these adducts undergo a crosslinking reaction with glutamic or aspartic acid side chains to form isopeptide bonds. The crosslinked proteins are visualized as high molecular weight aggregates on polyacrylamide gels. The scientific literature links protein aggregates to neurodegeneration by proposing that protein aggregates inhibit axonal transport. The consequence is slow loss of neuronal spine density, loss of connections between neurons, and clinical symptoms. Method: This proposal aims to establish the steps that initiate neurotoxicity. The plan is to use liquid chromatography-tandem mass spectrometry to show that crosslinked proteins correlate with loss of function. The functional test is inhibition of neurite outgrowth in cultured cells. The rate of formation and clearance of crosslinked proteins will be measured. Outcome: Mass spectrometry analysis is expected to identify crosslinks between neuronal cytoskeleton proteins in human neuroblastoma cells treated with chlorpyrifos oxon. The presence of crosslinked proteins is expected to correlate with inhibition of neurite outgrowth, thus showing a relationship between crosslinking and loss of function. Measurement of the rates of formation and clearance of crosslinked proteins is expected to support a mechanism where misfolded, crosslinked proteins accumulate in neurons. The consequence of accumulated crosslinked proteins is neurotoxicity. Innovation: We are the first to report that organophosphorus toxicants are crosslinking agents. Significance: The proposed work is expected to provide a mechanism to explain neurotoxicity from chronic low dose exposure to organophosphorus pesticides. Future studies may build on this work to understand neurotoxicity following exposure to a variety of chemicals.