Acetylcholinesterase (AChE), the enzyme that terminates cholinergic transmission in the nervous system, is the target of organophosphate nerve agents and pesticides. Inactivation of AChE can result in rapid debilitation or death, thus the use of easily synthesized nerve agents such as soman and sarin by terrorists looms as a potentially catastrophic event. Our laboratory has recently shown that amino terminal peptides derived from the non-catalytic AChE targeting subunits stabilize the newly-synthesized AChE molecules and prevent their degradation in skeletal muscle. This in turn dramatically increases active enzyme and specifically AChE at the neuromuscular synapse when administered to living mice. This increase is rapid and sufficient to protect the animals from exposure to 2x LD50 DFP. Recent preliminary studies in our lab have uncovered small molecules that can enhance AChE folding, and an additional study has provided a novel mechanism for replacing damaged AChE at the neuromuscular synapse. Our specific aims on this project are; 1) to continue our studies on AChE-inducing/stabilizing peptides in vivo using our mouse model to optimize dose and delivery when administered before or after exposure to the surrogate nerve agent DFP; additional studies will test the efficacy of the PRAD-KDEL peptides against soman and tabun; 2) We have identified several ligands that can increase expression of active AChE by enhancing its folding; these compounds will be studied in cultured cells to determine their mechanism of action and test the hypothesis that they act in a manner analogous but not identical to the PRAD-KDEL peptides; possible synergies will be determined; 3) test the hypothesis that these ligands can increase AChE expression in vivo by enhancing folding and stabilization using our mouse model; and 4) Determine the ability of our novel AChE replacement approach using organophosphate resistant enzyme to protect mice from exposure to organophosphate nerve agents and their ability to improve survival when administered after exposure. Together these studies will lead to novel methods for increasing AChE expression in vivo for protection against organophosphate compounds as well as enhancing the rate of recovery following exposure. PUBLIC HEATLTH RELEVANCE: Acetylcholinesterase (AChE) is the enzyme that terminates neuromuscular transmission and is the target of organophosphate nerve gases and some pesticides. Our research is aimed at developing novel therapies to restore active AChE following its inactivation using a combination of peptides and drugs that increase the production of enzyme in the brain and muscles, as well as replacing damaged molecules with new ones.