The Nerve Terminal as a Site of Acrylamide Action

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DESCRIPTION (provided by applicant): Exposure to acrylamide (ACR) produces ataxia and muscle weakness, which have been presumed to be caused by distal preterminal axon degeneration. However, research conducted during the current funding period has suggested that nerve terminals, and not axons, are primary sites of ACR action. This is in agreement with evidence suggesting that defective neurotransmission and terminal degeneration are early consequences of ACR intoxication. Whereas the nerve terminal might be a pathophysiologically relevant site, the mechanism of damage has not been addressed. Therefore, the goal of this project is to determine how ACR produces nerve terminal dysfunction and degeneration. We hypothesize that ACR impairs membrane fusion processes that mediate presynaptic exocytosis and docking of transport vesicles with nerve terminal plasmalemma. The fusion of opposing membranes is accomplished by formation of SNARE (Soluble NSF attachment protein receptors) core complexes that are subsequently disassembled by the actions of NSF (N-ethylmaleimide sensitive factor). NSF activity is exquisitely sensitive to inhibition by thiol oxidation and such inhibition has been shown to block membrane fusion. We propose that ACR binds to NSF through thiol adduction and that subsequent inhibition of NSF activity is responsible for functional and structural damage to nerve terminals. The following research has been designed to test this hypothesis. (1) Chemical interactions of ACR with different nerve terminals proteins (e.g., NSF, SNAP-25) will be characterized by mass spectroscopy. (2) The site of ACR inhibition within the synaptic vesicle cycle (e.g., docking, fusion, endocytosis) will be identified. (3) Formation of the SNARE core complex will be assessed in ACR-exposed synaptosomes. (4) SNARE core functionality will be determined during ACR exposure. (5) Effects of ACR on NSF-dependent dissolution of the SNARE complex will be examined. The hypothesis that ACR disrupts the SNARE core apparatus is novel. ACR is considered to be prototypical among chemicals that cause toxic neuropathies and, therefore, deciphering the corresponding molecular mechanism could provide insight into neurotoxicant mechanisms. Determining how these chemicals work will provide a rational basis for establishing occupational exposure conditions and for development of efficacious pharmacotherapeutic approaches.