The Nerve Terminal as a Site of Acrylamide Action

描述（由申请人提供）：暴露于丙烯酰胺（ACR）会产生共济失调和肌肉无力，这被认为是由末梢前轴突变性引起的。然而，在当前资助期内进行的研究表明，神经末梢而不是轴突是ACR作用的主要部位。这与证据表明神经传递缺陷和终末变性是ACR中毒的早期后果是一致的。尽管神经末梢可能是病理生理学上相关的部位，但其损伤机制尚未得到解决。因此，本项目的目标是确定ACR如何产生神经末梢功能障碍和变性。我们假设ACR损害了介导突触前胞分泌和转运囊泡与神经末梢质膜对接的膜融合过程。相反膜的融合是通过形成SNARE（可溶性NSF附着蛋白受体）核心复合物来完成的，该核心复合物随后被NSF （n -乙基马来酰亚胺敏感因子）的作用分解。NSF活性对巯基氧化的抑制非常敏感，这种抑制已被证明可以阻断膜融合。我们认为ACR通过巯基内聚与NSF结合，随后NSF活性的抑制是神经末梢功能和结构损伤的原因。下面的研究旨在验证这一假设。(1) ACR与不同神经末梢蛋白（如NSF、SNAP-25）的化学相互作用将通过质谱进行表征。(2)确定突触囊泡周期中ACR抑制的位点（如对接、融合、内吞作用）。(3)在acr暴露的突触体中，将评估SNARE核心复合物的形成。(4) SNARE核心功能将在ACR暴露期间确定。(5)将研究ACR对nsf依赖性SNARE复合物溶解的影响。ACR破坏SNARE核心装置的假设是新颖的。ACR被认为是引起毒性神经病的化学物质中的原型，因此，破译相应的分子机制可以为神经毒性机制提供深入了解。确定这些化学物质如何起作用将为建立职业暴露条件和开发有效的药物治疗方法提供合理的基础。

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.