The Nerve Terminal as the Site of Action for Type-2 Alkenes

描述（由申请方提供）：人类和实验室动物暴露于丙烯酰胺（ACR）会产生累积神经毒性，其特征为步态异常、肌无力和中枢-外周神经病。ACR是1，2-不饱和羰基衍生物，被分类为2型烯烃。这是一大类亲电子化学品，具有广泛的工业，农业和制药用途。这些化学物质也是公认的饮食污染物和环境污染物。在17-20年期间收集的数据提供了证据，表明ACR通过与功能重要蛋白上的亲核巯基形成不可逆的共价加合物来损害神经末梢功能。蛋白质组学分析表明，ACR和2型烯烃的蛋白质靶标也是一氧化氮（NO）信号传导的受体。NO是一种生物亲电体，并且已经被经典地认为通过鸟苷酸环化酶激活来影响细胞过程。然而，NO也可以通过与蛋白质催化三联体中的巯基化半胱氨酸形成可逆加合物来调节细胞生理学。在神经末梢，NO信号通过调节突触囊泡周期和其他突触前过程而关键地参与神经传递。因此，NO和ACR在共同的半胱氨酸巯基位点相互作用，因此，我们假设这些受体的ACR块可逆NO结合的不可逆加合。NO信号传导的中断和随后的神经调节控制的丧失产生突触前毒性。因此，具体目标#1研究将定义ACR与CNS神经末梢的S-亚硝基化（SNO）蛋白质组的相互作用。SNOSID（S-亚硝基化位点鉴定）蛋白质组学分析将用于证明神经末梢蛋白上SNO-半胱氨酸位点的ACR加合。具体目标#2研究将通过考虑替代作用机制来评估ACR-NO相互作用的特异性;即，我们将确定ACR对可溶性喹酰环化酶和一氧化氮合酶（NOS）活性/基因表达的影响。由于NO调节大多数细胞的生理过程，目前还不清楚为什么神经末梢NO信号可能被ACR选择性靶向。因此，特定目标3研究将考虑可能使神经末梢易受亲电攻击的几种解剖学和分子特征。确定ACR神经毒性的机制可以提供关于其他2型烯烃的毒理学过程的全面见解。拟议研究的结果还可以帮助我们了解阿尔茨海默病（AD）和其他慢性神经退行性疾病的发病机制，这些疾病可能涉及细胞氧化应激和丙烯醛和其他2型烯烃的内源性生成。人类暴露于共轭2型烯烃（例如，丙烯酰胺，丙烯酸甲酯，甲基乙烯基酮）通过普遍的环境来源（例如，工业接触、吸烟、汽车尾气、燃烧、药物），并可能导致神经组织和其他器官系统（肝脏、肾脏）的严重毒性。还有证据表明，2型烯烃的内源性产生（例如，丙烯醛，2-羟基-4-壬烯醛）在介导与意外神经创伤和某些人类神经变性病症如阿尔茨海默病相关的神经细胞损伤中起关键作用。因此，拟议的2型烯烃神经毒性研究可能会更好地了解环境毒物暴露或疾病过程引起的脑损伤，这最终将有助于开发有效的治疗方法。

DESCRIPTION (provided by applicant): Abstract Exposure of humans and laboratory animals to acrylamide (ACR) produces cumulative neurotoxicity characterized by gait abnormalities, muscle weakness and a central-peripheral neuropathy. ACR is an 1,2-unsaturated carbonyl derivative and is classified as a type-2 alkene. This is a large class of electrophilic chemicals that have broad industrial, agricultural and pharmaceutical uses. These chemicals are also well-recognized dietary contaminants and environmental pollutants. Data collected during yrs. 17-20 have provided evidence that ACR impairs nerve terminal function by forming irreversible covalent adducts with nucleophilic sulfhydryl groups on functionally important proteins. Proteomic analyses indicate that the protein targets of ACR and the type-2 alkenes are also acceptors for nitric oxide (NO) signaling. NO is a biological electrophile and has been classically thought to influence cell processes through guanylyl cyclase activation. However, NO can also modulate cell physiology by forming reversible adducts with cysteine thiolates in protein catalytic triads. At the nerve terminal, NO signaling is critically involved in neurotransmission through modulation of the synaptic vesicle cycle and other presynaptic processes. Thus, NO and ACR interact at common cysteine sulfhydryl sites and, therefore, we hypothesize that irreversible adduction of these receptors by ACR blocks reversible NO binding. The disruption of NO signaling and ensuing loss of neuromodulatory control produces presynaptic toxicity. Therefore, Specific Aim #1 research will define the interactions of ACR with the S-nitrosylated (SNO) proteome of CNS nerve terminals. SNOSID (S-nitrosylated site identification) proteomic analysis will be used to demonstrate ACR adduction of SNO-cysteine sites on nerve terminal proteins. Specific Aim #2 studies will evaluate the specificity of the ACR-NO interaction by considering alternative mechanisms of action; i.e., we will determine the effects of ACR on soluble quanylyl cyclase and nitric oxide synthase (NOS) activity/gene expression. Because NO modulates physiological processes in most cells, it is unclear why nerve terminal NO signaling might be selectively targeted by ACR. Therefore, Specific Aim #3 studies will consider several anatomical and molecular features that might predispose nerve terminals to electrophilic attack. Identifying the mechanism of ACR neurotoxicity could offer global insight regarding the toxicological processes of other type-2 alkenes. Results of the proposed research could also help us understand the pathogenesis of Alzheimer's disease (AD) and other chronic neurodegenerative conditions that presumably involve cellular oxidative stress and endogenous generation of acrolein and other type-2 alkenes. PUBLIC HEALTH RELEVANCE Human exposure to conjugated type-2 alkenes (e.g., acrylamide, methyl acrylate, methylvinyl ketone) occurs through pervasive environmental sources (e.g., industrial exposure, cigarette smoking, car exhaust, combustion, pharmaceuticals) and can result in significant toxicity in nervous tissue and other organ systems (liver, kidney). There is also evidence that endogenous production of type-2 alkenes (e.g., acrolein, 2-hydryoxy-4-nonenal) is critically involved in mediating nerve cell injury associated with accidental neurotrauma and certain human neurodegenerative conditions such as Alzheimer's disease. Therefore, the proposed studies of type-2 alkene neurotoxicity could lead to a better understanding of brain injuries caused by environmental toxicant exposure or disease processes, which would ultimately help in the development of effective therapeutic approaches.