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

人类和实验动物暴露于丙烯酰胺（ACR）会产生累积性神经毒性，其特征是步态异常、肌肉无力和中枢-周围神经病变。ACR是一种1,2不饱和羰基衍生物，被归类为2型烯烃。这是一类亲电化学物质，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。按年收集的数据。17-20提供的证据表明，ACR通过在功能重要的蛋白质上与亲核巯基形成不可逆的共价加合物而损害神经末梢功能。蛋白质组学分析表明，ACR和2型烯烃的靶蛋白也是一氧化氮（NO）信号的受体。一氧化氮是一种生物亲电试剂，传统上认为它通过胍基环化酶激活来影响细胞过程。然而，NO也可以通过在蛋白质催化三元组中与半胱氨酸硫酸盐形成可逆加合物来调节细胞生理。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ACR对这些受体的不可逆内合阻断了可逆的NO结合。一氧化氮信号的破坏和随之而来的神经调节控制的丧失产生突触前毒性。因此，Specific Aim #1研究将明确ACR与中枢神经末梢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的sno -半胱氨酸位点的ACR内聚。特异性Aim #2研究将通过考虑其他作用机制来评估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在大多数细胞中调节生理过程，因此尚不清楚为什么神经末梢NO信号可能被ACR选择性靶向。因此，Specific Aim #3研究将考虑一些可能使神经末梢易发生亲电攻击的解剖和分子特征。确定ACR的神经毒性机制可以为其他2型烯烃的毒理学过程提供全局视角。本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。人类通过普遍存在的环境来源（如工业接触、吸烟、汽车尾气、燃烧、药物）接触共轭2型烯烃（如丙烯酰胺、丙烯酸甲酯、甲基乙烯酮），并可对神经组织和其他器官系统（肝、肾）造成显著毒性。也有证据表明，内源性2型烯烃（如丙烯醛、2-羟基-4-壬烯醛）的产生在介导与意外神经创伤和某些人类神经退行性疾病（如阿尔茨海默病）相关的神经细胞损伤中起关键作用。因此，对2型烯烃神经毒性的研究可以更好地理解由环境毒物暴露或疾病过程引起的脑损伤，这将最终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.