Mechanisms of Methylmercury Induced Neuronal Toxicity

描述（由申请人提供）：甲基汞（MeHg）是一种全球性污染物和强效神经毒素，其在食物链中的丰度要求对其中枢神经系统毒性的后果和机制进行进一步研究。我们的新假设的形成是基于我们对以下方面的认识：(a)汞对阴离子形式的巯基（-SH）基团的显著亲和力，(b)硫醇在蛋白质生物化学中的重要作用，以及(c)分子伴侣蛋白，如热休克蛋白90 (Hsp90)，通过促进二硫桥的形成和断裂来调节蛋白质氧化还原状态。鉴于甲基汞对硫醇的高亲和力，我们假设甲基汞干扰Hsp90伴侣的功能，导致细胞稳态改变和神经毒性。已知与Hsp90结合的客户蛋白包括I型（神经元）一氧化氮合成酶（NOS）和胞浆前列腺素E合成酶（cPGESIp23）。此外，Hsp90降低线粒体电子传递蛋白细胞色素c （cyt c）。具体目的是证明MeHg与Hsp90物理结合，改变Hsp90伴侣的功能。确定MeHg是否通过改变Hsp90与PGES/p23的结合，增加cpla2 -环氧化酶-1 (COX-1)-PGES酶途径的活性，从而增加前列腺素E2 （PGE2）的产生。通过改变Hsp90与神经元NOS （nNOS）之间的联系，MeHg解除了nNOS的活性，导致NOS衍生的超氧化物（-O2）产生，降低了NO的生物利用度。[4]确定甲基汞诱导的氧化应激是否通过抑制线粒体电子传递链发生，从而导致-O2产生增加和细胞内抗氧化剂谷胱甘肽水平降低。实验方法包括无细胞裂解物，大鼠来源的体外星形胶质细胞，神经元，星形胶质细胞/神经元共培养，以及大鼠体内确证研究。甲基汞介导的Hsp90/客户蛋白相互作用的改变提供了一种创新和统一的机制，整合了甲基汞与含- sh配体形成复合物的已知倾向，以及甲基汞诱导的氧化应激、线粒体功能障碍和中枢神经系统毒性。这些研究结果将揭示甲基汞诱导神经毒性的有意义的机制，并为新的药物治疗方式铺平道路。此外，对Hsp90客户蛋白翻译后修饰的改变的研究可能为其他神经退行性疾病提供新的机制见解，因此它们具有广泛的生物学意义

DESCRIPTION (provided by applicant): Methylmercury (MeHg) is a global pollutant and potent neurotoxin whose abundance in the food chain mandates additional studies on the consequences and mechanisms of its CNS toxicity. Formulation of our new hypotheses was predicated on our appreciation for (a) the remarkable affinity of mercurials for the anionic form of sulfhydryl (-SH) groups, (b) the essential role of thiols in protein biochemistry, and (c) the role of molecular chaperone proteins, such as heat shock protein 90 (Hsp90), in the regulation of protein redox status by facilitating the formation and breakage of disulfide bridges. Given the high affinity of MeHg for thiols, we hypothesized that MeHg interferes with Hsp90 chaperone function resulting in altered cellular homeostasis and neurotoxicity. Among the client proteins known to bind to Hsp90 are type I (neuronal) nitric oxide synthase (NOS), and cytosolic prostaglandin E synthase (cPGESIp23). Additionally, Hsp90 reduces the mitochondrial electron transport protein, cytochrome c (cyt c). The specific aims are to [1] demonstrate that MeHg physically binds to Hsp90, altering Hsp90 chaperone function. [2] Determine whether MeHg increases the activity of the cPLA2-cyclooxygenase-1 (COX-1)-PGES enzymatic pathway resulting in enhanced prostaglandin E2 (PGE2) production by altering the binding of Hsp90 to PGES/p23. [3] Demonstrate that by altering the association between Hsp90 and neuronal NOS (nNOS) MeHg uncouples nNOS activity, resulting in NOS-derived superoxide (-O2) production and reduced NO bioavailability. [4] Determine whether MeHg-induced oxidant stress occurs via inhibition of the mitochondrial electron transport chain, causing increased -O2 production and reduced levels of the intracellular antioxidant, glutathione. The experimental approach includes cell-free lysates, rat-derived in vitro astrocyte, neuron, and astrocyte/neuron co-cultures, as well as in vivo corroborative studies in the rat. MeHg-mediated alterations in Hsp90/client protein interactions offers an innovative and unifying mechanism integrating the known propensity of MeHg to form complexes with -SH-containing ligands and MeHg-induced oxidant stress, mitochondrial dysfunction and CNS toxicity. The results of these studies will shed new light on meaningful mechanisms of MeHg-induced neurotoxicity, and pave the way for new pharmacological modalities for treatment. Additionally, studies on altered post-translational modification of Hsp90 client proteins might offer new mechanistic insight into other neurodegenerative disorders, and therefore they have broad biological implications