Mechanisms of Methylmercury Induced Neuronal Toxicity

说明(申请人提供)：甲基汞(MeHg)是一种全球性污染物和强有力的神经毒素，其在食物链中的丰富要求对其中枢神经系统毒性的后果和机制进行更多的研究。我们新假说的提出是基于我们对以下方面的认识：(A)汞化合物对-SH基团的阴离子形式具有显著的亲和力，(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)，通过促进二硫键的形成和断裂来调节蛋白质的氧化还原状态。鉴于甲基汞对硫醇的高亲和力，我们假设甲基汞干扰Hsp90伴侣功能，导致细胞内稳态和神经毒性改变。已知的与Hsp90结合的客户蛋白包括I型(神经性)一氧化氮合酶(NOS)和胞浆前列腺素E合成酶(CPGESIp23)。此外，Hsp90还减少了线粒体电子传输蛋白细胞色素c(Cytc)。其具体目的是[1]证明甲基汞在物理上与Hsp90结合，改变Hsp90的伴侣功能。[2]确定甲基汞是否通过改变Hsp90与PGES/p23的结合而增加cPLA2-环氧合酶-1(COX-1)-PGES酶途径的活性，从而增加前列腺素E2(PGE2)的产生。[3]证明通过改变Hsp90和神经元型一氧化氮合酶(NNOS)之间的联系，甲基汞使nNOS活性解偶联，导致一氧化氮合酶(-O2)的产生，并降低了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输链，导致氧产生增加和细胞内抗氧化剂谷胱甘肽水平降低而发生。实验方法包括无细胞裂解物、大鼠体外星形胶质细胞、神经元和星形胶质细胞/神经元共培养，以及在大鼠体内的确证研究。甲基汞介导的Hsp90/客户蛋白相互作用的改变提供了一个创新和统一的机制，将已知的甲基汞与含-SH的配体形成络合物的倾向与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结合在一起。这些研究的结果将为阐明甲基汞神经毒性的有意义的机制提供新的线索，并为新的药物治疗方法铺平道路。此外，对Hsp90客户蛋白翻译后修饰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