Environmental Metals, Excitotoxicity and ALS

 描述(申请人提供)：肌萎缩侧索硬化症(ALS)，或Lou Gehrig病，是一种进行性神经退行性疾病，导致运动神经变性和死亡。家族性(FALS)和散发性(SALS)两种形式都存在；后者占主导地位。其原因尚不清楚。谷氨酸能功能的过度刺激--兴奋性毒性--伴随着钙离子的增加和活性氧物种(ROS)的产生，即使不是必不可少的，也是一个重要的作用，尽管其他机制可能在ALS的发病中起作用。运动神经元(MN)和星形胶质细胞(AST)的相互作用似乎有助于疾病的发展。环境对肌萎缩侧索硬化症的贡献经常被假设，但到目前为止还没有进行系统的测试。我们建议研究环境毒物甲基汞(MeHg)与在患有ALS的人类中发现的两个基因突变的相互作用。出生后慢性暴露于超氧化物歧化酶-1(SOD1G93A，G93A)突变的小鼠，与未经处理的G93A小鼠或暴露于相同浓度的甲基汞的wt小鼠相比，可加速ALS表型的开始。我们现在将检验这一假设，即出生后开始长期、低剂量的甲基汞暴露通过增加[Ca2+]i、产生ROS并诱导继发于谷氨酸(Glu)释放或对AST Glu转运体(EAAT1-2)的作用而继而诱导线粒体毒性，从而增强G93A和G85R SOD1突变体中ALS表型的发生和进展。这两个SOD1突变株在ALS表型发展的时间进程和主要毒性部位上有所不同。G93A小鼠在4个月内出现表型，疾病是MN导向的。G85R小鼠在7-9个月内出现ALS表型，疾病是基于AST的。这一设计将使我们能够比较AST和MNS在甲基汞诱导的反应中的作用。具体目标1：研究慢性成人甲基汞暴露时ALS表型的发展及MNS和AST的相对作用。具体目标2：研究氧化应激对甲基汞诱导的肌萎缩侧索硬化症表型增强的贡献。具体目标3：检测MN功能对甲基汞反应的细胞自主性。将在wt和SOD-1细胞的嵌合培养中研究AST和MNS之间的相互作用，以评估它们在甲基汞诱导的ALS表型增强的兴奋性毒性发展中的潜在作用。将使用脊髓切片和来自G93A和G85R菌株的MNS和AST的共培养来检验ALS表型发展之前的早期发病效应。谷氨酸介导的兴奋性突触后电流、[Ca~(2+)]i升高和ROS水平将检测甲基汞诱导的兴奋性毒性。在ALS表型的发展过程中，将测量EAAT1-2和参与[Ca2+]i调节的蛋白质的稳态mRNA表达水平，以与在培养细胞中所做的研究相关联。来自SOD1小鼠或wt的培养中的MNS将允许单独检查MeHg对Glu功能、[Ca2+]i氧化应激或线粒体损伤的早期影响。这项拟议的研究结果应该能够评估甲基汞诱导的谷氨酸介导的兴奋性毒性在促进MN功能障碍发展中的作用，并为环境暴露是易感人群发生ALS的潜在危险因素这一假设提供验证。

 DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, is a progressive neurodegenerative disease resulting in motor nerve degeneration and death. Both familial (FALS) and sporadic (SALS) forms exist; the latter greatly predominate. Its cause is unknown. Overstimulation of glutamatergic function - excitotoxicity - with increased Ca2+ and generation of reactive oxygen species (ROS), plays an important, if not integral role, though other mechanisms likely contribute to ALS pathogenesis. Interaction of motor neurons (MNs) and astrocytes (ASTs) appear to contribute to disease development. Environmental contribution to ALS has been postulated often, but to date not systematically tested. We propose to examine the interaction of the environmental toxicant methylmercury (MeHg) with two genetic mutations found in humans with ALS. Chronic postnatal MeHg exposure of mice with a mutation in superoxide dismutase-1 (SOD1G93A, G93A) hastens the onset of ALS phenotype compared to untreated G93A mice or wt mice exposed to identical MeHg concentrations. We will now test the hypothesis that chronic, low dose, MeHg exposure beginning postnatally enhances development and progression of ALS phenotype in the G93A and G85R SOD1 mutants by increasing [Ca2+]i, generating ROS and inducing mitochondrial toxicity secondary to enhanced release of glutamate (Glu) or actions on AST Glu transporters (EAAT1-2). The two SOD1 mutant strains differ in their time course of development of ALS phenotype and in their primary toxic focus. The G93A mice develop phenotype in ~4 mos and disease is MN-directed. G85R mice develop ALS phenotype over 7-9 months and disease is AST-based. This design will allow us to compare the roles of ASTs and MNs to MeHg induced responses. Specific Aim 1: Examines the development of ALS phenotype and relative role of MNs and ASTs during chronic adult MeHg exposure. Specific Aim 2: Examines oxidative stress as a contributor to MeHg-induced enhancement of ALS phenotype. Specific Aim 3: Examines the cell autonomy of MN function in response to MeHg. Interactions between ASTs and MNs will be examined in chimeric cultures of wt and SOD-1 cells to assess their potential roles in the development of excitotoxicity in MeHg-induced enhancement of ALS phenotype. Early onset effects preceding development of ALS phenotype will be examined using spinal cord slices and co-cultures of MNs and ASTs, from the G93A and G85R strains. Glu-mediated excitatory postsynaptic currents and elevation of [Ca2+] i and levels of ROS will test for MeHg-induced excitotoxicity. Steady- state mRNA expression levels for EAAT1-2 and proteins involved in [Ca2+] i regulation will be measured during development of ALS phenotype to correlate with studies done in cells in culture. MNs in culture derived from SOD1 mice or wt will allow examination of early effects of MeHg on Glu function, [Ca2+] i oxidative stress or mitochondrial damage in isolation. Results of the proposed study should permit assessment of the role of MeHg-induced Glu- mediated excitotoxicity in facilitating development of MN dysfunction and provide verification for the postulate that environmental exposure to metals is a potential risk factor for susceptible populations in development of ALS.