Environmental Metals, Excitotoxicity and ALS

环境金属、兴奋性毒性和 ALS

• 批准号: 8909481
• 负责人: William D Atchison
• 金额: $ 46.32万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-12 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8909481
• 关键词: Address; Adult; Affect; Amino Acid Transporter; Amyotrophic Lateral Sclerosis; Antioxidants; Astrocytes; Cells; Cessation of life; Chimera organism; Chronic; Coculture Techniques; DNA Sequence Alteration; Development; Disease; Disease Progression; Divalent Cations; Dose; Environment; Environmental Exposure; Event; Excitatory Amino Acids; Exposure to; Functional disorder; Generations; Genes; Glutamate Transporter; Glutamates; Glutathione; Goals; Homeostasis; Human; Lesion; Measures; Mediating; Mercury; Metal exposure; Metals; Methylmercury Compounds; Mitochondria; Motor; Motor Neurons; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Injury; Neurons; Onset of illness; Oxidative Stress; Pathogenesis; Phenotype; Play; Population; Process; Production; Proteins; Reactive Oxygen Species; Regulation; Relative (related person); Risk Factors; Role; Secondary to; Single Nucleotide Polymorphism; Slice; Spinal Cord; Staging; System; Testing; Text; Time; Toxic Environmental Substances; Toxic effect; Transgenic Mice; base; cell type; comparative; design; early onset; environmental agent; excitotoxicity; gene environment interaction; genetic linkage; mRNA Expression; mitochondrial dysfunction; motor neuron development; motor neuron function; mouse model; mutant; overexpression; postnatal; postsynaptic; pre-clinical; progressive neurodegeneration; public health relevance; research study; response; superoxide dismutase 1; synaptic function; uptake

 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.

 描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）或Lou Gehrig病是一种导致运动神经变性和死亡的进行性神经退行性疾病。家族性（FALS）和散发性（SALS）形式都存在;后者占主导地位。其原因尚不清楚。过度刺激神经元能功能-兴奋性毒性-伴随Ca 2+增加和活性氧（ROS）的产生，即使不是不可或缺的作用，也起着重要的作用，尽管其他机制可能有助于ALS发病机制。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。环境因素对ALS的影响经常被假设，但迄今为止还没有系统的测试。我们建议研究环境毒物甲基汞（MeHg）与ALS患者中发现的两种基因突变的相互作用。与未经处理的G93 A小鼠或暴露于相同甲基汞浓度的野生型小鼠相比，超氧化物歧化酶-1（SOD 1G 93 A，G93 A）突变小鼠的慢性出生后甲基汞暴露加速了ALS表型的发病。我们现在将检验这一假设，即出生后开始的慢性低剂量甲基汞暴露通过增加[Ca 2 +]i、产生ROS和诱导继发于谷氨酸（Glu）释放增强的线粒体毒性或对AST Glu转运蛋白（EAAT 1 -2）的作用，增强G93 A和G85 R SOD 1突变体中ALS表型的发生和进展。这两种SOD 1突变株在ALS表型发展的时间进程和主要毒性焦点方面不同。G93 A小鼠在约4个月内形成表型，并且疾病是MN导向的。G85 R小鼠在7-9个月内形成ALS表型，并且疾病是基于AST的。这种设计将使我们能够比较ASTs和MN对甲基汞诱导反应的作用。具体目标1：检查ALS表型的发展和MN和AST在慢性成人甲基汞暴露过程中的相对作用。具体目标2：检查氧化应激作为甲基汞诱导的ALS表型增强的贡献者。具体目标3：检查细胞自主性的MN功能，以应对甲基汞。AST和MN之间的相互作用将在野生型和SOD-1细胞的嵌合培养物中进行检查，以评估它们在甲基汞诱导的ALS表型增强中的兴奋性毒性发展中的潜在作用。将使用脊髓切片和来自G93 A和G85 R菌株的MN和AST的共培养物来检查ALS表型发展之前的早发效应。Glu介导的兴奋性突触后电流和[Ca 2 +] i和ROS水平的升高将测试甲基汞诱导的兴奋性毒性。将在ALS表型发展期间测量EAAT 1 -2和参与[Ca 2 +] i调节的蛋白质的稳态mRNA表达水平，以与在培养的细胞中进行的研究相关联。来自SOD 1小鼠或野生型小鼠的培养物中的MN将允许单独检查甲基汞对Glu功能、[Ca 2 +] i氧化应激或线粒体损伤的早期影响。拟议研究的结果应允许评估甲基汞诱导的Glu介导的兴奋性毒性在促进MN功能障碍发展中的作用，并验证环境暴露于金属是易感人群发生ALS的潜在风险因素这一假设。