AGE-DEPENDENT CHANGES IN SYNAPTIC RAFT DOMAINS AND PLASMA MEMBRANE CA2+ - ATPASE

突触筏域和质膜 CA2 - ATP酶的年龄依赖性变化

• 批准号: 7347339
• 负责人: MARY L. MICHAELIS
• 金额: $ 25.23万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7347339
• 关键词: Affect; Age; Aging; Alzheimer' s Disease; Antibodies; Binding; Brain; Ca(2+)-Transporting ATPase; Calmodulin; Cell membrane; Cell model; Cells; Ceramides; Cholesterol; Chronic; Cognitive; Complex; Cultured Cells; Data; Dementia; Detergents; Disease; Disruption; Elderly; Environment; Enzymes; Experimental Models; Fluorescence; Gangliosides; Glycosphingolipids; Goals; Homeostasis; Image; Immunoprecipitation; Impaired cognition; In Situ; In Vitro; Intervention; Kinetics; Link; Lipid Peroxidation; Lipids; Localized; Management Information Systems; Membrane; Membrane Microdomains; Modeling; Modification; Molecular; Molecular Biology; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Oxidative Stress; Parkinson Disease; Pathologic Processes; Performance; Play; Population; Predisposition; Preparation; Principal Investigator; Prion Diseases; Process; Property; Proteins; RNA Interference; Rattus; Regulation; Relative (related person); Resistance; Role; Sampling; Signal Transduction; Site; Sphingolipids; Stress; Structure; Synapses; Synaptic Membranes; Synaptosomes; Techniques; Testing; Transcriptional Activation; Up-Regulation; Variant; age related; aged; aging brain; brain cell; brain tissue; enzyme activity; excitotoxicity; genetic manipulation; in vivo; insight; intracellular protein transport; macromolecule; middle age; oxidation; prevent; programs; protein localization location; research study; sarcopenia

Mechanisms underlying the age-related decline in cognitive performance and increased susceptibility to neurodegenerative diseases are not known, though dysregulation of Ca2+ homeostasis and enhanced oxidative stress appear to be primary contributors. We have found that a major Ca2+ regulator, the plasma membrane Ca2+ -ATPase (PMCA), is uniquely sensitive to oxidation and is progressively lost from specific membrane domains of brain neurons with age. These 'raft' domains create platforms for Ca2+- signaling and are also known to be sites for the processing of the abnormal proteins associated with neurodegenerative diseases. The overall goal of this project is to identify the mechanisms that regulate the localization of PMCA in raft domains and the age-dependent changes underlying the loss of PMCA activity from rafts. Our hypothesis is that this loss is due to enhanced oxidative stress that leads to agedependent changes in the proteins and lipids that regulate the activity and localization of PMCA within rafts. The Aims of this application are to: (1) characterize synaptic membrane rafts from 5, 22 and 34- mos F344/BNF1 rats in terms of protein and lipid oxidation, raft lipid composition, and effects of in vitro oxidative stress; (2) use pharmacological and genetic manipulations to elucidate the role of the raft lipid environment in the membrane localization and kinetic properties of PMCA; (3) determine the role of protein interactions in localization of PMCA in rafts by identifying PMCA binding partners in raft vs non-raft domains, determining their levels in membranes from 5, 22, and 34-mos rats, and altering expression of the major partners in cells to test directly their effects on PMCA localization and activity. Our strategy involves analysis of in vivo brain aging in parallel with in vitro neuronal models for testing specific mechanisms that may explain some of the age-dependent alterations. We make extensive use of expertise in lipidomics, protein identification, and molecular biology available in the Cores, and preliminary data support the feasibility of all aims. Despite the evidence for involvement of rafts in Ca2+ signaling and for Ca2+ dysregulation in agedependent neurodegenerative diseases, nothing is known about aging in neuronal rafts. Our studies will begin to fill that gap and provide new insights into links between aging, altered Ca2+ disposition, oxidative stress and the enhanced vulnerability of the aging brain to the devastating dementias that affect elderly populations. Lav Statement: The proposed studies will enhance our understanding of changes that occur in the aging brain that make it so vulnerable to cognitive impairment and diseases such as Alzheimer's. The ultimate goal is to identify what interventions might slow or prevent some of those changes with advancing age.

与年龄相关的认知能力下降和易感性增加的机制 神经退行性疾病尚不清楚，但 Ca2+ 稳态失调和增强 氧化应激似乎是主要因素。我们发现，血浆是一个主要的 Ca2+ 调节剂 膜 Ca2+ -ATP 酶 (PMCA) 对氧化特别敏感，并且会因特定的作用而逐渐丢失 随着年龄的增长，大脑神经元的膜域。这些“筏”结构域为 Ca2+- 信号传导和 还已知是处理与神经退行性疾病相关的异常蛋白质的位点 疾病。该项目的总体目标是确定调节本地化的机制 筏域中 PMCA 的含量以及 PMCA 活性丧失背后的年龄依赖性变化 木筏。我们的假设是，这种损失是由于氧化应激增强导致年龄依赖性 调节 PMCA 活性和定位的蛋白质和脂质的变化 筏内。本申请的目的是：(1) 表征 5、22 和 34-突触膜筏 mos F344/BNF1 大鼠的蛋白质和脂质氧化、筏脂质组成以及体外影响 氧化应激； (2)利用药理学和遗传学操作来阐明筏脂质的作用 PMCA的膜定位和动力学特性中的环境； (3)确定蛋白质的作用 通过识别筏域和非筏域中的 PMCA 结合伙伴，在筏中 PMCA 定位中的相互作用， 确定它们在 5、22 和 34 个月大鼠膜中的水平，并改变主要的表达 细胞中的合作伙伴直接测试它们对 PMCA 定位和活性的影响。我们的策略涉及分析 与体外神经元模型并行地研究体内脑老化，以测试可能的特定机制 解释一些与年龄相关的变化。我们广泛利用脂质组学、蛋白质组学方面的专业知识 核心中提供的鉴定和分子生物学，以及初步数据支持所有的可行性 目标。尽管有证据表明筏参与 Ca2+ 信号传导以及年龄依赖性的 Ca2+ 失调 神经退行性疾病，但我们对神经元筏的衰老一无所知。我们的研究将 开始填补这一空白，并为衰老、Ca2+分布改变、氧化之间的联系提供新的见解 压力以及老化大脑对影响老年人的毁灭性痴呆症的脆弱性增强 人口。 Lav声明：拟议的研究将增强我们对衰老过程中发生的变化的理解 大脑很容易受到认知障碍和阿尔茨海默氏症等疾病的影响。终极 目标是确定哪些干预措施可以减缓或防止随着年龄增长而发生的一些变化。