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

与年龄相关的认知能力下降和易患性增加的潜在机制 神经退行性疾病是未知的，尽管Ca 2+稳态失调和增强 氧化应激似乎是主要的贡献者。我们已经发现，一个主要的Ca 2+调节，血浆 膜Ca ~（2+）-ATP酶（PMCA）是唯一对氧化敏感的酶， 脑神经元膜结构域的变化。这些“筏”结构域为Ca 2+信号传导和 也已知是与神经退行性疾病相关的异常蛋白质的加工位点 疾病本项目的总体目标是确定规范本地化的机制 的PMCA在筏域和年龄依赖性的变化，潜在的损失PMCA的活动， 木筏我们的假设是，这种损失是由于增强氧化应激，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 在木筏上。本申请的目的是：（1）表征来自5、22和34的突触膜筏。 mos F344/BNF 1大鼠在蛋白质和脂质氧化、筏脂质组成以及体外 氧化应激;（2）使用药理学和遗传操作来阐明筏脂质的作用 PMCA在膜环境中的定位和动力学性质;（3）确定蛋白质的作用 通过鉴定筏与非筏结构域中的PMCA结合配偶体， 测定它们在5、22和34月龄大鼠细胞膜中的水平，并改变主要的 在细胞中的合作伙伴，直接测试其对PMCA的定位和活动的影响。我们的战略包括分析 体内脑老化与体外神经元模型平行，用于测试可能 解释了一些与年龄有关的变化。我们广泛利用脂质组学、蛋白质 鉴定和分子生物学的核心，和初步数据支持的可行性，所有 目标。尽管有证据表明筏参与了Ca 2+信号传导，并且在年龄依赖性的细胞中Ca 2+调节异常， 在神经退行性疾病中，对神经筏的衰老一无所知。我们的研究将 开始填补这一空白，并提供了新的见解之间的联系老化，改变钙离子的处置，氧化 压力和衰老的大脑对老年痴呆症的脆弱性 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。 这使得它很容易受到认知障碍和老年痴呆症等疾病的影响。最终 我们的目标是确定哪些干预措施可能会减缓或防止随着年龄的增长而发生的一些变化。

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.