CA2+ REGULATION AND MITOCHONDRIA IN BRAIN AGING/ AD

脑老化/ AD 中的 CA2 调节和线粒体

• 批准号: 6823630
• 负责人: PHILIP W. LANDFIELD
• 金额: $ 27.43万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6823630
• 关键词: Alzheimer' s disease; aging; amyloid proteins; antioxidants; calcium flux; disease /disorder etiology; embryo /fetus cell /tissue; gene mutation; genetically modified animals; hippocampus; intermolecular interaction; laboratory mouse; mitochondria; mitochondrial disease /disorder; nerve /myelin protein; neural degeneration; neurogenetics; neurons; neuropathology; neuroregulation; neurotoxins; nonhuman therapy evaluation; oxidation; oxidative stress; protein structure function; proteomics; tissue /cell culture

Amyloid beta-peptide (Abeta) is central to the pathogenesis of Alzheimer's disease (AD), and the AD brain is under extensive oxidative stress. Previous research from our laboratory combined these two observations into a model for neurodegeneration in AD, a model based on Abeta-associated free radical oxidative stress. In neurons, Abeta-associated free radical oxidative stress and toxicity depend critically on methionine residue 35 of Abeta(1-42), and both are prevented or modulated by exogenous antioxidants. We have used the emerging techniques of proteomics, coupled with immunoblottmg methods, to identify specifically oxidatively modified proteins in AD brain. We now propose to extend these observations to sporadic and inherited AD and rodent models thereof to gain insight into the mechanisms of Abeta (1-42) in the oxidative stress and neurotoxic properties of this peptide. In Specific Aim # 1, we will use proteomics to test the hypothesis that there will be a common set of oxidized proteins in inherited and sporadic AD that differ from those of normal aging, and that these oxidized proteins in common are important in the pathogenesis of AD. Similar studies in mice models of inherited AD are hypothesized to lead to the identity of the same common proteins. In Specific Aim #2, we will test the hypothesis that mitochondrial dysfunction and Ca 2+ accumulation in brain from genetic mutations relevant to AD will be found in adult brain mitochondria from the model chosen in Specific Aim #1 compared to adult brain from wild-type mice. Those proteins identified by proteomics in the systems studied will be expressed in SY5Y cells and challenged with Abeta(1-42) to determine if there is increased vulnerability to oxidative stress, mitochondrial dysfunction, Ca 2+ accumulation, and cytotoxicity. In Specific Aim #3, we will use proteomics to test the hypothesis that Abeta(1-42)-induced oxidative stress in embryonic neuronal cultures obtained from the rodent model of familial AD chosen in Specific Aim # 1 leads to the same oxidatively modified proteins as exists in adult brain from this animal and in human familial AD brain. In Specific Aim #4, we will investigate whether oxidative stress induces mitochondrial alterations in neuronal cultures obtained from the rodent model of inherited AD chosen in Specific Aim #1, or whether mitochondrial alterations induced in these cultures leads to oxidative stress. In Specific Aim # 5, we will test the hypothesis that endogenous or exogenous antioxidants protect brain in-vivo against oxidative stress in the rodent model chosen in Specific Aim # 1. This is a comprehensive set of proposed studies, employing novel approaches in systems ranging from sporadic and inherited AD brain, to genetic animal models of AD, to neuronal cultures, designed to gain insight into Abeta-associated free radical oxidative stress and neurotoxicity and their modulation by endogenous and exogenous antioxidants. The increased understanding that will result from these studies will provide insight into potential therapeutic interventions in this important dementing disorder that affects millions of Americans.

淀粉样β-肽（Abeta）是阿尔茨海默病（AD）发病机制的核心，并且AD脑处于广泛的氧化应激下。我们实验室以前的研究将这两种观察结果结合到AD神经变性模型中，该模型基于Abeta相关的自由基氧化应激。在神经元中，Abeta相关的自由基氧化应激和毒性严重依赖于Abeta（1-42）的甲硫氨酸残基35，两者都可以通过外源性抗氧化剂来预防或调节。我们已经使用了新兴的蛋白质组学技术，再加上免疫印迹方法，以确定特定的氧化修饰的蛋白质在AD脑。我们现在建议将这些观察结果扩展到散发性和遗传性AD及其啮齿动物模型，以深入了解Abeta（1-42）在该肽的氧化应激和神经毒性性质中的机制。在具体目标# 1中，我们将使用蛋白质组学来检验以下假设：在遗传性和散发性AD中存在一组共同的氧化蛋白，这些氧化蛋白与正常衰老的氧化蛋白不同，并且这些共同的氧化蛋白在AD的发病机制中很重要。在遗传性AD的小鼠模型中进行的类似研究被假设为导致相同的共同蛋白质的身份。在具体目标#2中，我们将检验以下假设：与野生型小鼠的成年脑相比，在具体目标#1中选择的模型的成年脑线粒体中将发现与AD相关的基因突变导致的脑中线粒体功能障碍和Ca 2+蓄积。在所研究的系统中通过蛋白质组学鉴定的那些蛋白质将在SY 5 Y细胞中表达并用Abeta（1-42）攻击以确定是否存在对氧化应激、线粒体功能障碍、Ca 2+积累和细胞毒性的增加的脆弱性。在具体目标#3中，我们将使用蛋白质组学来检验以下假设：在从具体目标#1中选择的家族性AD啮齿动物模型获得的胚胎神经元培养物中，Abeta（1-42）诱导的氧化应激导致与来自该动物的成人脑和人类家族性AD脑中存在的相同的氧化修饰蛋白。在具体目标#4中，我们将研究氧化应激是否诱导线粒体 从特定目标#1中选择的遗传性AD的啮齿动物模型获得的神经元培养物中的改变，或者在这些培养物中诱导的线粒体改变是否导致氧化应激。在具体目标5中，我们将在具体目标1中选择的啮齿动物模型中检验内源性或外源性抗氧化剂在体内保护脑免受氧化应激的假设。这是一套全面的拟议研究，采用新的方法，从散发性和遗传性AD脑，AD的遗传动物模型，神经元培养系统，旨在深入了解Abeta相关的自由基氧化应激和神经毒性及其调制内源性和外源性抗氧化剂。从这些研究中得到的更多的理解将为这种影响数百万美国人的重要痴呆症的潜在治疗干预提供深入了解。