Mitochondrial-dependent Mechanisms of Apolipoprotein E Gene Expression in the Mammalian Brain

项目概要 阿尔茨海默病 (AD) 是一种使人衰弱的常见痴呆症，也是人类死亡的主要原因 美国。淀粉样蛋白假说的测试一直是 AD 研究的重点。 根据这一假设，阿尔茨海默病前体蛋白 (APP) 的致病过程会导致 含有β淀粉样蛋白的斑块积聚是 AD 的标志和明显原因。尽管很多 但基于这一假设的研究尚未为 AD 提供有效的治疗靶点。因此，替代 提出线粒体以及脂质和胆固醇代谢在 AD 发病机制中因果作用的假设 也已被提出。虽然这些假设已经过淀粉样蛋白假说的检验，但 在确定线粒体和胆固醇/脂质是否具有因果作用方面仍存在关键的机制差距 AD发病机制中的代谢可能是相互联系的。脂质和胆固醇代谢的作用 AD 得到以下事实的支持：载脂蛋白 E (ApoE) 基因的等位基因，编码 ApoE 的主要载体 大脑中的脂质和胆固醇，是散发性 AD 最强的遗传危险因素。相比之下，研究 研究线粒体在 AD 中的因果作用的重点是生物能量学和氧化应激，尽管 线粒体在胆固醇和脂质代谢中也发挥着已知的作用。线粒体是否影响ApoE AD 背景下的表达和/或胆固醇稳态仍未被探索，这将是本研究的重点 提案。我们的初步数据表明线粒体膜转运蛋白的表达减少 SLC25A1 增加 ApoE 和 APP 的水平。 SLC25A1 将代谢物柠檬酸从线粒体运送到 它在细胞质中转化为脂质和胆固醇合成所需的乙酰辅酶A。中央 本提案将测试的假设是 SLC25A1 相互作用组（SLC25A1 以及与其物理相互作用的蛋白质网络）驱动 ApoE 表达增加和 胆固醇稳态，从而影响下游 APP 生产和加工。在目标 1 中， 受训者将确定 ApoE 表达增加是否是指示功能障碍的特定读数 SLC25A1 相互作用组的组成部分，或者是对线粒体功能障碍的一般反应，使用 原代神经元和细胞中的免疫印迹、定量聚合酶链反应 (RT-qPCR) 和脂质组学分析 神经胶质细胞。在目标 2 中，学员将确定 Slc25a1 基因剂量是否调节小鼠的 AD 病理学 AD 模型，使用基于平板的免疫测定和免疫细胞化学。完成这些目标将揭示 线粒体是否通过 ApoE 和/或胆固醇依赖性机制促进 AD 发病机制， 以及这种影响是否特定于参与脂质/胆固醇的线粒体蛋白的某个中心 新陈代谢。这些发现将增进我们对AD机制的理解，并提出新的建议 这种毁灭性疾病的药物靶标。

PROJECT SUMMARY Alzheimer’s disease (AD) is a debilitating and common form of dementia, and a leading cause of death in the United States. Testing of the amyloid hypothesis has overwhelmingly been the focus of research on AD. According to this hypothesis, pathogenic processing of the Alzheimer’s precursor protein (APP) leads to accumulation of plaques containing amyloid beta that are a hallmark, and apparent cause, of AD. Despite much effort, research based on this hypothesis has yet to provide effective therapeutic targets for AD. Thus, alternative hypotheses invoking causal roles for mitochondria and metabolism of lipids and cholesterol in AD pathogenesis have also been proposed. While these hypotheses have been tested in relation to the amyloid hypothesis, a critical mechanistic gap remains in determining whether causal roles for mitochondria and cholesterol/lipid metabolism in AD pathogenesis may be linked with one another. A role for lipid and cholesterol metabolism in AD is supported by the fact that an allele of the ApolipoproteinE (ApoE) gene, encoding the primary carrier of lipids and cholesterol in the brain, is the strongest genetic risk factor for sporadic AD. In contrast, research examining causal roles for mitochondria in AD has focused on bioenergetics and oxidative stress, although mitochondria also play known roles in cholesterol and lipid metabolism. Whether mitochondria influence ApoE expression and/or cholesterol homeostasis in the context of AD remains unexplored and will be the focus of this proposal. Our preliminary data indicates that reduced expression of the mitochondrial membrane transporter SLC25A1 increases levels of ApoE and APP. SLC25A1 shuttles the metabolite citrate from mitochondria to the cytoplasm, where it gets converted to acetyl-CoA that is required for lipid and cholesterol synthesis. The central hypothesis that will be tested in this proposal is that genetic disruption of the SLC25A1 interactome (SLC25A1 and the network of proteins with which it physically interacts) drives increased ApoE expression and changes in cholesterol homeostasis, consequently impacting downstream APP production and processing. In Aim 1, the trainee will determine whether increased ApoE expression is a specific readout indicating dysfunction of components of the SLC25A1 interactome, or is instead a general response to mitochondrial dysfunction, using immunoblots, quantitative polymerase chain reaction (RT-qPCR), and lipidomics profiling in primary neurons and glia. In Aim 2, the trainee will determine whether Slc25a1 gene dosage modulates AD pathology in a mouse model of AD, using plate-based immunoassays and immunocytochemistry. Completion of these aims will reveal whether mitochondria contribute to AD pathogenesis through ApoE and/or cholesterol-dependent mechanisms, as well as whether this influence is specific to a certain hub of mitochondrial proteins involved in lipid/cholesterol metabolism. These findings will improve our understanding of mechanisms contributing to AD and suggest novel drug targets for this devastating disease.