Endoplasmic Reticulum Mitochondrial membranes in Alzheimer's disease

描述(申请人提供)：阿尔茨海默病(AD)的特征是神经元丢失，特别是在皮质和海马区，伴随着细胞外含有B-淀粉样蛋白的神经炎斑(A？)和细胞内由过度磷酸化的tau蛋白组成的神经原纤维缠结。AD患者还表现出其他较少受到关注的特征，包括异常的胆固醇、磷脂和钙稳态，以及线粒体功能和动力学的改变。早老素-1(PS1)、早老素-2(PS2)和淀粉样前体蛋白(APP)的γ分泌酶活性都主要位于内质网的一个特殊的亚室中，该亚室与线粒体有物理和生物化学上的联系，称为线粒体相关膜。MAM参与调节胆固醇和磷脂代谢，钙稳态，以及线粒体的功能和动力学。最近，我们发现MAM是脂筏样结构域，AD患者的细胞大量上调MAM活性，增加内质网线粒体连接性，导致胆固醇、磷脂和钙稳态改变，线粒体动力学异常，这可能有助于解释AD患者的许多生化和形态特征。基于这些发现，我们认为MAM功能障碍和内质网-线粒体连接改变是AD发病的早期致病事件。我们现在提出的研究旨在从基础科学的角度理解MAM的功能，最终目的是将这一知识应用于翻译。具体地说，我们将(1)分析MAM在与AD更相关的细胞和组织中的功能，包括PS缺乏的人神经母细胞瘤细胞，分化为神经元的人诱导多能干细胞，以及从pS1基因敲除小鼠移植的组织和神经元；(2)分析MAM中的早老素和γ分泌酶调节相对于其他部分，如“散装”内质网或质膜，同时也评估γ分泌酶复合体的其他成分(例如APH-1、尼卡斯汀、PEN_2)和调节因子(例如CD147、GSAP和TMP21)的作用；以及(3)了解ER-线粒体通讯在AD发病机制中的作用，以不同的固定距离将ER与线粒体连接起来，使用新型的ER-线粒体“交联”质粒，以不依赖于衰老的方式模拟ER-线粒体的连接。

DESCRIPTION (provided by applicant): Alzheimer disease (AD) is characterized by neuronal loss, especially in the cortex and hippocampus, accompanied by accumulation in the brain of extracellular neuritic plaques containing ß-amyloid (Aß) and of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. AD patients also present with other features that have received less attention, including aberrant cholesterol, phospholipid, and calcium homeostasis, and altered mitochondrial function and dynamics. Presenilin-1 (PS1), presenilin-2 (PS2), and γ-secretase activity, which processes the amyloid precursor protein (APP) to generate Aß, are all located predominantly in a specialized subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAM). MAM is involved in the regulation of cholesterol and phospholipid metabolism, calcium homeostasis, and in mitochondrial function and dynamics. Recently, we showed that MAM is lipid raft-like domain and that cells from AD patients have massively upregulated MAM activity and increased ER mitochondrial connectivity, resulting in altered cholesterol, phospholipid and calcium homeostasis, and aberrant mitochondrial dynamics, which may help explain many of the biochemical and morphological features of the disease. Based on these findings, we believe that MAM dysfunction and altered ER-mitochondrial connectivity are early causative events in the pathogenesis of AD. We now propose studies aimed at understanding MAM function from a basic science standpoint, with the ultimate goal of applying this knowledge translationally. Specifically, we will (1) analyze MAM function in cells and tissues that are more "AD-relevant," including PS-deficient human neuroblastoma cells, human induced pluripotent stem cells differentiated into neurons, and tissues and neurons explanted from PS1 knock-in mice; (2) analyze presenilins and γ-secretase regulation in MAM versus other compartments, such as "bulk" ER or the plasma membrane, while also assessing the role of other components (e.g. APH-1, nicastrin, PEN2) and regulators (e.g. CD147, GSAP, and TMP21) of the γ-secretase complex; and (3) understand the role of ER-mitochondrial communication in the pathogenesis of AD, by tethering ER to mitochondria at various fixed distances, using novel ER-mitochondria "crosslinking" plasmids, in order to mimic ER-mitochondrial connectivity in a presenilin-independent manner.