APOE isoform affects protein structure and function in normal brain

载脂蛋白E基因型在阿尔茨海默病（AD）发病机制中的作用仍然是最关键的研究之一， AD研究中的问题。在过去的几年里，我们一直在研究一个有趣的假设，即APOE 基因型在生命早期改变了正常的大脑神经化学和结构，产生了一个环境， 老年痴呆症的风险。我们以前对APOE基因敲入小鼠的研究表明，即使在年轻的 对照小鼠脑，APOE基因型影响脑脂质生化，神经元结构和空间学习。 在探索这些不同影响的原因时，我们现在正在验证一个假设，即早期的 APOE 4个体的大脑中的差异是由于星形胶质细胞中正常脂质代谢的差异， 神经元在初步研究中，我们发现从大脑中分离的APOE存在于两个独立的 群体：修饰的和水溶性的，或未修饰的和膜缔合的。新的大众 光谱分析方法使我们能够确定可溶性APOE在几个位点被糖基化， 几种类型的聚糖; CNS中大多数修饰的APOE在血浆中未发现（特别是 APOE在其脂质结合结构域中的独特修饰）。体内研究表明，APOE 4的脂化程度较低， 与APOE 2和APOE 3相比，APOE 4小鼠的脑脂质改变 体内平衡最后，我们发现小鼠中的APOE 4相关表型被AD挽救， 预防性治疗。在这项工作中，我们建议定义如何脑脂质稳态是受载脂蛋白E 同种型，包括新鉴定的糖型。在目标1中，我们将定义APOE修饰如何改变 APOE在CNS中的代谢、运输和功能。在这些研究中，我们将确定和量化新的 APOE跨组织的翻译后修饰，并开发新的星形胶质细胞培养模型， 人糖基-APOE同种型的表达和调节。在目标2中，我们将确定APOE 4是否 导致体外和体内脂质流出不良，导致小鼠和人中LXR靶点的活化增加 大脑我们将使用计算机模拟来测试糖基APOE种类对APOE三级结构的影响 以及APOE与CNS脂蛋白的结合。在目标3中，我们将测试APOE 4是否会导致脂质摄取不良， 体外，导致神经元APOE代谢的变化。我们将使用神经胶质细胞和神经元培养来确定如何 APOE修饰随着APOE通过CNS隔室而改变。最后，在目标4中，我们将测试 预防小鼠和人类AD的方法（抗炎药和运动/环境）是否 富集）影响脑脂质代谢和脑结构/功能。我们将测试我们的新标记 受APOE 4存在影响的细胞，包括CNS特异性糖型，对 降低AD风险。因此，这一建议提供了新的机制洞察的翻译后修饰， 中枢神经系统中的载脂蛋白E以及大脑中复杂的载脂蛋白E亚型如何以多种方式调节脂质代谢 这可能会导致AD风险增加。

The role of APOE genotype in the pathogenesis of Alzheimer's disease (AD) remains one of the most critical questions in AD research. Over the past few years, we have pursued an interesting hypothesis that APOE genotype alters normal brain neurochemistry and structure early in life, generating an environment that alters the risk of AD with aging. Our previous studies of APOE knock-in mice have demonstrated that, even in young control mouse brains, APOE genotype affects brain lipid biochemistry, neuronal structure, and spatial learning. In exploring the cause of these various effects, we are now testing the hypothesis that early alterations of brains in APOE4 individuals are due to differences in normal lipid metabolism in astrocytes and neurons. In preliminary studies, we found that APOE isolated from the brain exists in two separate populations: either modified and water soluble, or unmodified and membrane associated. New mass spectrometry approaches allowed us to define that soluble APOE was glycosylated at several sites with several types of glycans; most of the modified APOE in the CNS was not found in the plasma (particularly with unique modification of APOE in its lipid binding domain). In vivo studies showed that APOE4 was less lipidated in CSF lipoproteins compared to APOE2 and APOE3, and that APOE4 mice have altered brain lipid homeostasis. Finally, we found that APOE4-associated phenotypes in mice were rescued by an AD preventative treatment. In this work, we propose to define how brain lipid homeostasis is affected by APOE isoforms, including the newly identified glycoforms. In Aim 1, we will define how APOE modifications alter APOE metabolism, trafficking, and function in the CNS. For these studies, we will identify and quantify new post-translational modifications of APOE across tissues, and develop new astrocyte culture models for expression and modulation of human glyco-APOE isoforms. In Aim 2, we will determine whether APOE4 causes poor lipid efflux in vitro and in vivo, leading to increased activation of LXR targets in mouse and human brains. We will use computer modeling to test the effects of glyco-APOE species on APOE tertiary structure and on APOE binding to CNS lipoproteins. In Aim 3, we will test whether APOE4 causes poor lipid uptake in vitro, leading to changes in neuronal APOE metabolism. We will use glial and neuronal cultures to define how APOE modification changes as APOE travels through CNS compartments. Finally, in Aim 4, we will test whether approaches that prevent AD in mice and humans (anti-inflammatory drugs and exercise/environmental enrichment) affect brain lipid metabolism and brain structure/function. We will test whether our new markers that are affected by the presence of APOE4, including the CNS-specific glycoforms, respond to conditions that reduce AD risk. Thus, this proposal provides new mechanistic insight into the post-translational modification of APOE in the CNS and how the complex set of APOE isoforms in the brain modulate lipid metabolism in ways that could lead to increased risk of AD.