CELLULAR BIOLOGY OF APOE CLEARANCE RECEPTOR IN CNS

中枢神经系统中 APO 清除受体的细胞生物学

• 批准号: 6295428
• 负责人: ALAN L SCHWARTZ
• 金额: $ 18.29万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-04 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6295428
• 关键词: Alzheimer's disease; RNase protection assay; aging; apolipoproteins; astrocytes; binding proteins; blood lipoprotein metabolism; gene expression; genetically modified animals; human tissue; immunocytochemistry; immunoelectron microscopy; in situ hybridization; intracellular transport; laboratory mouse; ligands; low density lipoprotein receptor; neurons; protein isoforms; protein structure function; receptor binding; receptor expression; receptor mediated endocytosis; tissue /cell culture

Recent studies have demonstrated an association between the gene dosage of apoE type 4 and late onset Alzheimer's disease (AD). Our long term goals are to elucidate the molecular mechanisms by which the apoE receptor system affects CNS development and function. ApoE transports lipoproteins via binding to low density lipoprotein (LDL) receptor and LDL receptor- related protein (LRP). ApoE is synthesized within the CNS. Additional ligands for LRP include tissue-type plasminogen activator and alpha2- macroglobulin, as well as a 39kDa protein which serves as a potent regulator of LRP function. We have recently demonstrated the expression, endocytotic function and regulation of LRP. the apoE clearance receptor, in rat brain using a variety of cellular and molecular approaches. Our reagents include isolated LRP, 39kDa protein and other ligands, as well as antibodies, cDNA's and both in vivo and isolated cellular systems. Thus the present aims are as follows: 1) Elucidate the pattern of expression of all components of the LRP system in brain specimens from the WUMS ADRC tissue bank. We shall examine LRP, 39kDa protein, apoE and other ligands using immunohistochemistry and quantitative colloidal gold immunoelectron microscopy. 2) Examine the function and regulation of the LRP system in murine primary neurons and astrocytes as well as differentiated cell lines. We shall define the biosynthesis, intracellular itinerary, cell surface function and endocytotic fate of LRP, the 39kDa protein and ligands using cellular biological approaches including biosynthetic labeling, receptor-ligand kinetics, chemical crosslinking, fluorescent microscopy, etc. 3) Define the expression and function of the LRP system in vivo and in primary CNS cello from young adult and aged a) normal mice, b) transgenic mice for apoE overexpression as well as c) apoE knockout mice. We shall use whole embryos as well as isolated tissues together with in situ hybridization and RNase protection assays, immunohistochemistry and Western analyses to define the components at the RNA and protein levels. 4) Define the molecular regions of apoE isoforms and of the 39kDa protein which recognize the ligand binding sites on the LRP molecule. We shall examine apoE isoforms and generate mutants of the 39kDa protein which will be examined both in binding competition studies as well as in direct binding studies to isolated LRP. Thus these studies taken together provide a basis for the initial approach to elucidating the molecular mechanisms by which apoE, the LRP clearance receptor and its regulator 39kDa protein contribute to CNS pathobiology in Alzheimer's disease.

最近的研究表明，基因剂量之间的关联， apoE 4型和迟发性阿尔茨海默病（AD）。我们的长期目标 旨在阐明apoE受体 系统影响CNS发育和功能。ApoE转运脂蛋白 通过结合低密度脂蛋白（LDL）受体和LDL受体- 相关蛋白（LRP）。ApoE在CNS内合成。额外 LRP的配体包括组织型纤溶酶原激活剂和α 2- 巨球蛋白，以及一种39 kDa的蛋白质， LRP功能的调节剂。我们最近证明了这个表达式， 内吞功能和LRP的调节。apoE清除受体， 在大鼠大脑中使用各种细胞和分子方法。我们 试剂包括分离的LRP、39 kDa蛋白和其他配体，以及 抗体、cDNA以及体内和分离的细胞系统。因此 本研究的目的是：1）阐明语言的表达模式， 来自WUMS ADRC的脑标本中LRP系统的所有组件 组织库我们将检测LRP，39 kDa蛋白，apoE和其他配体 应用免疫组织化学和胶体金免疫电子显微镜 显微镜2)检视本地注册程序系统的功能及规管， 小鼠原代神经元和星形胶质细胞以及分化细胞 线我们将定义生物合成，细胞内行程，细胞 LRP（39 kDa蛋白质）的表面功能和内吞命运 使用细胞生物学方法包括生物合成 标记，受体-配体动力学，化学交联，荧光 3）确定LRP系统的表达和功能 在体内和在来自年轻成年人和老年人的原代CNS细胞中a）正常小鼠， B）apoE过表达的转基因小鼠以及c）apoE敲除小鼠 小鼠我们将使用完整的胚胎以及分离的组织， 原位杂交和RNA酶保护试验，免疫组织化学 和Western分析来确定RNA和蛋白质的成分 程度. 4)定义apoE同工型和39 kDa 识别LRP分子上配体结合位点的蛋白质。我们 将检测apoE同种型并产生39 kDa蛋白的突变体 这将在约束性竞争研究以及 对分离的LRP进行直接结合研究。因此，这些研究结合在一起 提供了初步的方法来阐明分子的基础 apoE、LRP清除受体及其调节剂 39 kDa蛋白参与阿尔茨海默病的CNS病理生物学。