CELLULAR BIOLOGY OF APOE CLEARANCE RECEPTOR IN CNS

最近的研究表明，基因剂量之间存在关联。 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受体的分子机制 系统影响中枢神经系统的发育和功能。 ApoE 转运脂蛋白 通过与低密度脂蛋白（LDL）受体和LDL受体结合- 相关蛋白（LRP）。 ApoE 在 CNS 内合成。附加 LRP 的配体包括组织型纤溶酶原激活剂和 α2- 巨球蛋白以及 39kDa 的蛋白质，可作为有效的 LRP 功能的调节器。我们最近证明了这个表达式， LRP 的内吞功能和调节。 apoE清除受体， 使用各种细胞和分子方法在大鼠大脑中进行研究。我们的 试剂包括分离的LRP、39kDa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 目前的目标如下： 1）阐明表达模式 WUMS ADRC 脑标本中 LRP 系统的所有组成部分 组织库。我们将检查LRP、39kDa蛋白、apoE和其他配体 使用免疫组织化学和定量胶体金免疫电子 显微镜。 2）检查LRP系统的功能和调节 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（39kDa 蛋白）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）的配体 标记、受体-配体动力学、化学交联、荧光 3）定义LRP系统的表达和功能 a) 正常小鼠的体内和初级中枢神经系统大提琴中， b) apoE 过表达转基因小鼠以及 c) apoE 敲除 老鼠。我们将使用整个胚胎以及分离的组织 原位杂交和 RNase 保护测定、免疫组织化学 和 Western 分析来定义 RNA 和蛋白质的成分 水平。 4) 定义 apoE 异构体和 39kDa 的分子区域 识别 LRP 分子上配体结合位点的蛋白质。我们 应检查 apoE 同工型并生成 39kDa 蛋白的突变体 这将在具有约束力的竞争研究以及 与分离的 LRP 的直接结合研究。因此，这些研究综合起来 为阐明分子机制的初步方法提供了基础 apoE、LRP 清除受体及其调节剂的机制 39kDa 蛋白有助于阿尔茨海默病的中枢神经系统病理学。

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.