CELLULAR BIOLOGY OF APOE CLEARANCE RECEPTOR IN CNS

最近的研究已经证明了基因剂量与高血压之间的关系 APOE 4型和晚发性阿尔茨海默病(AD)。我们的长期目标 是为了阐明载脂蛋白E受体的分子机制 系统影响中枢神经系统的发育和功能。载脂蛋白E转运脂蛋白 通过与低密度脂蛋白受体和低密度脂蛋白受体结合- 相关蛋白(LRP)。APOE是在中枢神经系统内合成的。其他内容 LRP的配体包括组织型纤溶酶原激活剂和α2- 巨球蛋白，以及一种39 kDa的蛋白质，它是一种有效的 LRP功能的调节器。我们最近演示了这一表达， LRP的内吞功能及其调节。载脂蛋白E清除受体， 利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、39 kDa蛋白和其他配体以及 抗体，cDNAs，体内和分离的细胞系统。因此， 本研究的目的如下：1)阐明基因的表达模式 WUMS ADRC脑组织标本中LRP系统的所有成分 组织银行。我们将检测LRP、39 kDa蛋白、apoE和其他配体 免疫组织化学和胶体金定量免疫电子技术 显微镜。2)检查LRP系统的功能和规则 小鼠原代神经元、星形胶质细胞及分化细胞 台词。我们将定义生物合成、细胞内行程、细胞 LRP、39 kDa蛋白和LRP的表面功能和内吞命运 使用包括生物合成在内的细胞生物学方法的配体 标记、受体-配基动力学、化学交联、荧光 显微镜等)定义LRP系统的表达和功能 在活体和来自幼年和老年正常小鼠的原代CNS细胞中， B)apoE过表达的转基因小鼠以及c)apoE基因敲除 老鼠。我们将使用完整的胚胎以及分离的组织 原位杂交和核糖核酸酶保护分析、免疫组织化学 和Western分析来定义RNA和蛋白质的成分 级别。4)确定载脂蛋白E亚型和39 kDa的分子区域 识别LRP分子上的配体结合位置的蛋白质。我们 将检测apoE亚型并产生39 kDa蛋白的突变体 它将在约束性竞争研究中以及在 直接结合研究分离的LRP。因此，这些研究放在一起 为阐明分子的初步方法提供了基础 载脂蛋白E、LRP清除受体及其调节因子的作用机制 39 kDa蛋白在阿尔茨海默病中枢神经系统病理生物学中的作用。

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.