APOE3 AND APOE4 EFFECTS ON CELLULAR PATHOBIOLOGY

我们最近发现载脂蛋白(Apo)E3和E4 背根神经节神经元分化对轴突生长的影响 在体外，表明载脂蛋白E可能在调节 神经元发育或重塑。只有载脂蛋白E添加到细胞中的β- 迁移性极低密度脂蛋白(β-VLDL)或脂肪乳剂 一种效应，这种效应被apoE或 通过抗载脂蛋白E受体结合区的抗体。我们还有更多 证明了apoE4处理的细胞中微管的稳定性发生了变化。 这项建议的主要目标是：1)测定脂蛋白和 脂类需求介导载脂蛋白E3和载脂蛋白E4的不同作用 2)测定低密度脂蛋白(LDL) 受体，硫酸肝素蛋白多糖(HSPG)和/或低密度脂蛋白受体- 相关蛋白(LRP)是差异效应所必需的； 详细研究apoE3的结合、内化和降解 以及4)确定apoE3和apoE4 载脂蛋白E4对神经突起的生长有不同的影响。 为了实现这些目标，体外研究将与初级 神经元的培养，以及小鼠神经母细胞瘤细胞系Neuro-2a。这个 效果所需的脂类和脂蛋白将由以下因素决定 用apoE3或apoE4与天然的 出现脂蛋白低密度脂蛋白或载脂蛋白HDLc或与含有以下物质的脂肪乳剂一起使用 不同比例的胆固醇、胆固醇酯、三酰甘油和 磷脂，并测定对轴突生长的影响。这个 低密度脂蛋白受体、LRP和HSPG在差异效应中的重要性 载脂蛋白E亚型的研究将使用：来自低密度脂蛋白受体的神经元 基因敲除动物：与乳铁蛋白和39-kDa的竞争研究 ApoE的突变体(apo2、apoE(Arg142 YeeldsCys)、apoE Leiden) 有不同程度的与低密度脂蛋白受体，LRP， 和HSPG。载脂蛋白E变体的影响将在两种情况下进行检查 与β-极低密度脂蛋白一起外源添加，以及当添加β-极低密度脂蛋白时 稳定表达apoE变异体的细胞。内部化和 将研究用β-极低密度脂蛋白降解apoE3和apoE4 详细信息来确定我们观察到apoE3和apoE4的原因 将对极低密度脂蛋白进行详细检查，以确定我们 观察到apoE3在细胞中积累的程度比 载脂蛋白E4.此外，还将使用光能级和电子显微镜来 确定载脂蛋白E在细胞内的位置及其与 在轴突生长中起重要作用的细胞器。在最终的具体目标中 我们将确定截短的载脂蛋白E是否可以调节差异效应， 我们将确定载脂蛋白E影响微管的机制 队形。这些研究将提供对这些机制的理解。 这解释了apoE3和apoE4对轴突的不同影响 在体外生长，并可能解释为什么 载脂蛋白E4等位基因与阿尔茨海默病的相关性。

We have recently shown that apolipoproteins (apo) E3 and E4 have differential effects on neurite outgrowth from dorsal root ganglion neurons in vitro, suggesting that apoE could play a direct role in modulating neuron development or remodeling. Only apoE added to the cell with beta- migrating very low density lipoproteins (beta-VLDL) or lipid emulsions had an effect, and the effect was blocked by reductive methylation of apoE or by antibodies to the receptor-binding domain of apoE. We have further demonstrated that microtubule stability is altered in apoE4-treated cells. The major goals of this proposal are 1) to determine the lipoprotein and lipid requirements mediating the differential effects of apoE3 and apoE4 on neurite outgrowth; 2) to determine if the low density lipoprotein (LDL) receptor, heparan sulfate proteoglycans (HSPG) and/or the LDL receptor- related protein (LRP) are required for the differential effects; 3) to examine in detail the binding, internalization, and degradation of apoE3 and apoE4 by neurons; and 4) to determine the mechanism by which apoE3 and apoE4 exert differential effects on neurite outgrowth. To accomplish these goals, in vitro studies will be performed with primary cultures of neurons, and a murine neuroblastoma cell line, Neuro-2a. The lipid and lipoprotein requirements for the effect will be determined by incubating the cells with apoE3 or apoE4 together with the naturally occurring lipoproteins LDL or apoE HDLc or with lipid emulsions containing different ratios of cholesterol, cholesteryl ester, triacyglycerol, and phospholipid, and determining the effects on neurite outgrowth. The importance of the LDL receptor, LRP, and HSPG to the differential effects of the apoE isoforms will be explored using: neurons from LDL receptor knockout animals; competition studies with lactoferrin and the 39-kDa protein; and mutants of apoE (apoE2, apoE(Arg142 yieldsCys), apoE Leiden) with various degrees of defective binding to the LDL receptor, the LRP, and HSPG. The effect of the apoE variants will be examined both when they are added exogenously together with beta-VLDL and when beta-VLDL is added to cells stably expressing the apoE variants. Internalization and degradation of the apoE3 and apoE4 with beta-VLDL will be examined in detail to determine the reason for our observation that apoE3 and apoE4 with beta-VLDL will be examined in detail to determine the reason for our observation that apoE3 accumulates in the cells to a greater extent than apoE4. In addition, light-level and electron microscopy will be used to determine the location of intracellular apoE and its association with organelles of importance in neurite outgrowth. In the final Specific Aim we will determine if truncated apoE can mediate the differential effects, and we will determine the mechanism by which apoE affects microtubule formation. These studies will provide an understanding of the mechanisms that account for the differential effects of apoE3 and apoE4 on neurite outgrowth in vitro and potentially will explain the reason for the association of the apoE4 allele with Alzheimer's disease.