APOE3 AND APOE4 EFFECTS ON CELLULAR PATHOBIOLOGY

APOE3 和 APOE4 对细胞病理学的影响

• 批准号: 2442321
• 负责人: ROBERT E PITAS
• 金额: $ 31.27万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2442321
• 关键词: Alzheimer's disease; apolipoproteins; binding proteins; blood lipoprotein; cellular pathology; dogs; human tissue; laboratory mouse; laboratory rabbit; lipids; low density lipoprotein; low density lipoprotein receptor; microscopy; microtubule associated protein; nervous system regeneration; neuroblastoma; neurogenesis; neurons; nutrient requirement; protein degradation; protein isoforms; tissue /cell culture; very low density lipoprotein

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.

我们最近发现，载脂蛋白（apo）E3和E4具有 对背根神经节神经元突起生长的不同影响 在体外，这表明apoE可以发挥直接作用，调节 神经元发育或重塑。 只有apoE加入到细胞中， 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的还原甲基化或 通过apoE受体结合域的抗体。 我们进一步 表明微管稳定性在apoE 4处理的细胞中改变。 该建议的主要目标是1）确定脂蛋白， 脂质需求介导apoE 3和apoE 4对细胞凋亡的不同作用 神经突生长; 2）确定是否低密度脂蛋白（LDL） 受体、硫酸乙酰肝素蛋白聚糖（HSPG）和/或LDL受体- 相关蛋白（LRP）是差异效应所必需的; 3） 详细研究apoE 3的结合、内化和降解 和apoE 4的神经元;和4）确定apoE 3和apoE 4的机制， apoE 4对神经突生长具有不同的作用。 为了实现这些目标，将进行体外研究， 神经元培养物和鼠神经母细胞瘤细胞系Neuro-2a。 的 将通过以下方法确定该效应的脂质和脂蛋白需求： 将所述细胞与apoE 3或apoE 4以及天然的 存在脂蛋白LDL或apoE HDLc或含有以下成分的脂肪乳剂 不同比例的胆固醇、胆固醇酯、三酰基甘油和 磷脂，并确定对神经突生长的影响。 的 LDL受体、LRP和HSPG对差异效应的重要性 apoE亚型的研究将使用：LDL受体神经元 基因敲除动物;乳铁蛋白和39-kDa蛋白的竞争研究 蛋白;和apoE突变体（apoE 2、apoE（Arg 142 yieldsCys）、apoE Leiden） 与LDL受体，LRP， 和HSPG。 apoE变体的影响将在以下两种情况下进行检查： 与β-VLDL一起外源加入，当加入β-VLDL时， 稳定表达apoE变体的细胞。 内化和 apoE 3和apoE 4与β-VLDL的降解将在 详细说明我们观察到apoE 3和apoE 4 与β-VLDL将详细检查，以确定我们的原因 观察到apoE 3在细胞中积累的程度大于 载脂蛋白E4。 此外，光级和电子显微镜将用于 确定细胞内apoE的位置及其与 神经突生长中重要的细胞器。 在最后的具体目标 我们将确定截短的apoE是否可以介导差异效应， 我们将确定apoE影响微管的机制 阵 这些研究将提供一个了解的机制， 这解释了apoE 3和apoE 4对神经突的不同作用， 在体外生长，并可能解释的原因， apoE 4等位基因与阿尔茨海默病的关联。