Lipid droplet targeting and function of Apolipoprotein E in astrocytes

摘要 载脂蛋白E(ApoE)是晚发性阿尔茨海默病(AD)最强的遗传危险因素。个人 拥有一个或多个APOE4变体副本的人患AD的风险增加，并伴有 与那些更常见的APOE3等位基因的人相比，他们的发病年龄更早，认知能力下降更快。 载脂蛋白E主要在星形胶质细胞中表达，并在脂蛋白颗粒上分泌，以提供胆固醇和 不饱和磷脂对神经细胞膜扩张和突触形成的作用。然而，几乎没有什么是 已知载脂蛋白E在星形胶质细胞中发挥作用的基本分子机制，以及E4变体如何 会引发疾病。我发现载脂蛋白E既可以通过分泌途径也可以通过细胞质 星形胶质细胞内的脂滴。我的中心假设是，ApoE可以被刺激来靶向体内的脂滴 细胞质而不是分泌，它参与促进中性脂在体内的储存 脂滴。我还假设，在疾病相关的E4中，ApoE的脂滴功能发生了变化 和E2变异体。为了验证这一假设，我将进行结构-功能研究，以确定 这些蛋白质是ApoE靶向细胞质并定位于脂滴所必需的。这就做 通过星形胶质细胞-神经元共培养研究载脂蛋白E转运如何受神经元信号控制 化验。为了确定ApoE针对脂滴的功能，我将使用shRNA敲除ApoE和 观察对中性脂质在细胞内分布的影响。然后我将确定ApoE是否监管 通过结合时间推移的荧光脂质脉冲追逐分析研究脂滴的生物发生、生长或周转 显微镜和自动图像分析。我会比较不同变种的贩运和功能 以检测E2和E4的脂滴功能是否发生改变。该项目将揭示ApoE如何 变异体调节星形胶质细胞中的脂滴代谢。

ABSTRACT Apolipoprotein E (ApoE) is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). Individuals who possess one or more copies of the APOE4 variant have an increased risk of developing AD, with an earlier age of onset and more rapid cognitive decline, compared to those with the more common APOE3 allele. ApoE is expressed primarily in astrocytes, and secreted on lipoprotein particles to provide cholesterol and unsaturated phospholipids for membrane expansion and synapse formation in neurons. However, little is known regarding the basic molecular mechanisms behind ApoE function in astrocytes, and how the E4 variant promotes disease. I have discovered that ApoE can traffic either to the secretory pathway or to cytoplasmic lipid droplets in astrocytes. My central hypothesis is that ApoE can be stimulated to target lipid droplets in the cytoplasm rather than being secreted, and that it is involved in promoting the storage of neutral lipids within lipid droplets. I also hypothesize that the lipid droplet function of ApoE is altered in the disease-associated E4 and E2 variants. To test this hypothesis, I will perform structure-function studies to determine which regions of the protein are necessary for ApoE to target the cytoplasmic compartment and localize to lipid droplets. I will examine how ApoE trafficking is controlled by neuronal signals by performing astrocyte-neuron coculture assays. To determine the function of ApoE targeted to lipid droplets, I will use shRNA to knockdown ApoE and observe the effect on the distribution of neutral lipids in the cell. I will then determine whether ApoE regulates lipid droplet biogenesis, growth, or turnover via fluorescent lipid pulse-chase assays coupled with time-lapse microscopy and automated image analysis. I will compare the trafficking and function of the different variants of ApoE to test whether the lipid droplet function is altered in E2 and E4. This project will uncover how ApoE variants regulate lipid droplet metabolism in astrocytes.