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) 最强的遗传风险因素。个人 拥有一个或多个 APOE4 变体副本的人患 AD 的风险增加，其中 与携带更常见的 APOE3 等位基因的患者相比，发病年龄更早，认知能力下降更快。 ApoE 主要在星形胶质细胞中表达，并分泌在脂蛋白颗粒上，提供胆固醇和 不饱和磷脂用于神经元膜扩张和突触形成。然而，很少的是 已知星形胶质细胞中 ApoE 功能背后的基本分子机制，以及 E4 变体如何发挥作用 促进疾病。我发现 ApoE 可以转运至分泌途径或细胞质 星形胶质细胞中的脂滴。我的中心假设是 ApoE 可以被刺激以靶向脂质滴 细胞质而不是被分泌，并且它参与促进细胞内中性脂质的储存 脂滴。我还假设 ApoE 的脂滴功能在与疾病相关的 E4 中发生了改变 和 E2 变体。为了检验这个假设，我将进行结构功能研究以确定哪些区域 该蛋白质对于 ApoE 靶向细胞质区室并定位到脂滴是必需的。我会 通过进行星形胶质细胞-神经元共培养来检查 ApoE 运输如何受神经元信号控制 化验。为了确定 ApoE 靶向脂滴的功能，我将使用 shRNA 来敲低 ApoE 并 观察对细胞内中性脂质分布的影响。然后我将确定 ApoE 是否调节 通过荧光脂质脉冲追踪分析与延时相结合进行脂滴生物发生、生长或周转 显微镜和自动图像分析。我将比较不同变体的贩运和功能 ApoE 来测试 E2 和 E4 中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.