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APOE 4变体拷贝的人患AD的风险增加， 与携带更常见的APOE 3等位基因的患者相比，更早的发病年龄和更快的认知能力下降。 ApoE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脂蛋白。 不饱和磷脂用于膜扩张和神经元中的突触形成。然而， 关于ApoE在星形胶质细胞中功能背后的基本分子机制，以及E4变体 促进疾病。我发现ApoE既可以通过分泌途径，也可以通过细胞质途径， 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滴， 细胞质，而不是分泌，它是参与促进中性脂质的储存内 脂滴我还假设ApoE的脂滴功能在疾病相关的E4中改变， E2变种为了验证这一假设，我将进行结构-功能研究，以确定哪些区域的 这些蛋白质是ApoE靶向细胞质区室并定位于脂滴所必需的。我会 通过进行星形胶质细胞-神经元共培养，研究ApoE运输如何受神经元信号控制 测定。为了确定ApoE靶向脂滴的功能，我将使用shRNA敲低ApoE， 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测定结合时间推移法测定脂滴生物发生、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 的ApoE，以测试脂滴功能是否在E2和E4中改变。这个项目将揭示ApoE 变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.