权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Mevalonate Pathway Regulation of Astrocyte ApoE

星形胶质细胞 ApoE 的甲羟戊酸途径调节

基本信息

批准号：
10591079
负责人：
Heather Ferris
金额：
$ 49.95万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-15 至 2027-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10591079
关键词：
Abeta synthesis Address Alzheimer&apos s Disease Alzheimer&apos s disease model Alzheimer&apos s disease pathology Alzheimer&apos s disease risk Amyloid beta-Protein Amyloid beta-Protein Precursor Anticholesteremic Agents Apolipoprotein E Astrocytes Automobile Driving Behavior Brain Cell Death Cell physiology Chemicals Cholesterol Cholesterol Homeostasis Cholesterol Synthesis Inhibition Complement 1q Confocal Microscopy Data Drops Enzyme-Linked Immunosorbent Assay Ethidium Flow Cytometry Future Genetic Genetic Suppression Genotype Human Individual Interleukin-1 alpha Knock-in Mouse Knock-out Late Onset Alzheimer Disease Link Lipids Measures Mediating Membrane Biology Membrane Microdomains Mus Neurons Pathway interactions Pharmacology Phenotype Prevention Production Protein Isoforms Protein Isoprenylation Protein Secretion Proteins Regulation Risk Role Saturated Fatty Acids Senile Plaques Serum-Free Culture Media TNF gene Techniques Testing Therapeutic apolipoprotein E-2 apolipoprotein E-3 apolipoprotein E-4 arm chemoproteomics conditional knockout experimental study genetic risk factor improved in vivo inhibitor mevalonate mouse model neuron loss neuronal survival novel strategies novel therapeutics particle prenylation preservation prevent small hairpin RNA superresolution imaging tool

项目摘要

Abstract The ApoE 4 isoform is the strongest genetic link to Alzheimer’s disease for unclear reasons. ApoE is a lipid carrying protein. Both the content of the ApoE particles and their release from astrocytes varies by ApoE isoform. We have found that the mevalonate pathway can regulate both the release of ApoE from astrocytes and the lipid content of those ApoE particles. Depending on the part of the pathway that we inhibit we can separate these two functions. We hypothesize that targeting individual components of the mevalonate pathway can be used to manipulate the secretion and composition of ApoE in order to reduce Aβ synthesis and improve neuron survival. Further, we will explore if reducing the lipid content of the ApoE particles, without impacting secretion of the particles, can help shift the behavior of ApoE4 particles towards the behavior of lower risk ApoE genotypes. Through the proposed experiments we will test if: Aim 1. Secretion of ApoE is regulated via the mevalonate pathway in quiescent and activated astrocytes in an isoform specific manner. There are 3 well-characterized isoforms of ApoE in humans; 2, 3 and 4, which carry varying risks for developing AD. The quantity and composition of lipids carried by ApoE is influenced by ApoE genotype and astrocyte activation state. We will use mice expressing humanized ApoE 2, 3 or 4 or an ApoE knockout control to grow primary cultures of cortical astrocytes. Astrocytes will be grown in serum-free media to mimic the quiescent state or treated with TNF, IL-1α and C1q to mimic the activated state. Utilizing chemical inhibitors, shRNAs and conditional knockouts, we will determine the contributions of the cholesterol and prenylation arms of the mevalonate pathway to regulation of ApoE secretion by astrocytes. Aim 2. Inhibition of the mevalonate pathway can improve the abnormal lipid droplet accumulation and lipid secretion profile in ApoE4 astrocytes. Using the tools from Aim 1, we will manipulate the mevalonate pathway in astrocytes from ApoE knockout and ApoE 2, 3 or 4 knock-in mice. Intracellular ApoE and lipid droplets will be assessed by confocal microscopy. The lipid composition of astrocyte conditioned media from quiescent and activated astrocytes with and without inhibitors to cholesterol synthesis, prenylation or both will be measured. Changes in cholesterol-lipid carrier interactions will be determined using a cholesterol probe. Aim 3. Selective inhibition of the mevalonate pathway in astrocytes will reduce amyloid-beta cleavage and neuron death. We predict that inhibition of cholesterol, while preserving prenylation, will reduce AD pathology, with the greatest effect in ApoE4 astrocytes. Conditioned media from astrocytes treated with inhibitors of cholesterol synthesis, prenylation or both will be applied to primary neurons. We will then measure clustering of APP with lipid rafts by super resolution imaging, Aβ secretion by ELISA, activation of the lipoapoptotic pathway by western and cell death by ethidium incorporation.

抽象的 ApoE 4 亚型是与阿尔茨海默氏病最强的遗传联系，但原因尚不清楚。 ApoE是一种脂质携带蛋白质。 ApoE 颗粒的含量及其从星形胶质细胞中的释放随 ApoE 的不同而变化同工型。我们发现甲羟戊酸途径可以调节星形胶质细胞ApoE的释放以及这些 ApoE 颗粒的脂质含量。根据我们抑制的途径部分，我们可以将这两个功能分开。我们假设针对甲羟戊酸途径的各个成分可用于操纵 ApoE 的分泌和组成，以减少 Aβ 合成并提高神经元存活率。更远，我们将探索是否可以在不影响颗粒分泌的情况下降低 ApoE 颗粒的脂质含量，可以帮助将 ApoE4 颗粒的行为转向风险较低的 ApoE 基因型的行为。通过建议的实验，我们将测试：目标 1. ApoE 的分泌在静止和激活状态下通过甲羟戊酸途径进行调节星形胶质细胞以亚型特异性方式。人类 ApoE 有 3 种特征明确的亚型； 2, 3 和 4，它们具有不同程度的 AD 风险。 ApoE 携带的脂质的数量和组成为受ApoE基因型和星形胶质细胞激活状态的影响。我们将使用表达人源化 ApoE 2 的小鼠， 3 或 4 或 ApoE 敲除对照来生长皮质星形胶质细胞的原代培养物。星形胶质细胞将生长在无血清培养基模拟静止状态，或用 TNF、IL-1α 和 C1q 处理来模拟激活状态。利用化学抑制剂、shRNA 和条件敲除，我们将确定甲羟戊酸途径的胆固醇和异戊二烯化臂调节星形胶质细胞的 ApoE 分泌。目的2.抑制甲羟戊酸途径可以改善异常脂滴积累和 ApoE4 星形胶质细胞的脂质分泌谱。使用目标 1 中的工具，我们将操纵甲羟戊酸 ApoE 敲除和 ApoE 2、3 或 4 敲入小鼠的星形胶质细胞中的通路。细胞内 ApoE 和脂质液滴将通过共焦显微镜进行评估。星形胶质细胞条件培养基的脂质组成具有或不具有胆固醇合成、异戊二烯化或两者的抑制剂的静止和活化的星形胶质细胞将被测量。胆固醇-脂质载体相互作用的变化将使用胆固醇探针来确定。目标 3. 星形胶质细胞中甲羟戊酸途径的选择性抑制将减少淀粉样蛋白-β 裂解和神经元死亡。我们预测，抑制胆固醇，同时保留异戊二烯化，将减少 AD 病理学方面，对 ApoE4 星形胶质细胞影响最大。来自经处理的星形胶质细胞的条件培养基胆固醇合成、异戊二烯化或两者的抑制剂将应用于初级神经元。然后我们将测量通过超分辨率成像将 APP 与脂筏聚集，通过 ELISA 分泌 Aβ，激活西方的脂肪凋亡途径和乙锭掺入的细胞死亡。