Reversing Microglial Inflammarafts and Mitochondrial Dysfunction in Alzheimer's Disease

项目摘要 神经炎症是阿尔茨海默病(AD)进展的一个主要因素。炎症性脑小胶质细胞 以胆固醇和脂类代谢改变为特征。胆固醇和多种受体调控 炎症反应共同分布在有序的膜微域中，通常被称为脂筏。vt.在.的基础上 激活，脂筏驻留和招募的分子组装并启动信号级联导致 发炎。我们已经确定apoA-I结合蛋白(AIBP，由APOA1BP基因编码)是一个关键 细胞胆固醇代谢调节剂，可选择性地靶向炎症细胞中的脂筏 (炎症)通过其与TLR4的结合。而细胞外AIBP调节血浆中胆固醇的消耗 膜和控制脂筏，细胞内AIBP定位于线粒体，促进有丝分裂并帮助 维持正常的线粒体功能，控制氧化应激。Apoa1BP-/-APP/PS1小鼠呈现更多 淀粉样β蛋白(Aβ)斑块，一种加重的功能障碍的小胶质细胞表型，并显示细胞死亡增加 与APP/PS1小鼠相比。AIBP缺乏的小胶质细胞线粒体形态扭曲， 一种典型的超枝状和杯状，通常见于氧化应激之后。由AAV介导的 Apoa1BP-/-APP/PS1小鼠脑内表达分泌型AIBP可恢复脑内环境平衡 小胶质细胞形态。这项提案的目的是描述AIBP保护作用的机制 在阿尔茨海默病的大脑中，主要关注小胶质脂筏和线粒体功能障碍。具体地说，在目标1中我们 建议检验细胞外AIBP逆转小胶质细胞病理性脂筏减少的假说 在阿尔茨海默病小鼠模型中，神经炎症和防止神经变性。我们发现了一种TLR4- 并证明了不与TLR4结合的AIBP(ΔTLR4)变异体， 不能逆转脂筏改变。使用AAV递送，我们计划恢复分泌的变体的表达 ApoA1BP-/-APP/PS1小鼠脑中的AIBP，预计AIBP(Wt)而不是AIBP(ΔTLR4)将会减少 神经炎症、Aβ斑块负荷和磷酸化tau蓄积。我们还希望在以下方面有所改善 记忆和学习。在目标2中，我们将检验细胞内AIBP保护线粒体的假设 阿尔茨海默病小鼠模型的动力学和功能。线粒体是显示病毒浓度的主要部位。 细胞内AIBP，初步研究表明AIBP参与了线粒体功能的控制， 有丝分裂和氧化应激。方法将包括相关光学显微镜和跨尺度的3D EM， 利用连续块面扫描EM和EM断层扫描技术的进步，以及 海马生物能量学。为了测试建议的机制与人类AD的相关性，在目标3中，我们将 尸检和活检脑组织中脂筏和线粒体功能障碍的AIBP相关标志物的特征 AD受试者的切片。

Project Summary Neuroinflammation is a major factor in the progression of Alzheimer's disease (AD). Inflammatory brain microglia are characterized by altered cholesterol and lipid metabolism. Cholesterol and many receptors governing inflammatory responses colocalize in the ordered membrane microdomains often designated as lipid rafts. Upon activation, lipid raft resident and recruited molecules assemble and initiate signaling cascades leading to inflammation. We have identified the apoA-I binding protein (AIBP, encoded by the APOA1BP gene) as a key regulator of cellular cholesterol metabolism, which can selectively target lipid rafts in inflammatory cells (inflammarafts) via its binding to TLR4. While extracellular AIBP regulates cholesterol depletion from the plasma membrane and controls lipid rafts, intracellular AIBP localizes to mitochondria, facilitates mitophagy and helps maintain normal mitochondrial function and control oxidative stress. Apoa1bp-/- APP/PS1 mice present more amyloid beta (Aβ) plaques, an exacerbated dysfunctional microglia phenotype and show increases in cell death when compared to APP/PS1 mice. Mitochondria in AIBP-deficient microglia are morphologically distorted, with a characteristic hyper-branched and cupped shape, typically seen following oxidative stress. The AAV-mediated expression of a secreted form of AIBP in the brain of Apoa1bp-/- APP/PS1 mice restored the homeostatic microglia morphology. The goal of this proposal is to delineate mechanisms governing protective effects of AIBP in the AD brain, focusing on microglial lipid rafts and on mitochondrial dysfunction. Specifically, in Aim 1 we propose to test the hypothesis that extracellular AIBP reverses pathological lipid rafts in microglia to reduce neuroinflammation and protect against neurodegeneration in a mouse model of AD. We have identified a TLR4- binding domain in the AIBP molecule and demonstrated that an AIBP(ΔTLR4) variant, which does not bind TLR4, cannot reverse lipid raft alterations. Using AAV delivery, we plan to restore expression of secreted variants of AIBP in the brain of Apoa1bp-/- APP/PS1 mice and expect that AIBP(wt) but not AIBP(ΔTLR4) will lessen neuroinflammation, the Aβ plaque burden and accumulation of phospho-tau. We also expect improvements in memory and learning. In Aim 2, we will be testing the hypothesis that intracellular AIBP protects mitochondrial dynamics and function in a mouse model of AD. Mitochondria are the major sites displaying concentration of intracellular AIBP, and preliminary studies suggest AIBP involvement in control of mitochondrial function, mitophagy and oxidative stress. Methods will include correlated light microscopy and 3D EM across scales, leveraging advances in serial blockface scanning EM and EM tomography, along with correlated measures of bioenergetics by Seahorse. To test relevance of the proposed mechanisms to human AD, in Aim 3 we will characterize AIBP-related markers of lipid rafts and mitochondrial dysfunction in postmortem and biopsy brain sections from AD subjects.