Neuroprotective Mechanisms of S-Nitrosylated ApoE2 in Aging and Alzheimer's Disease

载脂蛋白-E（ApoE）是已知的维持脂蛋白颗粒的结构和介导脂质转运。 在三种ApoE同种型（即，ApoE 2、ApoE 3和ApoE 4），ApoE 4被鉴定为一种 ApoE 2是晚发性阿尔茨海默病（AD）的主要遗传风险因素，而ApoE 2被认为是保护性的。 ApoE 3代表最普遍的形式，因此被认为是一般人群的标准。 ApoE的三种变体仅在112和158处的Cys/Arg组成上不同。也就是说，ApoE 2含有 ApoE 3包括Cys 112和Arg 158; ApoE 4具有Arg 112和Arg 158。尽管如何 ApoE 2对AD发作的保护作用仍不完全清楚，ApoE 2中两个Cys残基的存在， ApoE 2似乎产生一种独特的结构（例如，二硫键连接的二聚体）和功能，可能解释 ApoE 2对AD的保护作用。值得注意的是，最近的研究表明，ApoE作为一种 TREM 2的配体，另一种在小胶质细胞中表达的AD风险因子，从而促进 聚集的Aβ。在本申请中，我们提出氧化还原介导的翻译后修饰， S-亚硝基化通过形成SNO-ApoE 2而有助于ApoE的保护作用。蛋白质S-亚硝基化是一种 涉及共价加成一氧化氮（NO）相关物质（例如，亚硝阳离子 [NO+]）连接到半胱氨酸的硫醇根阴离子。体内产生SNO蛋白的关键机制需要 NO相关物质的转移（即，转亚硝基化）从一种蛋白质到另一种蛋白质。我们集团和其他 已经广泛地证明了由生理水平的NO介导的S-亚硝基化 在神经退行性变模型中，过量的NO可通过异常的神经元凋亡而增强神经毒性， S-亚硝基化。值得注意的是，我们的初步研究和发表的报告表明，与ApoE 4不同，ApoE 2 有效地进行S-亚硝基化，导致构象变化。SNO-ApoE的变化 构象影响与相互作用配偶体如TREM 2和其他受体的结合。因此我们 发现SNO-ApoE形成影响hiPSC衍生的小胶质细胞中的Ab摄取和降解。此外，委员会认为， 我们的初步研究表明，SNO-ApoE 2可以转亚硝基化TREM 2（产生SNO-TREM 2），这意味着 ApoE 2可能通过这种转亚硝基化作用影响TREM 2依赖性神经保护信号通路， 反应在目前的R 01应用中，我们建议研究S-亚硝基化的机制， 影响ApoE 2的生化特性以调节TREM 2介导的Ab摄取和降解（特异性 目标1）。最后，我们将描述SNO-ApoE 2-TREM 2转亚硝基化对神经细胞的保护作用。 AD转基因小鼠模型以及hiPSC AD模型中的突触完整性和神经元存活 （具体目标2和3）。

Apolipoprotein-E (ApoE) is known to maintain the structure of lipoprotein particles and mediate lipid transport. Among three ApoE isoforms (i.e., ApoE2, ApoE3, and ApoE4) present in human, ApoE4 was identified as a major genetic risk factor for late onset Alzheimer’s disease (AD), whereas ApoE2 is thought to be protective. ApoE3 represents the most prevalent form and is thereby considered the standard for the general population. The three variants of ApoE differ only in their Cys/Arg composition at 112 and 158. Namely, ApoE2 contains Cys112 and Cys158; ApoE3 includes Cys112 and Arg158; and ApoE4 has Arg112 and Arg158. Although how ApoE2 protects against AD onset remains incompletely understood, the presence of two Cys residues in ApoE2 seems to engender a distinct structure (e.g., disulfide-linked dimers) and function, possibly explaining the protective effects that ApoE2 exerts towards AD. Notably, recent studies demonstrated that ApoE acts as a ligand for TREM2, another AD risk factor expressed in microglia, thus facilitating uptake and degradation of aggregated Aβ. In the current application, we propose that the redox-mediated posttranslational modification, S-nitrosylation, contributes to the protective effect of ApoE by forming SNO-ApoE2. Protein S-nitrosylation is a modification involving the covalent addition of a nitric oxide (NO)-related species (e.g., nitrosonium cation [NO+]) to a thiolate anion of cysteine. A critical mechanism for generation of SNO-proteins in vivo entails transfer of an NO-related species (i.e., transnitrosylation) from one protein to another. Our group and others have extensively demonstrated that S-nitrosylation produced by physiological levels of NO mediates neuroprotective effects, while, NO in excess elicits neurotoxicity in models of neurodegeneration via aberrant S-nitrosylation. Notably, our Preliminary Studies and published reports show that, unlike ApoE4, ApoE2 efficiently undergoes S-nitrosylation, leading to conformational changes. The change in SNO-ApoE conformation affects binding to interacting partners such as TREM2 and other receptors. Accordingly, we found that SNO-ApoE formation influences Ab uptake and degradation in hiPSC-derived microglia. Moreover, our Preliminary Studies show that SNO-ApoE2 can transnitrosylate TREM2 (producing SNO-TREM2), implying that ApoE2 may affect TREM2-dependent neuroprotective signaling pathways via this transnitrosylation reaction. In the current R01 application, we propose to investigate the mechanism whereby S-nitrosylation affects the biochemical properties of ApoE2 to regulate TREM2-mediated Ab uptake and degradation (Specific Aim 1). Finally, we will characterize the neuroprotective effects of SNO-ApoE2-TREM2 transnitrosylation on synaptic integrity and neuronal survival in AD transgenic mouse models as well as in hiPSC AD models (Specific Aims 2 and 3).