Mechanisms of APOE-Induced Preclinical Alzheimer's Pathophysiology in Human Olfactory System

载脂蛋白E（APOE）是一种主要的胆固醇载体，支持脂质转运和损伤修复， 大脑在中枢神经系统中，APOE主要由星形胶质细胞产生，并通过 APOE受体，是低密度脂蛋白受体（LDLR）的成员。然而，在压力或 损伤后，神经元产生的APOE蛋白增加。APOE多态性等位基因是主要遗传因素， 晚发性阿尔茨海默病（LOAD）风险的决定因素：携带ε4等位基因的个体 与携带更常见的ε3等位基因的人相比，LOAD的风险更高，而ε2等位基因降低风险。 APOE ε4等位基因的存在也与脑淀粉样血管病和年龄相关。 与正常衰老相关的认知能力下降。尽管有这些发现，APOE 具体影响AD病理生理学的机制知之甚少。更重要的是，为什么APOE ε4 与负荷在一些人，但不是在其他人仍然未知。最近，我们检查了参与者- 与非携带者相比，APOE 4携带者的衍生嗅觉细胞， 有丝分裂后（GAP 43+）/成熟（OMP+）密度，线粒体功能障碍，衰老细胞中的细胞死亡， ε4与非ε4亚型比较。重要的是，虽然ε4亚型对Aβ寡聚体表现出更大的易感性， （ABO）诱导的损伤，我们观察到DNA polβ（DNA修复基因）表达的个体差异， 调节ABO对线粒体功能障碍、异常蛋白质稳态和细胞死亡的影响。这是 与我们先前的出版物（Misiak et. 2017）证明DNA polβ的单倍不足 加重AD小鼠的线粒体功能障碍和细胞死亡。因此，我们假设（1）APOE 4 通过异常的神经元分化导致临床前AD的嗅觉功能障碍， 更大的线粒体和突触功能障碍，DNA损伤和神经元死亡的易感性，因为 神经元随着年龄的增长而增长，并暴露于越来越多的毒素（例如Aβ）;以及（2）个体 DNA修复能力的差异导致了这些神经元结果的个体差异。 我们提出了一项为期5年的前瞻性研究，通过非侵入性采样200个嗅觉细胞， 非痴呆老年人（60-75岁; 100名ε4和100名非ε4），具有良好的嗅觉功能， 研究临床前AD病理生理学严重程度异质性的分子机制 标记。在这个提议中的分子研究是转录组学、基于RNA的 表观基因组，蛋白质验证，线粒体分析和酶的研究，旨在揭示新的 临床前机制在目标1中，我们将研究APOE亚型特异性表达的分子机制。 对嗅觉的影响-（a）分化（B）神经突形态和轴突生长（c）全细胞反应 氧物种（ROS）（d）线粒体功能（e）突触蛋白水平和（f）蛋白质稳态-跨 细胞年龄（DIV 7，14和21）。在目标2中，我们将研究 每个APOE亚型对ABO诱导的应激暴露的异质性反应。在目标3中，我们将 每年对所有参与者进行嗅觉心理物理和认知任务，以确定APOE 4的影响 在3年的随访中，观察嗅觉和认知轨迹的状态。鉴于10%的流失率，我们将 目标是250名学员。最后，我们将验证40例嗅觉细胞中APOE 4的嗅觉和分子标记。 轻度认知障碍（MCI）和AD个体，以确定哪些标志物预测MCI或AD状态。 这项研究的完成可能会导致在最早的病理生理标志的识别 临床前AD阶段，这为有效的疾病修饰提供了独特的机会。

Apolipoprotein E (APOE) is a major cholesterol carrier that supports lipid transport and injury repair in the brain. In the CNS, APOE is mainly produced by astrocytes, and transports cholesterol to neurons via APOE receptors, which are members of the low-density lipoprotein receptor (LDLR). However, during stress or injury, neuronal production of APOE protein increases. APOE polymorphic alleles are the main genetic determinants of late-onset Alzheimer disease (LOAD) risk: individuals carrying the ε4 allele are at increased risk of LOAD compared to those carrying the more common ε3 allele, whereas the ε2 allele decreases risk. Presence of the APOE ε4 allele is also associated with increased risk for cerebral amyloid angiopathy and age- related cognitive decline during normal aging. Despite these findings, the mechanism by which APOE specifically influences AD pathophysiology is poorly understood. Even more importantly, why APOE ε4 is associated with LOAD in some people, but not in others remains unknown. Recently, we examined participant- derived olfactory cells of APOE4 carriers compared to non-carriers and observed differentially higher postmitotic (GAP43+)/mature (OMP+) densities, mitochondrial dysfunctions, cell deaths among aging cells of ε4 compared non-ε4 isoforms. Importantly, while ε4 isoforms showed greater susceptibility to Aβ oligomer (ABO)-induced injury, we observed that individual differences in the expression of DNA polβ (DNA repair gene) moderated the impact of ABO on mitochondrial dysfunction, abnormal proteostasis and cell death. This is consistent with our previous publication (Misiak et. al. 2017) demonstrating that haploinsufficency of DNA polβ exacerbated mitochondrial dysfunction and cell death in AD mice. Therefore, we hypothesize that (1) APOE4 contributes to olfactory dysfunction in preclinical AD through aberrant neuronal differentiation, with resultant susceptibility to greater mitochondrial and synaptic dysfunctions, DNA damages and neuronal death, as the neurons advance in age and become exposed to increasing amount of toxins (e.g. Aβ); and (2) individual differences in proficiency for DNA repairs contribute to individual differences on these neuronal outcomes. We propose a 5-year prospective study to acquire olfactory cells through non-invasive sampling of 200 non-demented older (60-75 years; 100 ε4 and 100 non-ε4) adults with well characterized olfactory functions to investigate molecular mechanisms underlying heterogeneity in severity of preclinical AD pathophysiologic markers. Molecular studies in this proposal are a multimodal combination of transcriptomic, RNA-based epigenomic, protein validation, mitochondrial profiling and enzymatic studies, aimed at uncovering novel preclinical mechanisms. In Aim 1, we will investigate molecular mechanisms underlying APOE isoform-specific effects on olfactory – (a) differentiation (b) neurite morphology and axonal outgrowth (c) whole-cell reactive oxygen species (ROS) (d) mitochondrial function (e) synaptic protein levels and (f) proteostasis – across cellular ages (DIV 7, 14 and 21). In Aim 2, we will examine the molecular mechanisms underlying heterogeneous responses of each APOE isoform to ABO-induced stress exposures. In Aim 3, we will conduct olfactory psychophysical and cognitive tasks on all participants annually to determine the influence of APOE4 status on both olfactory and cognitive trajectory over a 3-year follow-up. In view of 10% attrition rate, we will target 250 enrollees. Finally, we will validate olfactory and molecular markers of APOE4 in olfactory cells of 40 mildly cognitively impaired (MCI) and AD individuals to determine which markers predict MCI or AD status. Accomplishment of this study could result in the identification of pathophysiologic hallmarks at the earliest phases of preclinical AD, which offers a unique opportunity for effective disease modification.