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

摘要 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，载脂蛋白E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，在压力或压力下 损伤后，神经元产生的APOE蛋白增多。载脂蛋白E的多态等位基因是主要的遗传 晚发性阿尔茨海默病(LOAD)风险的决定因素：携带ε4等位基因的个体风险增加 与携带更常见的ε3等位基因的人相比，携带ε2等位基因的人风险更低。 载脂蛋白Eε4等位基因的存在也与脑淀粉样血管病的风险增加和年龄相关。 正常衰老过程中相关的认知功能下降。尽管有这些发现，载脂蛋白E 具体影响阿尔茨海默病的病理生理学还知之甚少。更重要的是，为什么APOEε4 在一些人中与负荷有关，而在另一些人中则不是，目前仍不清楚。最近，我们检查了参与者- APOE4携带者来源的嗅觉细胞与非携带者的比较和观察到的差异 老年细胞中有丝分裂后(GAP43+)/成熟(OMP+)密度、线粒体功能障碍和细胞死亡 ε-4比较了非ε-4亚型。重要的是，虽然ε4亚型对β寡聚体表现出更大的敏感性 (ABO)诱导的损伤后，我们观察到个体差异表达的dna pollβ(dna修复基因)。 缓解ABO对线粒体功能障碍、异常蛋白平衡和细胞死亡的影响。这是 与我们之前的出版物(Misiak et.艾尔2017)表明DNAPollβ的单倍性不足 加重AD小鼠线粒体功能障碍和细胞死亡。因此，我们假设(1)APOE4 通过异常的神经元分化导致临床前阿尔茨海默病患者的嗅觉功能障碍 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，如 神经元随着年龄的增长而暴露在越来越多的毒素中(例如Aβ)；以及(2)个体 DNA修复熟练程度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，通过非侵入性采样获得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的成年人 探讨临床前阿尔茨海默病严重程度异质性的分子机制 记号笔。这项建议中的分子研究是转录、基于RNA的多模式组合 表观基因组学、蛋白质验证、线粒体图谱和酶研究，旨在发现新的 临床前机制。在目标1中，我们将研究apoE异构体特异性的分子机制 对嗅觉的影响-(A)分化(B)轴突形态和轴突生长(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)蛋白稳定 细胞年龄(DIV 7、14和21)。在目标2中，我们将研究潜在的分子机制。 各APOE亚型对ABO诱导的应激暴露的异质性反应。在目标3中，我们将进行 每年对所有参与者进行嗅觉、心理物理和认知任务，以确定APOE4的影响 3年随访的嗅觉和认知轨迹状况。鉴于10%的流失率，我们将 目标注册人数为250人。最后，我们将在40个嗅觉细胞中验证APOE4的嗅觉和分子标记 轻度认知受损(MCI)和阿尔茨海默病(AD)患者，以确定哪些标志物预测MCI或AD状态。 这项研究的完成可能导致最早识别出病理生理标志 临床前阿尔茨海默病的各个阶段，这为有效的疾病改进提供了独特的机会。

ABSTRACT 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.