Human Cerebrospinal Fluid Extracellular Vesicles: Utility as Disease Specific Biomarkers and Impact on Alzheimer's Disease Pathology

项目总结 阿尔茨海默病(AD)是痴呆症最常见的形式，也是导致这些人死亡的第五大原因 65岁及以上。阿尔茨海默病患者的护理费用很高：2022年的总付款估计为 3210亿美元，而且成本每年将增加10亿美元。AD的危险因素包括年龄、性别和遗传。的 患有AD的美国人中，400万为女性，250万为男性，载脂蛋白E(APOE)-e4 等位基因是散发性AD最重要的遗传危险因素。此外，女性载脂蛋白E-e4携带者更多 可能从轻度认知障碍(MCI)进展到AD，有更多的脑萎缩和记忆力丧失，以及 与年龄匹配的男性相比，患阿尔茨海默病的几率更高。AD的显著特征包括淀粉样蛋白的积聚 β(Aβ)和Tau的异常磷酸化，导致大脑中的斑块和神经原纤维缠结。 在AD中，重要的是细胞外小泡(EV)生物发生的中断导致EV miRNA和蛋白质的改变 在AD中，货物和神经元EV同时含有和传播Aβ和Tau。我们之前的研究发现 活体供者脑脊液中miRNAs对AD患者与健康人的鉴别作用 对照(CTL)参与者，我们发现结合CSFmiRNAs和CSFAβ42：总TAU测量 改进了AD和MCI与CTL的分类。在本文提出的研究中，我们将重点放在csf的效用上。 电动汽车及其货物作为AD的更强大的生物标志物，显示出女性与男性和 APOE-E4型与APOE-E3型比较。我们在人类脑脊液、EVS和尸检脑中的试点数据 结果表明：1)AD与CTL-CSF相比，膜颗粒较大；2)脑脊液EV具有表面标志 与CTL相比，AD和/或帕金森病(PD)特有的蛋白质；III)脑脊液EV miRNAs得到改善 AD与总脑脊液的预测能力；IV)脑脊液EV miRNAs显示性别和载脂蛋白E依赖的变化 V)与CTL相比，女性AD-CSF EV中miR-16-5p的表达增加；vi)预测的基因靶点 MIR-16-5P、SNAP-25在女性AD海马区表达降低。总而言之，这些研究支持性行为和 APOE-e4均可导致AD-CSFEV的改变，这可能是较高的AD-CSFEV患病率的部分原因 ApoE-e4基因携带者患AD的风险增加。因此，我们建议 在性别和性别背景下，建立AD-CSF EVS与CTL的物理特征和分子载量 APOE基因。我们将使用信息论方法来识别最强的依赖关系， 根据CTL区分AD和PD，并确定高危AD的候选生物标志物。我们包括男性和 女性PD以识别脑脊液EV表面标志物和AD独有的货物。然后，我们将确定和 验证脑脊液EV miRNAs与AD相关的基因靶点。共同努力，我们的研究将建立具体的 通过结合物理特征、表面标记谱、miRNAs和脑脊液中的蛋白质来预测AD的生物标志物 电动汽车。他们还将在人脑中建立AD EV miRNAs的神经学相关基因靶点，这 可能参与AD的发病机制，并可作为治疗AD的新靶点。

PROJECT SUMMARY Alzheimer's disease (AD) is the most common form of dementia and the fifth leading cause of death for those age 65 and older. Costs of care for individuals with AD are substantial: total payments in 2022 are estimated at $321 billion, and costs will increase by $1 billion each year. AD risk factors include age, sex, and genetics. Of Americans living with AD, 4 million are women and 2.5 million are men, and the apolipoprotein E (APOE) -e4 allele is the most important genetic risk factor for sporadic AD. In addition, female APOE-e4 carriers are more likely to progress from mild cognitive impairment (MCI) to AD, have more brain atrophy and memory loss, and develop AD more frequently than age matched males. Hallmark features of AD include the buildup of amyloid beta (Aβ) and abnormal phosphorylation of Tau, resulting in plaques and neurofibrillary tangles in the brain. Importantly in AD, disruptions to extracellular vesicle (EV) biogenesis result in altered EV miRNA and protein cargo, and neuronal EVs in AD contain and propagate both Aβ and Tau. Our previous studies identified miRNAs in human cerebrospinal fluid (CSF) from living donors that discriminate AD patients from healthy control (CTL) participants, and we found that combining CSF miRNAs plus CSF Aβ42:total Tau measurements improves classification of AD and MCI vs. CTL. In the studies proposed herein, we focus on the utility of CSF EVs and their cargo as more robust biomarkers for AD, that show distinct differences in females vs. males and those with the APOE-e4 vs. APOE-e3 genotype. Our pilot data in human CSF EVs and postmortem brain shows that: i) the size of membranous particles is larger in AD vs. CTL CSF; ii) CSF EVs have surface marker proteins that are unique to AD and/or Parkinson's disease (PD) vs. CTL; iii) CSF EV miRNAs have improved predictive power for AD vs. total CSF; iv) CSF EV miRNAs show sex and APOE dependent changes in expression; v) there is increased miR-16-5p in female AD CSF EVs vs. CTL; and vi) a predicted gene target of miR-16-5p, SNAP-25, is decreased in female AD hippocampus. Together, these studies support that sex and APOE-e4 both contribute to changes in AD CSF EVs, which may account in part for the higher prevalence of AD in females, and the increased risk for AD in people with the APOE-e4 genotype. Thus, we propose to establish the physical features and molecular cargo of AD CSF EVs vs. CTL, within the context of sex and APOE genotype. We will use our Information Theory method to identify the strongest dependencies that can distinguish AD from PD from CTL, and to identify candidate biomarkers for high-risk AD. We include male and female PD to identify CSF EV surface markers and cargo that are unique from AD. We will then identify and verify gene targets of CSF EV miRNAs that are relevant to AD. Together, our studies will establish specific biomarkers for AD by combining the physical features, surface marker profiles, miRNAs, and proteins in CSF EVs. They will also establish the neurologically relevant gene targets of AD EV miRNAs in human brain, which may contribute to the processes underlying AD pathogenesis and serve as novel therapeutic targets for AD.