MicroRNAs in CNS-derived extracellular microvesicles as peripheral blood biomarkers for Alzheimer disease

摘要 在这项研究中，我们建议解决当前阿尔茨海默病(AD)的几个主要挑战 生物标志物研究，具体地说：1)可获得性有限，费用高昂 神经影像测量，2)开发基于抗体的定量蛋白质的困难 针对大多数新候选对象的分析以及现有免疫分析方法之间的显著差异，3) 侵入性采样抑制了脑脊液生物标志物的广泛使用，4)敏感性/特异性低 和/或在相对较大的基于血液的标记物的队列中缺乏有效性，以及，5)检测 AD处于早期甚至临床前阶段。为此，我们将针对在中央包装的microRNA 神经系统(CNS)来源于血浆中的细胞外微囊泡(EMVS)。MicroRNA是 一类小的、非编码的RNA，通过后编码在许多细胞过程中发挥重要作用。 蛋白质水平的转录调控和异常的microRNA表达已成为一种 各种疾病的新主题，包括阿尔茨海默病和其他神经退行性疾病 精神错乱。在体液中发现的microRNA可以通过油井常规和可靠地测量- 已建立的方法(例如，RT-PCR)，数据显示EMVS在 中枢神经系统和外周血液及其货物蛋白质和核酸因来源细胞而异。 对这类EMVS中货物蛋白的测量在识别血液方面显示出特别的前景- AD和轻度认知障碍(MCI，或前驱AD)的生物标志物。在飞行员R21中 研究中，我们对中枢神经系统来源的相对神经元特异性的microRNAs进行了全局图谱分析 (L1CAM阳性)血浆中的EMVS，并鉴定了许多microRNA和其他小RNA转录本 在AD和健康对照、AD和帕金森之间的差异表达 疾病，或在疾病阶段之间。 在这里，我们的目标是确认和验证已确定的AD/MCI诊断、区分 在几个大型、成熟的队列中的诊断和疾病进展，包括 附属于华盛顿大学的阿尔茨海默病研究中心 和ADNI(阿尔茨海默病神经成像倡议)，与交叉- 收集的横断面和纵向样本，以及广泛的临床特征。在……里面 同时，我们将使用全局分析方法在不同的 血浆中的CNS细胞类型或神经元亚群特异性EMVS。最后，要及早改进或 临床前诊断，我们将在非常早期的MCI队列中评估潜在的microRNA生物标志物， AD风险升高的受试者。我们的项目设计是为了生产一块 生物标记物比目前可用的更强大，更可重复，临床上更容易获得。

Summary In this study we propose to address several major challenges in current Alzheimer disease (AD) biomarker research, specifically: 1) limited accessibility and high expense associated with neuroimaging measurements, 2) difficulties in developing antibody-based, quantitative protein assays for most novel candidates and significant variability among existing immunoassays, 3) invasive sampling inhibiting widespread use of CSF-based biomarkers, 4) low sensitivity/specificity and/or lack of validation in relatively large cohorts for blood-based markers, and, 5) detection of AD at early or even preclinical stages. To these ends we will target microRNAs packaged in central nervous system (CNS) derived extracellular microvesicles (EMVs) in blood plasma. microRNAs are a class of small, non-coding RNAs playing important roles in many cellular processes by post- transcriptional regulation of protein levels, and aberrant microRNA expression has become an emerging theme for a wide variety of diseases including AD and other neurodegenerative disorders. microRNAs found in body fluids can be routinely and reliably measured by well- established methods (e.g., RT-PCR), and data shows EMVs transport biomolecules between the CNS and peripheral blood and their cargo proteins and nucleic acids vary by cell of origin. Measurement of cargo proteins in such EMVs has shown particular promise in identifying blood- based biomarkers for AD and mild cognitive impairment (MCI, or prodromal AD). In a pilot R21 study, we employed global profiling of microRNAs in CNS-derived, relatively neuron-specific (L1CAM-positive) EMVs in plasma and identified many microRNA and other small RNA transcripts to be differentially expressed between AD and healthy controls, between AD and Parkinson disease, or between disease stages. Here, we aim to confirm and validate the identified candidates for AD/MCI diagnosis, differential diagnosis, and disease progression in several large, well-established cohorts, including Alzheimer's Disease Research Centers (ADRCs) affiliated with the University of Washington and University of Pennsylvania, and ADNI (Alzheimer's Disease Neuroimaging Initiative), with cross- sectional and longitudinal samples collected, along with extensive clinical characterization. In parallel, we will use global profiling methods to identify additional microRNA candidates in different CNS cell type- or neuronal subpopulation- specific EMVs in plasma. Finally, to improve early or pre-clinical diagnosis, we will evaluate potential microRNA biomarkers in a very early MCI cohort, subjects at elevated risk for AD. Our project design is geared towards the production of a panel of biomarkers that is more robust, repeatable, and clinically accessible than currently available.