Single Exosome Technology for Alzheimer's Disease

项目摘要 该项目响应了改善阿尔茨海默病（AD）诊断的重要需求。现有 基于生物标志物如来自脑脊液（CSF）的Ab和tau蛋白比率的方法是有用的， 不完整此外，CSF采样需要腰椎穿刺，并且对于广泛筛查来说过于昂贵。一 一类细胞外囊泡（EV），外泌体，为AD诊断提供了有吸引力的靶标。外来体 自由地穿过血脑屏障，并且可以容易地在外周血中取样，使得能够进行基于血液的 液体活检外泌体还为疾病检测提供了丰富的特征，包括蛋白质和核酸 核酸（mRNA，miRNA和其他非编码RNA）。大多数关于EV和AD的研究都是 进行批量或批次分析。批量分析有一个基本的局限性，因为相对罕见的 中枢神经系统（CNS）特异性的外来体容易被外来体混淆（淹没 外周细胞的贡献。为了克服这一限制，我们正在开发方法， 在单个外来体中进行蛋白质/核酸分析。我们的创新、高含量、高通量方法 旨在一次性同时分析多达107个样品中多达10种潜在的AD生物标志物货物， 单个CNS标记的血液外泌体。这些独特的功能提供了多个优势， 接近。我们的方法可以快速地：1）区分并分别分析外泌体和其他EV; 2） 区分并同时评价多种CNS特异性外泌体表面标志物，而 传统的方法一次只能评估一种CNS特异性表面标志物， 鉴定CNS来源的外泌体的能力; 3）单独地询问样品中的每个外泌体， 与传统方法相比， 货物必须合并;和4）在独特的、CNS特异性的生物标志物中寻找独特的生物标志物组合。 外泌体种群，这是常规方法不可能实现的。因此，我们提出以下建议 第一阶段的目标。首先，优化组合的蛋白质/核酸分析， 由人SH-SY 5 Y细胞产生的外泌体。第二，测定脑匀浆样品中的外来体 从40例AD、40例轻度认知障碍（MCI）、40例非AD神经系统疾病的快速尸检中， （nADneuro）（例如，帕金森病、肌萎缩侧索硬化症）和40名正常老年人对照组（NC） 科目第三，测定来自AD、MCI、nADneuro和NC受试者的快速尸检血液样品中的外来体 为目标二提供大脑样本 我们预计我们的新型成像平台有潜力成为一种新的研究/诊断/ 用于AD或其他病理的临床管理的预后工具，其中EV/外泌体分析可以 提供临床有用的信息，如其他神经退行性疾病，癌症和心血管疾病 疾病这种能力甚至可以帮助开发针对这些病理的基于外泌体的疗法。

PROJECT SUMMARY This project responds to the important need for improved diagnostics for Alzheimer’s disease (AD). Existing methods based on biomarkers such as Ab and tau protein ratios from cerebrospinal fluid (CSF) are useful but incomplete. Furthermore, CSF sampling requires lumbar puncture and is too expensive for broad screening. A class of extracellular vesicles (EVs), exosomes, provide an attractive target for AD diagnostics. Exosomes freely cross the blood-brain barrier and can be readily sampled in peripheral blood, enabling a blood-based liquid biopsy. Exosomes also provide rich signatures for disease detection, including both proteins and nucleic acids (mRNA, miRNA, and other non-coding RNAs). The majority of studies on EVs and AD have been performed with bulk or batch analyses. Bulk analysis has a fundamental limitation because the relatively rare exosomes specific to the central nervous system (CNS) are easily confounded (swamped) by the exosome contributions of peripheral cells. To overcome this limitation, we are developing methods for combined protein/nucleic acid analysis in single exosomes. Our innovative, high-content, high-throughput method is designed to simultaneously analyze, in one pass, up to 10 potential AD biomarker cargoes in as many as 107 individual, CNS-tagged blood exosomes. These unique capabilities provide multiple advantages over previous approaches. Our method can rapidly: 1) distinguish and separately analyze both exosomes and other EVs; 2) discriminate and simultaneously evaluate multiple CNS-specific exosome surface markers, whereas conventional approaches can only evaluate one CNS-specific surface marker at a time, significantly limiting the ability to identify exosomes of CNS origin; 3) individually interrogate each and every exosome in a sample for its cargoes, dramatically raising information content compared to conventional methods where exosome cargoes must be pooled; and 4) search for unique combinations of biomarkers within unique, CNS-specific exosomal populations, an impossibility with conventional approaches. We therefore propose the following stepwise objectives for this Phase I project. First, to optimize the combined protein/nucleic acid analysis of exosomes produced by human SH-SY5Y cells. Second, to assay exosomes in brain homogenate samples from rapid autopsies of 40 AD, 40 mild cognitive impairment (MCI), 40 non-AD neurological conditions (nADneuro) (e.g., Parkinson’s disease, amyotrophic lateral sclerosis), and 40 normal elderly control (NC) subjects. Third, to assay exosomes in rapid autopsy blood samples from AD, MCI, nADneuro, and NC subjects that provided brain samples used in Objective 2. We anticipate that our novel imaging platform has the potential to become a new research/diagnostic/ prognostic tool for the clinical management of AD or other pathologies in which EV/exosomal analysis could provide clinically useful information, such as other neurodegenerative diseases, cancer, and cardiovascular disease. This capability may even assist in developing exosome-based therapies for these pathologies.