Single Exosome Technology for Alzheimer's Disease

项目总结 该项目响应了改善阿尔茨海默病(AD)诊断的重要需求。现有 基于生物标记物的方法，如从脑脊液中提取抗体和tau蛋白比率是有用的，但 不完整。此外，脑脊液采样需要腰椎穿刺术，而且对于广泛的筛查来说太昂贵了。一个 胞外囊泡(EVS)是一类外切体，为AD的诊断提供了一个有吸引力的靶点。外切体 可自由穿越血脑屏障，并可在外周血液中轻松采样，使基于血液的 液体活组织检查。外切体也为疾病检测提供了丰富的信号，包括蛋白质和核。 酸(mRNA、miRNA和其他非编码RNA)。大多数关于电动汽车和AD的研究都是 使用批量或批处理分析执行。批量分析有一个基本限制，因为相对较少的 中枢神经系统(CNS)特有的外切体很容易被外切体混淆(淹没)。 外周细胞的贡献。为了克服这一限制，我们正在开发组合的方法 单个外切体中的蛋白质/核酸分析。我们创新的、高含量、高通量的方法是 设计用于同时分析多达107个样品中的10个潜在AD生物标记物 个体，中枢神经系统标记的血液外切体。这些独特的功能提供了比以前的 接近了。我们的方法可以快速地：1)区分并分别分析外切体和其他EV；2) 区分并同时评估多个CNS特异性外切体表面标记，而 传统的方法一次只能评估一个中枢神经系统特异的表面标志，这大大限制了 识别中枢神经系统起源的外切体的能力；3)单独询问样本中的每个外切体 它的货物，与传统的方法相比，极大地提高了信息含量 货物必须汇集在一起；以及4)在唯一的、特定于中枢神经系统的区域内搜索生物标志物的唯一组合 外体种群，这是传统方法所不可能的。因此，我们提出以下建议 这个第一阶段项目的逐步目标。第一，优化蛋白质/核酸联合分析 人SH-SY5Y细胞产生的外切体。第二，检测脑组织匀浆样本中的外切体 从40例AD、40例轻度认知障碍(MCI)和40例非AD神经疾病的快速尸检中 (NADnero)(例如帕金森氏病、肌萎缩侧索硬化症)，以及40名正常老年人对照组(NC) 研究对象。第三，检测AD、MCI、nADnero和NC受试者的快速尸检血液样本中的外切体 提供了目标2中使用的大脑样本。 我们期待我们的新成像平台有潜力成为新的研究/诊断/ 用于临床处理AD或其他病理的预后工具，在这些疾病中，EV/Exosomal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。这种能力甚至可能有助于为这些病理疾病开发基于外切体的疗法。

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.