Characterization and quantification of CNS cell specific extracellular microvesicles in blood

血液中中枢神经系统细胞特异性细胞外微泡的表征和定量

• 批准号: 9789945
• 负责人: Tessandra Stewart
• 金额: $ 19.28万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-21 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789945
• 关键词: 2' ,3' -Cyclic-Nucleotide Phosphodiesterases; Address; Adoption; Affect; Age; Alzheimer' s Disease; Antibodies; Astrocytes; Attention; Biological Markers; Blood; Brain; Brain Diseases; Categories; Cell Communication; Cells; Clinical; Clinical Data; Collection; Consumption; DNA sequencing; Data; Development; Devices; Disease; Fingerprint; GLAST Protein; Goals; Health; Human; Individual; Lewy Body Dementia; Measurement; Measures; Membrane; Membrane Proteins; Methods; MicroRNAs; Microfluidic Microchips; Molecular; Neural Cell Adhesion Molecule L1; Neurodegenerative Disorders; Neuroglia; Neurons; Oligodendroglia; Parents; Parkinson Disease; Parkinson' s Dementia; Pathologic; Patients; Plasma; Population; Population Sizes; Precipitation; Process; Proteins; Proteomics; Protocols documentation; Research; Role; Sampling; Sorting - Cell Movement; Specificity; Surface; Techniques; Technology; Temperature; Testing; Time; Traumatic Brain Injury; Traumatic injury; Ultracentrifugation; Validation; Vesicle; alpha synuclein; base; blood-based biomarker; brain cell; brain dysfunction; cell type; cellular targeting; cohort; exosome; extracellular; high throughput screening; human subject; improved; interest; microvesicles; new technology; novel; novel marker; novel strategies; protein aggregate; prototype; scale up; tau Proteins; theories; vesicular release

Extracellular microvesicles (EMVs) are small, membrane-bound vesicles released by most cell types, and can be found circulating in the blood and other biofluids. The proteins, miRNAs, and other molecular components they carry as cargo have become a target for the development of novel biomarkers that reflect the EMV parent cell types. In particular, a strategy of targeting cellular markers carried on their membrane surfaces has been used to probe the state of the brain by examining EMVs carrying L1CAM, a relatively CNS-specific neuronal marker. Measurement of cargo proteins in such EMVs has shown particular promise in identifying blood-based biomarkers for neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Their utility in probing the state of the brain in other pathological conditions, such as after a traumatic injury, remains to be determined. Additional targets are now being developed to identify EMVs from non-neuronal brain cell types, including GLAST and GLT-1 for astrocytes and CNPase for oligodendrocytes. Despite this progress, identification of cell-specific markers remains crude, focused only on markers that tend to be present across a broad cell type. The cells themselves, in contrast, encompass multiple sub-types with different functional niches, likely differentially affected in pathological states. Thus, it should be possible to identify sub- types of EMVs and examine their composition for more specific reflections of brain processes. However, the appropriate surface markers, or combinations of markers, to target have not yet been identified. Despite the promise of EMV-based biomarkers for CNS conditions, serious challenges to their widespread adoption for clinical usage remain. The current strategies for EMV isolation are largely centered on ultracentrifugation, yield vesicle samples with contamination by large protein aggregates, and usually require large sample volumes. The newly developed immunocapture method that has allowed specific measurement of L1CAM EMV cargos is far more specific, as it targets surface markers, but is expensive, time consuming, and tends to have poor yield. Therefore, novel technologies are needed to identify EMVs of interest, isolate them, and quantify cargos. Here, we will address these challenges by developing novel strategies and technologies to better quantify and characterize brain-derived plasma EMVs in the R21 stage, and then validating them in a large cohort of human subjects in the R33 stage. First, we will optimize two new EMV capture and sorting strategies, precipitation using Smart Beads, and sorting using a microfluidics device, to isolate specific categories of EMVs based on surface markers. Second, we will identify new, more specific targets, to isolate sub-populations of EMVs that might better represent disease-relevant cells of interest. Next, in the R33 stage, we will scale up these new techniques to examine large cohorts, and measure cargo proteins of interest within these specific EMV populations as biomarkers of brain dysfunction caused by AD, PD, and traumatic brain injury.

细胞外微囊泡(EMVS)是一种小的、膜结合的囊泡，由大多数细胞类型释放，可以 在血液和其他生物体液中循环。蛋白质、miRNAs和其他分子成分 它们作为货物携带已成为反映EMV母体的新型生物标志物开发的目标 单元类型。特别是，针对携带在其膜表面的细胞标记物的策略已经被 用于通过检查携带L1CAM的EMV来探测大脑的状态，L1CAM是一种相对中枢神经特异的神经元 记号笔。对这类EMVS中货物蛋白的测量在识别血源性疾病方面显示出了特别的前景 神经退行性疾病的生物标记物，如阿尔茨海默病(AD)和帕金森病(PD)。 它们在探测其他病理条件下的大脑状态方面的作用，例如在创伤后， 仍有待确定。现在正在开发更多的靶点来识别EMV和非神经元的EMV 脑细胞类型，包括星形胶质细胞的GLAST和GLT-1，少突胶质细胞的CNPase。尽管如此 尽管取得了一些进展，但细胞特异性标志物的鉴定仍然很粗糙，只关注那些倾向于存在的标志物 横跨一种广泛的细胞类型。相比之下，单元格本身包含多个子类型，具有不同的 功能生态位，可能在病理状态下受到不同的影响。因此，应该有可能确定亚- 并检查它们的组成，以更具体地反映大脑过程。然而， 目标的适当表面标记或标记组合尚未确定。 尽管基于EMV的生物标记物有望用于中枢神经系统疾病，但其广泛存在的严重挑战 临床使用的采用率仍然存在。目前的EMV隔离策略主要集中在 超速离心，产生被大蛋白质聚集体污染的囊泡样品，通常需要 样本量大。新开发的免疫捕获方法允许特定的测量 L1CAM EMV货物要具体得多，因为它针对表面标记，但昂贵、耗时 而且产量往往很低。因此，需要新的技术来识别感兴趣的EMV、分离 他们，并量化货物。 在这里，我们将通过开发新的战略和技术来更好地量化和 确定R21阶段脑源性血浆EMV的特征，然后在一大群人中验证它们 R33阶段的受试者。首先，我们将优化两个新的EMV捕获和排序策略，即沉淀 使用智能珠，并使用微流控设备进行分类，以根据以下条件分离特定类别的EMV 曲面标记。其次，我们将确定新的、更具体的目标，以隔离符合以下条件的EMV亚群 可能更好地代表了与疾病相关的感兴趣细胞。接下来，在R33阶段，我们将扩展这些新的 检查大量队列，并测量这些特定EMV中感兴趣的货物蛋白质的技术 人群作为AD、PD和创伤性脑损伤引起的脑功能障碍的生物标志物。