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.

细胞外微泡（EMV）是大多数细胞类型释放的膜结合的小囊泡，可以 在血液和其他生物流体中发现循环。蛋白质，miRNA和其他分子成分 他们随着货物而携带的货物已成为反映EMV父母的新型生物标志物的目标 细胞类型。特别是，针对膜表面上的细胞标记的策略一直是 用于通过检查携带L1CAM的EMV（一种相对CNS特异性神经元）来探测大脑状态 标记。在这种EMV中的货物蛋白的测量在鉴定基于血液方面表现出了特别的希望 神经退行性疾病的生物标志物，例如阿尔茨海默氏病（AD）和帕金森氏病（PD）。 它们在其他病理状况（例如创伤性损伤之后）探测大脑状态方面的效用 仍有待确定。现在正在开发其他目标来识别非神经元的EMV 脑细胞类型，包括用于星形胶质细胞的GLAST和GLT-1，以及用于少突胶质细胞的CNPase。尽管如此 进度，细胞特异性标记的识别仍然是粗略的，仅集中在往往存在的标记上 跨越广泛的细胞类型。相比之下，单元本身包括多个具有不同的子类型 功能性位，可能在病理状态下受到差异影响。因此，应该可以识别 EMV的类型并检查其组成，以了解对大脑过程的更具体的反思。但是， 尚未鉴定出适当的表面标记或标记的组合标记。 尽管有基于EMV的生物标志物在中枢神经系统条件下的承诺，但对其广泛的挑战 保留临床使用的采用。当前的EMV隔离策略主要集中在 超速离心，大蛋白质聚集体污染的囊泡样品，通常需要 大型样品量。新开发的免疫接种方法，允许特定的测量 L1CAM EMV Cargos的特异性更为具体，因为它针对地表标记，但昂贵，耗时， 并且往往的产量差。因此，需要新颖的技术来识别感兴趣的EMV，分离 它们并量化货物。 在这里，我们将通过制定新颖的策略和技术来更好地量化和 在R21阶段表征脑衍生的等离子体EMV，然后在大量人类中验证它们 R33阶段的受试者。首先，我们将优化两种新的EMV捕获和分类策略，降水 使用智能珠和使用微流体设备进行排序，以基于EMV的特定类别 表面标记。其次，我们将确定新的，更具体的目标，以隔离EMV的子群 可以更好地代表与疾病相关的感兴趣的细胞。接下来，在R33阶段，我们将扩展这些新的 检查大型队列并测量这些特定EMV中感兴趣的货物蛋白的技术 人群是由AD，PD和脑部损伤引起的脑功能障碍的生物标志物。