Brain-derived extracellular vesicles as a novel source of biomarkers for disease progression and environmental exposure in ALS

摘要 肌萎缩侧索硬化症(ALS)是一种主要是散发性的且总是致命的麻痹性疾病。ALS致病 机制难以捉摸，其病因也知之甚少，即使环境暴露，包括有毒物质 金属被认为起到了关键作用。肌萎缩侧索硬化症的治疗和预防研究进展缓慢 由于缺乏明确的生物标志物，无法指示患者的疾病进展和中枢神经系统(CNS) 暴露在环境毒物中。在这里，我们建议测试血液中的细胞外物质 囊泡(EVS)，即微小的膜结合胶囊，将生物分子运送出中枢神经系统，可用作 中枢神经系统金属暴露和肌萎缩侧索硬化疾病进展的新生物标记物。电动汽车携带核酸(包括 MicroRNAs[miRNAs])、蛋白质和其他元素，同时在全身循环中进行器官间循环 沟通。电动汽车还可以充当“垃圾袋”，让电池清除多余的电池。 材料，包括有毒金属和蛋白质。CNS-EV可以从血液中分离出来，因为它们表达在 它们的表面有神经元特异性蛋白L1CAM或星形胶质细胞特异性蛋白GLAST。血液CNS-EVS 可以从外围打开观察ALS大脑的直接窗口，并提供不同类型的 生物标志物。首先，中枢神经系统肠道病毒可能通过递增携带致病蛋白来反映疾病进展。 在肌萎缩侧索硬化症的神经元和星形胶质细胞中蓄积，如TDP-43(TDP-43)，或通过 显示miRNA图谱的渐进性变化。其次，CNS-EV金属水平可以直接替代 CNS金属负载，金属对miRNA图谱的影响表明，它们可以提供一个分子 当不能直接测量时，患者金属暴露的指纹。为了检验这些假设，我们 将利用来自三个表型良好的ALS患者队列的独特生物样品收集和一组 140份对照样本：(1)COSMOS多中心队列(n=269名患者；2-6次连续访问；血液)； Neals对ALS生物标记物的纵向研究(实验室)(N=150)，2-3次访问；以及(3)退伍军人事务部 生物库(VAB)(n=147；配对血液和死后中枢神经系统组织)。在每个阶段评估临床分期 通过ALS功能评定量表抽血-修订。我们的具体目标是：目标-1：测试 血清L1CAM/GLAST-EVS中TDP-43水平是ALS进展的生物标志物 TDP-43在连续宇宙和实验室抽血，并检测其与ALS进展的关系。目标-- 2：通过测量血液中CNS-EV中的金属含量来评估其作为患者暴露的生物标志物 在配对的CNS-EVS和CNS组织(VAB)中与ALS相关的7种金属，并测试它们与ALS的相关性 进展(实验室)。目的-3：检测血液中的miRNA图谱是否为两种ALS的生物标志物 用miRNA测序研究进展和金属暴露及其是否介导Tdp-43‘S和 金属对肌萎缩侧索硬化症进展的影响。在这里测试的新型微创EV生物标记物可以大大 完善肌萎缩侧索硬化症治疗效果评价，革新金属暴露评价。

SUMMARY Amyotrophic lateral sclerosis (ALS) is a mostly sporadic and invariably fatal paralytic disorder. ALS pathogenic mechanisms are elusive and its etiology is poorly understood, even if environmental exposures, including toxic metals, are thought to play a key role. Progresses in both therapy and prevention for ALS have been held back by the lack of clear biomarkers, indicative of patients' disease progression and central nervous system (CNS) exposure to environmental toxicants. Here, we propose to test whether the cargoes of blood extra-cellular vesicles (EVs), i.e., tiny membrane-bound capsules that shuttle biomolecules out of the CNS, can serve as novel biomarkers of CNS metal exposure and disease progression in ALS. EVs carry nucleic acids (including microRNAs [miRNAs]), proteins, and other elements, while circulating throughout the body for inter- organ communication. EVs also operate as "trash bags" allowing cells to eliminate excesses of unwanted cellular materials, including toxic metals and proteins. CNS-EVs can be isolated from blood because they express on their surface the neuron-specific protein L1CAM or the astrocyte-specific protein GLAST. Blood CNS-EVs could open a direct window of observation into the ALS brain from the periphery and provide different types of biomarkers. First, CNS-EVs may reflect disease progression by carrying pathogenic proteins progressively accumulating in neurons and astrocytes in ALS, such as the TAR DNA-binding protein 43 (TDP-43), or by showing progressive changes in miRNA profiles. Second, CNS-EV metal levels could be a direct surrogate of CNS metal load, and as suggested by metals' effects on miRNA profiles, they could provide a molecular fingerprint of patient metal exposure, when direct measurement is not possible. To test these hypotheses, we will leverage unique biospecimen collections from three well-phenotyped cohorts of ALS patients and a set of 140 control samples: (1) the COSMOS multi-center cohort (n=269 patients; 2-6 serial visits; blood); (2) the NEALS longitudinal study for ALS biomarkers (LABB) (N=150) with 2-3 visits; and (3) the Veterans Affairs Biorepository (VAB) (n=147; paired blood and postmortem CNS tissues). Clinical stage was assessed at each blood draw through the ALS Functional Rating Scale-Revised. Our specific aims are to: Aim-1: Test whether TDP-43 level in blood L1CAM/GLAST-EVs is a biomarker of ALS progression by ELISA quantification of TDP-43 in the serial COSMOS and LABB blood draws, and testing its association with ALS progression. Aim- 2: Assess metal content in blood CNS-EV as a biomarker of patient's exposure by measuring the levels of 7 metals linked to ALS in paired CNS-EVs and CNS tissues (VAB), and testing their association with ALS progression (LABB). Aim-3: Test whether miRNA profiles in blood CNS-EVs are biomarkers of both ALS progression and metal exposure by miRNA-sequencing and assessing whether they mediate TDP-43's and metals' effect on ALS progression. The novel and minimally invasive EV biomarkers tested here could greatly improve the evaluation of ALS treatment efficacy, and revolutionize metal exposure assessment.