Novel extracellular vesicle and molecular biomarkers of environmental exposure and disease progression in ALS

项目总结 肌萎缩侧索硬化症(ALS)是一种主要影响运动神经元(MNS)的散发性疾病。它的 病因尚不清楚，但已有几种环境神经毒物与ALS有关。不过，一个也没有 具有明确的致病作用。在之前调查环境因素的作用的研究中，只有少数几项 在肌萎缩侧索硬化症患者中评估了暴露的个体生物标记物(主要是持久性污染物)，但没有人 证实了神经毒性暴露产生的信号通路与 与肌萎缩侧索硬化症有牵连的人。到目前为止，这一领域的进展由于缺乏与中枢神经系统有关的具体信息而受到阻碍 用于监测环境中神经毒物暴露和疾病进展的生物标记物。 最近，我们在体外筛选了一系列与肌萎缩侧索硬化症相关的高流行神经毒物，发现 小鼠和人的锰对金属锰表现出优先的和剂量依赖的易损性。 和砷(As)，以及几种有机磷(OP)和拟除虫菊酯(PT)杀虫剂(例如毒死蜱[CPS]) 和氯氰菊酯)。我们还发现，表达一种不完全穿透性的家族性ALS变种TAR的MN DNA结合蛋白43(G298S TDP-43)在极低剂量下易受As、Mn和CPS的影响 对野生型MNS无害。TDP-43病理是绝大多数ALS病例的标志，这两种病例都是家族性的 而且是零星的，所以我们的数据表明，这些毒物可能是 大多数形式的肌萎缩侧索硬化症。有趣的是，过量的金属和病理蛋白如TDP-43可以 它们都是通过一种动态平衡机制从细胞中排出的，这种机制包括释放微小的 膜结合的隔室称为细胞外小泡(EVS)。电动汽车现在特别令人感兴趣，因为 其他领域的疾病生物标志物。中枢神经系统来源的EV可以从血液中分离出来，因为它们是异位的 L1CAM的膜表达。在这项研究中，我们将使用美国肌萎缩侧索硬化症国家实验室提供的生物素 生物库和目标ALS脑库实现以下目标：1)我们将验证头发 作为ALS中一种有用的生物示踪剂，用于测量研究较少的非持久性污染物，如有机磷农药 和PTS(200名患者x2；在两个ALS阶段采集)；2)我们将检查分离的CNS-L1CAM-EVS的使用 从相同的200名患者(2个ALS阶段)的血液中提取，作为环境暴露的生物标志物 (比较金属水平和分布)，以及通过积累 TDP-43；3)我们的流行病学和统计分析将确定 环境毒物、TDP-43和ALS进展；4)我们将检查 CPS和Mn对小鼠基因表达谱的毒物特异性暴露 和从我们的目标ALS联盟获得的患者致病转录签名，以识别 肌萎缩侧索硬化症的潜在治疗途径。通过阐明分子机制和 对于环境ALS的生物标记物，我们的研究将具有几个深远的临床和治疗意义。

Project summary Amyotrophic lateral sclerosis (ALS) is a predominantly sporadic condition affecting motor neurons (MNs). Its etiology is unknown but several environmental neurotoxicants have been associated with ALS. Though, none have a clear pathogenic role. Only a few of the previous studies investigating the role of environmental factors in ALS have assessed individual biomarkers of exposure (mostly to persistent pollutants) and none has demonstrated a direct concordance between signaling pathways produced by neurotoxic exposure and those implicated in ALS. So far, the progress in this field is hampered by the lack of CNS-relevant specific biomarkers for monitoring both environmental exposure to neurotoxicants and disease progression. Recently, we screened a series of highly prevalent neurotoxicants associated with ALS in vitro and found that mouse and human MNs exhibit a preferential and dose-dependent vulnerability to the metals manganese (Mn) and arsenic (As), and several organophosphate (OP) and pyrethroid (PT) pesticides (e.g. chlorpyrifos [CPS] and cypermethrin). Also, we found that MNs expressing an incompletely penetrant familial ALS variant of TAR DNA-binding protein 43 (G298S TDP-43) are vulnerable to As, Mn and CPS at very low doses that are innocuous to wild-type MNs. TDP-43 pathology is a hallmark of the large majority of ALS cases, both familial and sporadic, so altogether our data suggest that these toxicants could be environmental modifiers of most of the forms of ALS. Intriguingly, excess metals and pathological proteins such as TDP-43 can both be extruded from cells through a homeostatic mechanism that involves the release of tiny membrane-bound compartments called extracellular vesicles (EVs). EVs are now of particular interest as disease biomarkers in other fields. CNS-derived EVs can be isolated from blood because of their ectopic membrane expression of L1CAM. In this study, we will use biospecimens available in the US ALS National Biorepository and from the Target ALS brain bank to reach the following objectives: 1) we will validate hair as a useful biospecimen in ALS for the measure of poorly investigated non-persistent pollutants like OPs and PTs (200 patients x2; taken at 2 ALS stages); 2) we will examine the use of CNS-L1CAM-EVs isolated from the blood of the same 200 patients (2 ALS stages) as a biomarker of environmental exposure (compare metal levels and profiles), as well as a biomarker of disease progression via the accumulation of TDP-43; 3) our epidemiological and statistical analysis will determine association between environmental toxicants, TDP-43 and ALS progression; 4) we will examine concordance between toxicant-specific exposure extracted from gene-expression profiling in mice exposed to CPS and Mn and patient pathogenic transcriptional signatures obtained from our Target ALS consortium to identify disease pathways with therapeutic potential for ALS. By elucidating molecular mechanisms and biomarkers of environmental ALS, our study will have several far-reaching clinical and therapeutic implications.