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

项目摘要 肌萎缩侧索硬化症（ALS）是一种影响运动神经元（MN）的主要散发性疾病。其 病因学尚不清楚，但几种环境神经毒物与ALS有关。不过， 具有明显的致病作用。只有少数以前的研究调查环境因素的作用 已经评估了暴露的个体生物标志物（主要是持久性污染物）， 证明了神经毒性暴露产生的信号通路之间的直接一致性， 与ALS有关的人到目前为止，由于缺乏与中枢神经系统相关的具体信息， 用于监测神经毒物的环境暴露和疾病进展的生物标志物。 最近，我们在体外筛选了一系列与ALS相关的高度流行的神经毒物，发现 小鼠和人类MN对金属锰（Mn）表现出优先和剂量依赖性的脆弱性 和砷（As），以及几种有机磷（OP）和拟除虫菊酯（PT）农药（如毒死蜱[CPS] 和氯氰菊酯）。此外，我们发现表达TAR的不完全外显家族性ALS变体的MN， DNA结合蛋白43（G298 S TDP-43）在非常低的剂量下易受As，Mn和CPS的影响， 对野生型MN无害。TDP-43病理学是大多数ALS病例的标志，无论是家族性的还是非家族性的。 和零星的，所以总的来说，我们的数据表明，这些有毒物质可能是环境的调节剂， 大多数的ALS。有趣的是，过量的金属和病理性蛋白质，如TDP-43， 两者都通过一种体内平衡机制从细胞中挤出， 细胞外囊泡（EV）。电动汽车现在特别感兴趣， 其他领域的疾病生物标志物。CNS衍生的EV可以从血液中分离，因为它们的异位表达。 L1 CAM的膜表达。在这项研究中，我们将使用美国ALS国家实验室提供的生物标本。 生物储存库和目标ALS脑银行，以达到以下目标：1）我们将验证头发 作为ALS中有用的生物标本，用于测量研究不足的非持久性污染物，如OP 和PT（200例患者x2;在2个ALS阶段采集）; 2）我们将检查分离的CNS-L1 CAM-EV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 （比较金属水平和概况），以及通过积累的疾病进展的生物标志物 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 从暴露于CPS和Mn的小鼠的基因表达谱中提取的毒物特异性暴露 和从我们的靶向ALS联盟获得的患者致病性转录特征，以鉴定 具有治疗ALS潜力的疾病途径。通过阐明分子机制， 环境ALS的生物标志物，我们的研究将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.