Neuronal exosomes to identify biomarkers and pathology of deployment-related TBI

创伤性脑损伤是OIF/OEF退伍军人的标志性损伤。超过36万名军人服役 成员们在2000年至2016年的战斗和训练中遭受了TBI。目前没有诊断 创伤性脑损伤的生物标记物，目前的诊断工具也无法识别慢性脑损伤的最大风险个体 颅脑损伤后的神经和随后的功能损害。神经源性外体(NDE) 从外周血中获取可能是开发可获得的中枢神经系统生物标志物的有力工具 与神经元功能障碍有关，特别是与长期脑损伤和 神经退行性变。我们最近发表的研究表明，神经病理蛋白(例如 NDE中的β-淀粉样蛋白、A？和tau)可以预测从轻度认知障碍到 阿尔茨海默氏症(AD)，而血浆水平则不是。对近地天体的研究现在正在作为同伴进行测试。 AD临床试验中的生物标记物有助于降低筛查失败率和增加登记人数。我们最近做了 发现部署相关脑损伤患者细胞骨架和突触蛋白也异常 在TBI之后。具体地说，在以下情况下，参与者的血浆NDE中的A？和神经颗粒素都发生了变化 经历了与部署相关的TBI。综上所述，我们的数据支持这样一种假设，即血浆NDE 可能是一个强大的工具，以确定可获得的和中枢神经系统特异性的蛋白生物标志物的脑损伤。NDE也是 富含短长度“微型”核糖核酸(MiRNA)货物。每个miRNA都可以调节蛋白质的表达 数百个靶信使RNA，提供了一种有效的机制来实施全基因组调控 同时影响几条细胞通路。MiRNAs赋予组织特异性，最近已经 成为神经退行性变的潜在生物标志物和治疗靶点。因此，确定 与NDES相关的中枢神经系统特异性miRNAs可能为了解慢性脑梗塞的发病过程提供了一个窗口 用于未来干预的TBI。我们假设NDE中与神经退行性变相关的蛋白质， 以及与NDE相关的miRNAs有可能成为TBI和 相关症状。为了验证这一假设，我们将利用我们的前瞻性纵向研究 1200名海军陆战队士兵的战斗部署效果，其中176人与轻度/中度部署相关 TBI。本研究采集了1个月的身心健康、神经认知功能和血样 在阿富汗作战部署之前和之后4-6个月。我们将使用150个TBI样本，其中150个 无脑损伤病史的匹配对照样本完成2个目标。目标1将检查以下各项的效用 NDES中细胞骨架和神经元蛋白在脑震荡后持续性脑损伤鉴别中的作用 症状和认知能力下降。候选神经退行性蛋白包括tau、A？、神经颗粒素、 神经丝轻链和Calain裂解的αII-Spectrin N末端片段。研究也将利用 这些预期样本用于检查损伤前后NDE蛋白的变化是否反映了症状 改变，为这些蛋白在脑损伤的致病过程中提供潜在的因果推断。目标2将 使用无偏见的发现方法识别与TBI相关的新miRNAs和 症状和神经认知能力下降。小RNA测序将在外体RNA上进行。 与TBI相关的miRNA将使用RT-qPCR进行验证，并在独立的样本集中复制。 顶尖候选人将在受伤前和受伤后接受检查，以了解这些变化的贡献 损伤后致病过程的标志物。这项研究具有很强的潜力，可以提供方便、 脑损伤和相关症状的定量生物标记物，以及识别潜在的功能靶点 干预。好了！ 好了！

Traumatic brain injury (TBI) is a signature injury of OIF/OEF Veterans. More than 360,000 armed service members sustained TBI during combat and training from 2000 to 2016. There is currently no diagnostic biological marker for TBI nor can current diagnostic tools identify individuals at greatest risk for chronic neurological and subsequent functional impairments after TBI. Neuronally-derived exosomes (NDEs) obtained from peripheral blood may be a powerful tool to develop accessible CNS-based biomarkers associated with neuronal dysfunction, particularly in relation to long-term brain injury and neurodegeneration. Our recently published work demonstrates that neuropathological proteins (e.g betaamyloid, Aß, and tau) within NDEs can predict conversion from mild cognitive impairment to Alzheimer's Disease (AD) while plasma levels do not. Studies of NDEs are now being tested as companion biomarkers in AD clinical trials to help reduce screen fail rates and increased enrollment. We have recently found that cytoskeletal and synaptic proteins are also abnormal in deployment-related TBI patients >3 mo after TBI. Specifically, both Aß and neurogranin are altered in plasma NDEs from participants who experienced a deployment-related TBI. Taken together, our data support the hypothesis that plasma NDEs may be a powerful tool to identify accessible and CNS-specific protein biomarkers for TBI. NDEs are also enriched for short length “micro” RNA (miRNA) cargo. Each miRNA can regulate protein expression from hundreds of target messenger RNAs, providing an efficient mechanism to exert genome-wide regulation and simultaneously affect several cellular pathways. miRNAs confer tissue specificity and have recently emerged as potential biomarkers and therapeutic targets for neurodegeneration. Hence, identification of CNS-specific miRNAs associated with NDEs may provide a window to the pathogenic processes in chronic TBI for future intervention. We hypothesize that proteins related to neurodegeneration within NDEs, such as Aß, as well as miRNAs associated with NDEs have the potential to be biomarkers of TBI and associated symptoms. To test this hypothesis we will leverage our prospective longitudinal study of combat deployment effects in >1200 Marines, of which 176 experienced mild/moderate deployment-related TBI. This study collected physical and mental health, neurocognitive performance and blood samples 1 mo before and 4-6 mo after a combat deployment to Afghanistan. We will use 150 TBI samples with 150 samples of matched controls with no TBI history to complete 2 aims. Aim 1 will examine utility of cytoskeletal and neuronal proteins in NDEs to identify TBI in addition to persistence of post-concussive symptoms and cognitive decline. Candidate neurodegenerative proteins include tau, Aß, neurogranin, neurofilament light chain and calpain-cleaved αII-spectrin N-terminal fragment. Studies will also leverage these prospective samples to examine if change in NDE proteins from pre-and post injury reflects symptom change, providing potential causal inferences for these proteins in the pathogenic process of TBI. Aim 2 will use an unbiased discovery approach to identify novel miRNAs associated with TBI and associated symptoms and neurocognitive decline. Small RNA sequencing will be conducted on exosomal RNA. miRNA associated with TBI will be validated using RT-qPCR and replicated in an independent sample set. Top candidates will be examined for changes pre and post injury to understand the contribution of these markers to the pathogenic process after injury. This study has strong potential to provide accessible, quantitative biomarkers for TBI and associated symptoms, as well as identify potential functional targets for intervention. ! !