FASD Inhibition of ASPH-Notch Mediates Adolescent Cerebral White Matter Pathology-Potential Utility of Non-invasive Extracellular Vesicle Assays

胎儿酒精谱系障碍（FASD）对中枢神经系统的长期影响随暴露剂量、持续时间和 时机尽管进行了数十年的研究，我们仍然缺乏有效的手段来检测和治疗靶向 损害然而，由于我们未能注意到大脑病理学的全方位， 与神经行为功能障碍有关例如，除了神经元，酒精暴露在整个生命周期，但 特别是在发育过程中，发挥影响白色物质（WM）髓鞘完整性的毒性/代谢作用， 轴突和少突胶质细胞（OL），但其机制知之甚少。OL功能障碍或丢失 损害髓鞘的结构和功能，损害轴突传导性。初步研究表明， 发育期暴露于乙醇会降低成熟髓鞘相关基因和蛋白的表达， 部分原因是寡祖细胞成熟受损和/或成熟OL存活。伴随髓鞘形成不足 和髓鞘形成障碍使得WM轴突易受毒性/代谢损伤和变性的影响， 连接.这种竞争性的更新R37应用程序建立在我们以前的收益基础上， 乙醇通过PI 3 K-Akt和下游途径损害胰岛素和IGF-1信号传导，导致神经元损伤。 在发展过程中的生存和迁移。这些实验的另一个关键成功是， 乙醇抑制乙酰天冬酰胺酰-β-羟化酶（ASPH）和Notch如何损害存活、成熟和 未成熟CNS神经元的迁移。然而，经验观察和随后的初步研究导致 这一假说认为，WMOLs可能类似地被乙醇通过破坏Akt-mTOR-mTORC 1/2而受损， 改变WM髓鞘的组成和完整性。我们现在建议通过确定如何扩大这些收益， 乙醇抑制胰岛素/IGF-Akt-mTOR-mTORC 1/2信号传导，并降低ASPH-Notch激活， 出生后和青春期发育过程中的WM结构和功能。目的1将阐明 通过检查发育阶段相关的信号传导损伤， 通过胰岛素/IGF-1-PI 3 K-Akt-mTOR-mTORC 1/2和ASPH-Notch激活。目标2将评估FASD剂量- 与OL成熟受损相关的硫苷脂（ST）依赖性抑制;在许多CNS疾病中，ST WM的消耗介导认知运动缺陷。目标3源于初步研究表明乙醇 对WM鞘脂谱的影响，包括ST，在外周血循环细胞外 囊泡（EV）。因此，我们建议检查MALDI质谱（MS）EV测定的实用性， WM病理学的非侵入性检测和对恢复所需ASPH-Notch通路的治疗的反应 OL/WM成熟和功能。我们的假设将在体内，切片培养，和孤立的OL解决 模型，定量免疫组织化学，电子显微镜，多重ELISA，mRNA分析，和 MALDI-MS。该研究是新颖的、机械的、稳健的、透明的，并且包括剂量效应和两性。 通过非侵入性MALDI EV检测将这项研究扩展到人类的前景非常高。

Long-term effects of fetal alcohol spectrum disorder (FASD) on the CNS vary with exposure dose, duration, and timing. Despite decades of research, we still lack efficient means of detecting and therapeutically targeting the damage. However, progress may have been limited by our failure to attend to the full spectrum of brain pathology linked to neurobehavioral dysfunction. For example, besides neurons, alcohol exposures across the lifespan, but especially during development, exert toxic/metabolic effects that impact the integrity of white matter (WM) myelin, axons, and oligodendrocytes (OLs), yet the mechanisms are poorly understood. Dysfunction or loss of OLs compromises the structure and function of myelin, impairing axonal conductivity. Preliminary studies showed that developmental exposures to ethanol reduce the expression of mature myelin-associated genes and proteins, in part due to impaired maturation of oligoprogenitor cells and/or survival of mature OLs. Attendant hypomyelination and dysmyelination render WM axons vulnerable to toxic/metabolic injury and degeneration with loss of synaptic connections. This competing renewal R37 application builds on our previous gains that mechanistically linked ethanol-impaired insulin and IGF-1 signaling through PI3K-Akt and downstream pathways to deficits in neuronal survival and migration during development. Another critical success of those experiments was that they revealed how ethanol inhibition of aspartyl-asparaginyl-β-hydroxylase (ASPH) and Notch impair survival, maturation and migration of immature CNS neurons. However, empirical observations and subsequent preliminary studies led to the hypothesis that WM OLs may be similarly impaired by ethanol via disruption of Akt-mTOR-mTORC1/2 which alters the composition and integrity of WM myelin. We now propose to extend these gains by determining how ethanol inhibits insulin/IGF-Akt-mTOR-mTORC1/2 signaling and reduces ASPH-Notch activation in relation to WM structure and function during postnatal and adolescent development. Aim 1 will delineate mechanisms of long term FASD WM and OL pathologies by examining developmental stage-associated impairments in signaling through insulin/IGF-1-PI3K-Akt-mTOR-mTORC1/2, and ASPH-Notch activation. Aim 2 will assess FASD dose- dependent inhibition of sulfatide (ST) in relation to impairments in OL maturation; in many CNS diseases, ST depletion in WM mediates cognitive-motor deficits. Aim 3 stems from preliminary studies showing that ethanol’s effects on WM sphingolipid profiles, including STs, are detectable in peripheral blood circulating extracellular vesicles (EVs). Therefore, we propose to examine the utility of MALDI Mass Spectrometry (MS) EV assays for non-invasive detection of WM pathology and responses to treatments that restore ASPH-Notch pathways needed for OL/WM maturation and function. Our hypotheses will be addressed with in vivo, slice culture, and isolated OL models, quantitative immunohistochemistry, electron microscopy, multiplex ELISAs, mRNA analyses, and MALDI-MS. The research is novel, mechanistic, robust, transparent, and inclusive of dose effects and both sexes. The prospects for extending this research to humans via non-invasive MALDI EV assays are extremely high.