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因暴露剂量、持续时间和 时机到了。尽管经过了几十年的研究，我们仍然缺乏有效的手段来检测和治疗靶向 损坏。然而，进展可能由于我们未能注意到大脑病理的全部范围而受到限制。 与神经行为障碍有关。例如，除了神经元，酒精暴露在整个生命周期中，但 尤其是在发育过程中，会产生毒性/代谢效应，影响白质(WM)髓鞘的完整性， 轴突和少突胶质细胞(OLs)，但其机制尚不清楚。OLS功能障碍或缺失 损害髓鞘的结构和功能，损害轴突的传导性。初步研究表明， 在发育过程中，酒精暴露会减少成熟的髓鞘相关基因和蛋白的表达 部分原因是寡祖细胞的成熟受损和/或成熟OL的存活。随之而来的骨髓灰质化 和髓鞘功能障碍使WM轴突容易受到毒性/代谢性损伤和变性并失去突触 关系。这一竞争性续订R37应用程序建立在我们之前机械链接的收益的基础上 乙醇损伤的胰岛素和胰岛素样生长因子-1通过PI3K-Akt及其下游通路导致神经元功能缺陷 发展中的生存和迁徙。这些实验的另一个关键成功是他们揭示了 乙醇抑制天冬氨酰天冬酰胺β羟基酶和Notch对存活、成熟和死亡的影响 未成熟中枢神经系统神经元的迁移。然而，经验观察和随后的初步研究导致了 假设WM OL可能类似地被乙醇通过破坏Akt-mTOR-mTORC1/2而受损，Akt-mTOR-mTORC1/2 改变WM髓磷脂的组成和完整性。我们现在建议通过确定如何 乙醇抑制胰岛素/IGF-Akt-mTOR-mTORC1/2信号转导，降低ASPH-Notch激活 WM在出生后和青少年发育过程中的结构和功能。目标1将描述以下机制 长期FASD、WM和OL的病理--通过检测发育阶段相关的信号损伤 通过胰岛素/IGF-1-PI3K-Akt-mTOR-mTORC1/2和ASPH-Notch激活。AIM 2将评估FASD剂量- 硫脂(ST)的依赖抑制与OL成熟障碍的关系；在许多中枢神经系统疾病中，ST WM中的耗竭会导致认知运动障碍。目标3源于初步研究表明，乙醇的 在外周血液细胞外循环中可以检测到对包括STS在内的WM鞘脂谱的影响 水泡(EVS)。因此，我们建议研究MALDI质谱学(MS)EV分析在 非侵入性检测WM病理和恢复ASPH-Notch通路所需治疗的反应 对于OL/WM的成熟和功能。我们的假设将通过活体、切片培养和分离的OL来解决 模型，定量免疫组织化学，电子显微镜，多重ELISA，mRNA分析，以及 MALDI-MS这项研究是新颖的、机械性的、稳健的、透明的，包括剂量效应和性别。 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.