Mass Spectrometric Analysis of Alcohol-Induced White Matter Degeneration

描述（由申请人提供）：与酒精相关的脑部疾病可导致神经认知和行为缺陷，与髓鞘和纤维丢失的白质变性有关。乙醇抑制胰岛素/IGF信号传导，调节脂质代谢和少突胶质细胞功能（髓磷脂维持、成熟和基因表达）。相应的，成年Long Evans大鼠的长期乙醇喂养（36%热量）导致额叶白质中有髓鞘轴突脱髓鞘和变性。通过我在de la Monte实验室的培训，使用MALDI成像质谱（IMS）和UPLC-MS/MS进行的初步研究揭示了乙醇对额叶白质脂质生化的显著影响，包括鞘脂含量和谱。将额叶切片培养物暴露于长期暴露于高水平乙醇的大脑中积累的神经酰胺物种中，可抑制通过Akt通路、线粒体功能和髓磷脂相关糖蛋白表达的胰岛素/IGF-1信号。独立的报告进一步表明，有毒的神经酰胺促进细胞死亡、神经炎症和氧化应激，而肉豆蔻素，一种神经酰胺抑制剂，可以逆转乙醇在肝脏和大脑中的许多不利影响。我的初步研究表明，乙醇诱导的白质生化异常可以用MALDI-IMS检测到，也可以用UPLC-MS/MS量化，并且可以原位研究感兴趣的特定区域，如胼胝体。结果可能与白质病理、胰岛素/IGF信号损伤、少突胶质髓鞘相关基因/蛋白表达和神经认知缺陷有关。我假设慢性乙醇暴露将在结构损伤之前产生白质鞘脂含量和谱的剂量依赖性改变，并且这些影响将与脑胰岛素/IGF抵抗的出现和髓磷脂相关基因/蛋白质的少突胶质细胞表达损伤相关。我的研究分为两个目标：1)利用MALDI-IMS和UPLC-MS/MS表征慢性乙醇暴露对原位（胼胝体和额叶白质）鞘脂谱的影响，并将结果与胰岛素信号、髓磷脂基因/蛋白表达和认知功能的损伤联系起来；2)评估胰岛素增敏剂（PPAR-δ+γ激动剂）和肉豆豆素对乙醇相关鞘脂谱（MALDI-IMS/UPLC-MS/MS）、少突胶质细胞功能（mRNA和蛋白）以及空间学习和记忆（Morris水迷宫）的治疗作用。MALDI-IMS与UPLC-MS/MS结合使用是一种创新，因为它可以生成乙醇诱导的白质结构、分子和信号转导异常的生化特征。这些临床前方法可以适用于人类酒精脑研究。我的研究计划，加上生命大学和布朗大学出色的教育机会、指导和酒精相关研究人员的社区，将为我提供多学科转化科学方面的最先进的培训和教育，这是我在酒精相关研究方面展开独立职业生涯所需要的。

DESCRIPTION (provided by applicant): Alcohol-related brain disease can cause neurocognitive and behavioral deficits linked to white matter degeneration with loss of myelin and fibers. Ethanol inhibits insulin/IGF signaling and dysregulates lipid metabolism and oligodendrocyte function (myelin maintenance, maturation, and gene expression). Correspondingly, chronic ethanol feeding (36% caloric) of adult Long Evans rats causes demyelination and degeneration of myelinated axons in frontal white matter. Preliminary studies enabled by my training in the de la Monte lab using MALDI imaging mass spectrometry (IMS) and UPLC-MS/MS revealed striking effects of ethanol on frontal lobe white matter lipid biochemistry, including sphingolipid content and profiles. Exposure of frontal lobe slice cultures to the ceramide species that accumulate in brains chronically exposed to high levels of ethanol inhibits insulin/IGF-1 signaling through Akt pathways, mitochondrial function, and myelin-associated glycoprotein expression. Independent reports further showed that toxic ceramides promote cell death neuro- inflammation, and oxidative stress, while Myriocin, a ceramide inhibitor, reverses many adverse effects of ethanol in liver and brain. My preliminary studies suggest that ethanol-induced biochemical abnormalities in white matter are detectable by MALDI-IMS and quantifiable by UPLC-MS/MS, and that specific regions of interest, such as corpus callosum, can be studied in situ. Results can be correlated with white matter pathology, impairments in insulin/IGF signaling, oligodendroglial myelin-associated gene/protein expression, and neurocognitive deficits. I hypothesize that chronic ethanol exposures will produce dose-dependent alterations in white matter sphingolipid content and profiles prior to structural damage, and that these effects will correlate with emergence of brain insulin/IGF resistance and impairments in oligodendrocyte expression of myelin-associated genes/proteins. My research is organized under 2 aims to: 1) characterize effects of chronic ethanol exposures on in situ (corpus callosum and frontal white matter) sphingolipid profiles using MALDI-IMS and UPLC-MS/MS, and correlate results with impairments in insulin signaling, myelin gene/protein expression, and cognitive function; and 2) assess therapeutic effects of insulin sensitizers (PPAR-δ+γ agonists) and Myriocin on ethanol-associated sphingolipid profiles (MALDI-IMS/UPLC-MS/MS), oligodendrocyte function (mRNA and protein), and spatial learning and memory (Morris Water Maze). The use of MALDI-IMS with UPLC-MS/MS is innovative because it can generate biochemical signatures of the ethanol-induced structural, molecular, and signal transduction abnormalities in white matter. These preclinical approaches could be adapted to human alcoholic brain studies. My research plan, together with the outstanding educational opportunities, mentoring, and community of alcohol-related researchers at Lifespan and Brown, will provide me with state-of-the-art training and education in multidisciplinary translational science which I need to launch my independent career in alcohol-related research.