Mass Spectrometric Analysis of Alcohol-Induced White Matter Degeneration

描述（由申请人提供）：酒精相关性脑病可导致神经认知和行为缺陷，与白色物质变性相关，伴有髓鞘和纤维丢失。乙醇抑制胰岛素/IGF信号传导并失调脂质代谢和少突胶质细胞功能（髓鞘维持、成熟和基因表达）。相应地，成年Long Evans大鼠慢性乙醇喂养（36%热量）导致额叶白色物质中有髓轴突脱髓鞘和变性。通过我在de la Monte实验室使用MALDI成像质谱（IMS）和UPLC-MS/MS进行的培训，初步研究揭示了乙醇对额叶白色物质脂质生物化学的显著影响，包括鞘脂含量和概况。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质，通过Akt途径、线粒体功能和髓鞘相关糖蛋白表达抑制胰岛素/IGF-1信号传导。独立报告进一步表明，有毒的神经酰胺促进细胞死亡、神经炎症和氧化应激，而神经酰胺抑制剂多粒壳菌素逆转了乙醇在肝脏和大脑中的许多不利影响。我的初步研究表明，乙醇诱导的生化异常的白色物质是可检测的MALDI-IMS和定量的UPLC-MS/MS，和特定区域的利益，如胼胝体，可以在原位研究。结果可能与白色病变、胰岛素/IGF信号传导障碍、少突胶质细胞髓鞘相关基因/蛋白表达和神经认知缺陷相关。我推测，慢性乙醇暴露将产生剂量依赖性的改变，在白色物质鞘脂含量和配置文件之前的结构损伤，这些影响将与出现脑胰岛素/胰岛素样生长因子抵抗和髓鞘相关基因/蛋白的少突胶质细胞表达受损。我的研究有两个目的：1）描述慢性乙醇暴露对原位（胼胝体和额叶白色物质）鞘脂谱，并将结果与胰岛素信号传导、髓鞘基因/蛋白表达和认知功能的损伤相关联;和2）评估胰岛素增敏剂的治疗效果（PPAR-δ+γ激动剂）和多壳菌素对乙醇相关鞘脂谱的影响（MALDI-IMS/UPLC-MS/MS）、少突胶质细胞功能（mRNA和蛋白质）和空间学习和记忆（Morris Water迷宫）。使用MALDI-IMS与UPLC-MS/MS是创新的，因为它可以产生乙醇诱导的结构，分子和信号转导异常的白色物质的生化签名。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，加上出色的教育机会，指导，以及在Lifespan和Brown的酒精相关研究人员社区，将为我提供最先进的多学科转化科学培训和教育，我需要在酒精相关研究中开展我的独立职业生涯。

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.