Mass Spectrometric Analysis of Alcohol-Induced White Matter Degeneration

描述(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，与白质变性有关，并伴有髓鞘和纤维的丧失。乙醇抑制胰岛素/胰岛素样生长因子信号转导，影响脂代谢和少突胶质细胞功能(髓鞘的维持、成熟和基因表达)。相应地，成年Long Evans大鼠长期摄入酒精(36%卡路里)会导致额叶白质脱髓鞘和有髓轴突变性。我在德拉蒙特实验室接受的培训使用MALDI成像质谱仪(IMS)和UPLC-MS/MS进行的初步研究表明，乙醇对额叶白质脂类生化指标(包括鞘磷脂含量和分布)有显著影响。将额叶脑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类物质，可通过Akt途径、线粒体功能和髓鞘相关糖蛋白的表达抑制胰岛素/IGF-1信号。独立报告进一步表明，有毒神经酰胺会促进细胞死亡、神经炎症和氧化应激，而神经酰胺抑制剂豆蔻菌素可以逆转乙醇对肝脏和大脑的许多不利影响。我的初步研究表明，酒精诱导的白质生化异常可以通过MALDI-IMS检测到，并可以通过UPLC-MS/MS进行量化，并且可以在原位研究特定的感兴趣区域，如胼胝体。结果可能与脑白质病理、胰岛素/胰岛素样生长因子信号受损、少突胶质髓鞘相关基因/蛋白表达和神经认知缺陷有关。我假设慢性酒精暴露将导致脑白质鞘脂含量和轮廓在结构损伤之前发生剂量依赖性的变化，这些影响将与脑胰岛素/IGF抵抗的出现和髓鞘相关基因/蛋白的少突胶质细胞表达受损相关。我的研究安排在2个目标下：1)利用MALDI-IMS和UPLC-MS/MS表征慢性乙醇暴露对原位(穹隆和额叶白质)鞘脂谱的影响，并将结果与胰岛素信号、髓鞘基因/蛋白表达和认知功能障碍相关联；2)评估胰岛素增敏剂(PPAR-δ+γ激动剂)和Myriocin对乙醇相关鞘脂谱(MALDI-IMS/UPLC-MS/MS)、少突胶质细胞功能(mRNA和蛋白质)以及空间学习和记忆(Morris Water Maze)的治疗效果。MALDI-IMS与UPLC-MS/MS的结合使用具有创新性，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。这些临床前方法可能适用于人类酒精中毒的大脑研究。我的研究计划，加上LIFESPAN和Brown的出色的教育机会、指导和酒精相关研究人员的社区，将为我提供最先进的多学科翻译科学培训和教育，我需要这些培训和教育来开始我在酒精相关研究的独立职业生涯。

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.