7-Dehydrocholesterol-derived oxysterols in SLOS: role and therapy

描述（由申请人提供）：候选人。我的博士后研究重点是脂质过氧化的速率和机制，因为它与许多人类神经系统疾病密切相关，如阿尔茨海默病、帕金森病、亨廷顿病等。随着我发现7-脱氢胆固醇（7-DHC）是迄今为止已知的最容易氧化的脂质分子，我的研究重点转移到Smith-Lemli-Opitz综合征（SLOS），因为在SLOS患者的组织和体液中观察到7-DHC水平显著升高（同时胆固醇水平降低）。随后在溶液、细胞和体内发现了20种新的7-DHC氧化产物。在溶液中形成的7-DHC氧甾醇具有细胞毒性，并诱导细胞中有害的基因表达变化。我在K99期的短期目标（Specific Aim 1）是研究体内形成的7-DHC氧甾醇对sls细胞模型中基因表达和脂质谱（脂质组）的生物学作用，同时接受神经科学细胞和分子生物学以及尖端离子迁移质谱（IM-MS）的培训。我在R00阶段的目标（具体目标2和3）是将相同的研究集扩展到基于我现有和新获得的技能集的SLOS动物模型。我的长期职业目标是运用我在化学结构、反应性、机理、合成和分析方面的知识来了解脂类相关的生物学

DESCRIPTION (provided by applicant): The candidate. My postdoctoral research centered on rate and mechanism of lipid peroxidation as it is closely associated with a number of human neurological disorders, such as Alzheimer's, Parkinson's, Huntington's disease, etc. With my discovery that 7-dehydrocholesterol (7-DHC) is the most readily oxidizable lipid molecule known to date, my research focus shifted to Smith-Lemli-Opitz syndrome (SLOS) as markedly elevated levels of 7-DHC (along with decreased levels of cholesterol) were observed in tissues and fluids of SLOS patients. Twenty novel oxysterols were subsequently discovered as products of oxidation of 7-DHC in solution, in cell and in vivo. 7-DHC oxysterols formed in solution were found to be cytotoxic and induce deleterious gene expression changes in cells. My short-term objective during the K99 phase (Specific Aim 1) is to study the biological actions of the in vivo-formed 7-DHC oxysterols on gene expression and lipid profiles (lipidomes) in cellular models of SLOS while receiving training in cell and molecular biology in neuroscience and the cutting-edge ion mobility-mass spectrometry (IM-MS). My objective for the R00 phase (Specific Aims 2 and 3) is to expand the same set of studies to animal models of SLOS building on my existing and newly acquired skill sets. My long-term career goal is to apply my knowledge in chemical structure, reactivity, mechanism, synthesis, and analysis to understanding lipid-related biological processes and developing translational approaches to- ward human diseases involving abnormal lipid metabolism. The environment. My Mentoring Committee is composed of five outstanding mentors and collabora- tors/consultants with complimentary expertise in lipid peroxidation, neuroscience, mass spectrometry, lipidomics, SLOS, cholesterol metabolism, gene expression, etc. The training institution, Vanderbilt University, has rich intellectual and physical resources, including institutes such as Vanderbilt Institute of Chemical Biolo- gy (VICB) and Vanderbilt Kennedy Center that are closely related to the proposed research, a full line of core laboratories, and the designated Office of Career Development. VICB has a strong and collaborative group on lipid research, which is available for consultation and establishing new collaboration. Overall, the commitment from my Mentoring Committee and the institution, along with the rich academic environment at Vanderbilt, will ensure the successful implementation of my training plans and proposed research. The research. SLOS is an autosomal recessive metabolic disorder that is caused by an inborn error of cholesterol biosynthesis. SLOS manifests a broad spectrum of phenotypes including multiple congenital malformations, neurological defects, mental retardation, and behavior problems. Over 50% of the SLOS children display autism-like behavior. Conventional therapy of SLOS is cholesterol supplementation, but the outcomes are inconsistent and controversial. Studies that focus on 7-DHC-derived metabolites are lacking, which is the gap that the proposed research is expected to fill. The central hypothesis of this project is that 7-DHC-derived oxysterols are key causal agents in the underlying molecular and pathophysiological mechanisms of SLOS. In Specific Aims 1 and 2, gene expression will be assayed by qPCR and lipidomes will be analyzed by IM-MS in cell and/or animal models of SLOS to examine the biological activities of 7-DHC oxysterols. IM-MS is a rapid two-dimensional separation technique that resolves biomolecular ions on the basis of mobility drift time and mass-to-charge ratio within micro to milliseconds. Application of the IM-MS technique in lipidomic studies is innovative because this methodology requires minimum amount of biological materials and is efficient in sample processing and data acquisition. Specific Aim 3 focuses on developing therapeutic interventions of SLOS through the inhibition of the formation of 7-DHC oxysterols. As both free radical and enzymatic oxidation con- tribute to the formation of 7-DHC oxysterols in vivo, approaches to inhibit both pathways will be explored in a rat model of SLOS. Oxysterol levels, gene expression, and lipidome will be assayed to evaluate the effectiveness of these therapies. The proposed research is expected to contribute to the elucidation of the roles of 7-DHC-derived oxysterols in the pathophysiology of SLOS, ultimately lead to a rapid and thorough diagnostic method by examining blood lipidomes of SLOS patients with IM-MS and lay the groundwork for a combination therapy through inhibiting the formation of 7-DHC oxysterol while supplementing cholesterol. The knowledge on gene expression and lipidome and the therapeutic approaches generated from this study are expected to have significant impact on other diseases that are related to abnormal cholesterol biosynthesis or metabolism, such as X-linked dominant chondrodysplasia punctata (CDPX2), cerebrotendinous xanthomatosis (CTX), and autism. PUBLIC HEALTH RELEVANCE: The proposed research is expected to enrich our understanding of the roles of 7-dehydrocholesterol-derived oxysterols in the pathophysiology of Smith-Lemli-Opitz syndrome (SLOS) - a cholesterol biosynthesis disorder that affects nervous system development, and develop therapeutic approaches through the inhibition of the formation of these oxysterols. This project relates to the mission of NICHD toward understanding intellectual and developmental disabilities, particularly disorders of metabolism that affect brain function and development.