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

7-脱氢胆固醇衍生的氧甾醇在 SLOS 中的作用和治疗

• 批准号: 8966938
• 负责人: Libin Xu
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8966938
• 关键词: 7-dehydrocholesterol; 7-dehydrocholesterol reductase; 7-ketocholesterol; AY9944; Affect; Alzheimer' s Disease; Animal Model; Antioxidants; Astrocytes; Autistic Disorder; Behavior; Bile Acid Biosynthesis Pathway; Bile Acids; Biocompatible Materials; Biological; Biological Assay; Biological Process; Biology; Blood; Brain; CYP7A1 gene; Cell Line; Cell model; Cells; Cellular biology; Cerebrotendinous Xanthomatosis; Charge; Chemical Structure; Chemicals; Child; Cholesterol; Cholesterol 7-alpha-Monooxygenase; Cholesterol Homeostasis; Chondrodysplasia Punctata; Collaborations; Combined Modality Therapy; Complementary therapies; Congenital Abnormality; Consultations; Defect; Development; Diagnostic Procedure; Disease; Effectiveness; Ensure; Environment; Enzymes; Fibroblasts; Free Radicals; Functional disorder; Gene Expression; Genes; Goals; High Pressure Liquid Chromatography; Human; Huntington Disease; Inborn Genetic Diseases; Individual; Institutes; Institution; Intellectual functioning disability; Ions; Knowledge; Laboratories; Lead; Light; Link; Lip structure; Lipid Peroxidation; Lipids; Liquid substance; Liver; Mass Spectrum Analysis; Mental Retardation; Mentors; Metabolic Diseases; Methodology; Mission; Modeling; Molecular; Molecular Biology; Mus; Mutation; National Institute of Child Health and Human Development; Neurologic; Neurons; Neurosciences; Outcome; Oxidoreductase; Parkinson Disease; Pathway interactions; Patients; Phase; Phenotype; Problem behavior; Process; Rattus; Relative (related person); Research; Resources; Rodent Model; Role; Sampling; Smith-Lemli-Opitz Syndrome; Solutions; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Supplementation; Techniques; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Training; Universities; base; career; career development; cell growth; cholesterol biosynthesis; conventional therapy; cytotoxic; data acquisition; human disease; in vivo; inhibitor/antagonist; innovation; ion mobility; lipid biosynthesis; lipid metabolism; millisecond; nervous system development; nervous system disorder; neuron development; novel; novel therapeutic intervention; oxidation; skills; translational approach; two-dimensional; ward

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, Hunting- ton'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 ele- vated 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 bio- logical actions of the in vivo-formed 7-DHC oxysterols on gene expression and lipid profiles (lipidomes) in cel- lular 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, lip- idomics, 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 mal- formations, 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 sam- ple 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 effective- ness of these therapies. The proposed research is expected to contribute to the elucidation of the roles of 7-DHC-derived oxys- terols 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 inhib- iting 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 im- pact 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.

我的博士后研究重点是脂质过氧化的速率和机制。 与许多人类神经系统疾病密切相关，如阿尔茨海默氏症、帕金森氏症、狩猎症 吨氏病等。我发现7-脱氢胆固醇（7-DHC）是最容易氧化的脂质 由于迄今为止已知的分子，我的研究重点转移到 Smith-Lemli-Opitz 综合征 (SLOS) 在 SLOS 的组织和体液中观察到 7-DHC 水平升高（同时胆固醇水平降低） 随后发现了 20 种新的氧甾醇，它们是溶液中 7-DHC 的氧化产物， 在细胞和体内，在溶液中形成的7-DHC氧化甾醇被发现具有细胞毒性并诱导有害基因。 我在 K99 阶段的短期目标（具体目标 1）是研究生物表达变化。 体内形成的 7-DHC 氧甾醇对细胞中基因表达和脂质谱（脂质体）的逻辑作用 SLOS 的卢拉模型，同时接受神经科学和前沿领域细胞和分子生物学的培训 边缘离子淌度质谱 (IM-MS) 我的 R00 相（具体目标 2 和 3）的目标是 基于我现有的和新获得的技能，将同一组研究扩展到 SLOS 动物模型。 我的长期职业目标是应用我在化学结构、反应性、机理、合成、 和分析以了解脂质相关的生物过程并开发转化方法： 病房涉及脂质代谢异常的人类疾病。 我的指导委员会由五位杰出的导师和协作者组成—— 在脂质过氧化、神经科学、质谱、唇- idomics、SLOS、胆固醇代谢、基因表达等。培训机构，范德比尔特大学， 拥有丰富的智力和物质资源，包括范德比尔特化学生物研究所等机构 gy (VICB) 和范德比尔特肯尼迪中心与拟议的研究密切相关，全线核心 实验室和指定的职业发展办公室有一个强大的协作小组。 脂质研究，可用于咨询和建立新的合作。 我的指导委员会和机构的帮助，以及范德堡大学丰富的学术环境，将 确保我的培训计划和拟议研究的成功实施。 该研究表明，SLOS 是一种常染色体隐性遗传代谢性疾病，由先天性缺陷引起。 胆固醇生物合成表现出广泛的表型，包括多种先天性缺陷。 超过 50% 的 SLOS 儿童存在神经系统缺陷、智力低下和行为问题。 SLOS 的传统治疗方法是补充胆固醇，但结果却显示出类似自闭症的行为。 缺乏针对 7-DHC 衍生代谢物的研究，这也是不一致的且有争议的。 该项目的中心假设是 7-DHC 衍生的。 氧甾醇是 SLOS 潜在分子和病理生理机制的关键致病因素。 具体目标 1 和 2，将通过 qPCR 测定基因表达，并通过 IM-MS 分析脂质组 使用 SLOS 细胞和/或动物模型来检查 7-DHC 氧化甾醇的生物活性是一种快速的方法。 基于迁移率漂移时间和分离生物分子离子的二维分离技术 IM-MS 技术在脂质组学研究中的应用是在微到毫秒内实现质荷比。 创新是因为这种方法需要最少量的生物材料并且在样品中是有效的 具体目标 3 侧重于开发 SLOS 的治疗干预措施。 通过抑制 7-DHC 氧化甾醇的形成，作为自由基和酶氧化作用。 由于体内 7-DHC 氧化甾醇的形成，抑制这两种途径的方法将在 将测定 SLOS 大鼠模型的氧甾醇水平、基因表达和脂质组以评估有效性。 这些疗法的有效性。 拟议的研究预计将有助于阐明 7-DHC 衍生的 oxys- terols 在 SLOS 病理生理学中的作用，通过检查最终形成快速、彻底的诊断方法 通过 IM-MS 检测 SLOS 患者的血脂，并为通过抑制进行联合治疗奠定基础 补充胆固醇的同时形成7-DHC氧甾醇。关于基因表达的知识。 andome 脂质和本研究产生的治疗方法预计将产生重大影响 与异常胆固醇生物合成或代谢相关的其他疾病（例如 X 连锁疾病）的协议 显性点状软骨发育不良 (CDPX2)、脑腱黄瘤病 (CTX) 和自闭症。