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.

候选人。我的博士后研究以脂质过氧化的速率和机制为中心 与许多人类神经系统疾病密切相关，例如阿尔茨海默氏症，帕金森氏症，狩猎 - 我发现，7-脱氢胆固醇（7-DHC）是最容易氧化的脂质的发现， 迄今已知的分子，我的研究重点转移到了史密斯 - 莱姆利 - 奥普兹综合征（SLO）中，显着 在SLOS的组织和流体中观察到了传播的7-DHC水平（以及提高的胆固醇水平） 患者。随后发现了二十种新型氧化酚作为溶液中7-DHC的氧化产物， 在细胞和体内。发现在溶液中形成的7-DHC氧蛋白酶是细胞毒性的，并诱导有害基因 细胞的表达变化。我在K99阶段的短期目标（特定目的1）是研究生物 体内形成的7-DHC氧蛋白酶对基因表达和脂质谱（脂质）的逻辑作用 在接受神经科学的细胞和分子生物学培训时，SLO的lular模型和神经科学的训练 边缘离子迁移率质谱法（IM-MS）。我对R00阶段的目标（特定目的2和3）是 将相同的研究扩展到我现有和新获得的技能的动物模型。 我的长期职业目标是将我的知识应用于化学结构，反应性，机制，合成， 和分析以了解与脂质相关的生物学过程并开发转化方法 病房人类疾病涉及异常脂质代谢。 环境。我的指导委员会由五位出色的导师和合作组成 具有脂质过氧化，神经科学，质谱法，唇部的TOR/顾问的TOR/顾问 成语，SLO，胆固醇代谢，基因表达等。范德比尔特大学培训机构， 拥有丰富的智力和物理资源，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（Vanderbilt Kennedy Center）与拟议研究密切相关，这是一系列核心线 实验室和指定的职业发展办公室。 VICB有一个强大而协作的小组 脂质研究，可用于咨询和建立新的合作。总体而言，承诺 从我的指导委员会和机构以及范德比尔特丰富的学术环境中 确保成功实施我的培训计划和拟议的研究。 研究。 SLO是一种常染色体隐性代谢疾病，是由天生错误引起的 胆固醇生物合成。 SLO表现出广泛的表型，包括多个先天性恶麦 形成，神经系统缺陷，智力低下和行为问题。超过50％的SLO儿童 显示自闭症的行为。 SLO的常规疗法是补充胆固醇，但结果是 是不一致的和有争议的。缺乏关注7-DHC衍生代谢产物的研究，这就是 预计拟议研究将填补的差距。该项目的中心假设是7-DHC衍生 氧化酚是SLO的基本分子和病理生理机制中的关键因果剂。在 具体目的1和2，基因表达将由qPCR分配，脂质组将通过IM-MS分析 SLO的细胞和/或动物模型检查7-DHC氧甲醇的生物学活性。 IM-MS很快 二维分离技术，基于移动性漂移时间和 微型与毫秒内的质量比率。 IM-MS技术在脂质组研究中的应用是 创新性，因为这种方法需要最少的生物材料，并且有效 PLE处理和数据获取。特定目标3着重于开发SLO的治疗干预措施 通过抑制7-DHC氧蛋白酶的形成。作为自由基和酶促氧化的综合 为在体内形成7-DHC氧蛋白醇的形成，抑制这两种途径的方法将在A中探索 大鼠SLO的模型。将分配氧化酚水平，基因表达和脂肪组，以评估有效的 这些疗法的NES。 预计拟议的研究将有助于阐明7-DHC衍生的Oxys- SLO的病理生理学中的Terols，最终通过检查而导致快速而彻底的诊断方法 IM-MS的SLOS患者的血液脂质组，并通过抑制作用为联合治疗奠定基础 在补充胆固醇的同时，它会形成7-DHC氧蛋白酶。基因表达的知识 预计该研究产生的脂肪组和治疗方法将具有明显的影响 关于与异常胆固醇生物合成或代谢有关的其他疾病的契约，例如X连接 主要的软骨发育不良（CDPX2），脑齿状异常病（CTX）和自闭症。