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。我的博士后研究集中在脂质过氧化的速率和机制，因为它是 与一些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- 随着我发现7-脱氢胆固醇（7-DHC）是最容易氧化的脂质， 分子已知的日期，我的研究重点转移到Smith-Lemli-Opitz综合征（SLOS），作为显着的ele-peptide。 在SLOS的组织和体液中观察到7-DHC水平降低（沿着胆固醇水平降低 患者随后发现了20种新的氧甾醇作为7-DHC在溶液中氧化的产物， 在细胞和体内。7-发现在溶液中形成的DHC氧化固醇具有细胞毒性，并诱导有害基因 细胞中的表达变化。我在K99阶段的短期目标（具体目标1）是研究生物学， 体内形成的7-DHC氧固醇对细胞基因表达和脂质谱（脂质体）的逻辑作用， 在接受神经科学细胞和分子生物学培训的同时， 边缘离子迁移率-质谱（IM-MS）。R 00阶段（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的动物模型。 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和发展翻译方法， 防止脂质代谢异常的人类疾病。 环境保护我的导师委员会由五位杰出的导师和合作者组成- 在脂质过氧化、神经科学、质谱、唇- 体素组学、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， 戈伊（VICB）和范德比尔特肯尼迪中心即密切相关，拟进行全线核心研究 实验室，和职业发展的指定办公室。国际合作中心有一个强大的协作小组， 脂质研究，可供咨询和建立新的合作。总的来说，承诺 来自我的指导委员会和机构，沿着在范德比尔特丰富的学术环境，将 确保我的培训计划和拟议研究的成功实施。 研究SLOS是一种常染色体隐性遗传代谢紊乱，由先天性缺陷引起， 胆固醇生物合成SLOS表现出广泛的表型，包括多种先天性畸形， 形成，神经缺陷，智力迟钝和行为问题。超过50%的SLOS儿童 表现出类似自闭症的行为SLOS的常规治疗是补充胆固醇，但结果 是不一致和有争议的。研究，重点是7-DHC衍生的代谢物是缺乏的，这是 这项研究有望填补的空白。该项目的中心假设是7-DHC衍生的 氧固醇是SLOS潜在分子和病理生理机制的关键致病因子。在 具体目的1和2，将通过qPCR测定基因表达，并通过IM-MS分析脂质体， 细胞和/或SLOS的动物模型，以检查7-DHC氧固醇的生物活性。IM-MS是一种快速 二维分离技术，其基于迁移率漂移时间解析生物分子离子， 质荷比在微秒到毫秒之间。介绍了IM-MS技术在脂质组学研究中的应用 创新的，因为这种方法需要最少量的生物材料，并在相同的， 并行处理和数据采集。具体目标3侧重于制定SLOS的治疗干预措施 通过抑制7-DHC氧化固醇的形成。由于自由基和酶促氧化作用都与 由于7-DHC氧固醇在体内的形成，抑制这两种途径的方法将在一个 SLOS大鼠模型。将测定氧固醇水平、基因表达和脂质组，以评估有效的 这些疗法的功效。 该研究有望有助于阐明7-DHC衍生的oxys- 固醇在SLOS的病理生理学中的作用，最终通过检查 血液脂质体的SLOS患者与IM-MS，并奠定了基础的联合治疗， 在补充胆固醇的同时刺激7-DHC氧固醇的形成。关于基因表达的知识 和脂质体和本研究产生的治疗方法预计将有显着的改善， 对其他与胆固醇生物合成或代谢异常有关的疾病，如X连锁 显性点状软骨发育不良（CDPX 2）、腱性黄瘤病（CTX）和自闭症。