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

描述(申请人提供)：候选人。我的博士后研究集中在脂质过氧化的速度和机制上，因为它与许多人类神经疾病密切相关，如阿尔茨海默氏症、帕金森氏症、亨廷顿病等。随着我发现7-脱氢胆固醇(7-DHC)是迄今已知的最容易氧化的脂质分子，我的研究重点转移到了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(同时胆固醇水平下降)。随后发现了20种新的氧化甾醇，它们是7-DHC在溶液中、细胞内和体内氧化的产物。在溶液中形成的7-DHC氧化甾醇被发现具有细胞毒性，并诱导细胞内有害的基因表达变化。我在K99阶段的短期目标(具体目标1)是在接受神经科学和尖端离子迁移率-质谱仪(IM-MS)方面的细胞和分子生物学培训的同时，研究体内形成的7-DHC氧合甾醇对SLOS细胞模型中基因表达和脂类(脂体)的生物学作用。我在R00阶段(具体目标2和3)的目标是在我现有和新获得的技能的基础上，将同一套研究扩展到SLOS的动物模型。我长期的职业目标是应用我在化学结构、反应性、机理、合成和分析方面的知识来理解与脂质相关的生物学。 治疗涉及脂代谢异常的人类疾病的过程和发展的翻译方法。环境问题。我的指导委员会由五位杰出的导师和协作者/顾问组成，他们在脂质过氧化、神经科学、质谱学、脂类组学、SLOS、胆固醇代谢、基因表达等方面拥有免费的专业知识。培训机构范德比尔特大学拥有丰富的智力和体力资源，包括与拟议研究密切相关的机构，如范德比尔特化学生物研究所(VICB)和范德比尔特肯尼迪中心，全系列核心实验室，以及指定的职业发展办公室。VICB在脂类研究方面有一个强大的合作小组，可以进行咨询和建立新的合作。总体而言，我的指导委员会和学校的承诺，以及范德比尔特丰富的学术环境，将确保我的培训计划和拟议的研究成功实施。这项研究。SLOS是一种常染色体隐性代谢性疾病，由先天性胆固醇生物合成错误引起。SLOS表现出广泛的表型，包括多发性先天性畸形、神经缺陷、智力低下和行为问题。超过50%的SLOS儿童表现出自闭症样行为。系统性红斑狼疮的传统治疗方法是补充胆固醇，但结果不一致，也存在争议。目前缺乏针对7-DHC衍生代谢物的研究，这是拟议中的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潜在的分子和病理生理机制中的关键致病因素。在特定的目标1和2中，将用定量聚合酶链式反应检测基因表达，用IM-MS分析SLOS细胞和/或动物模型中的脂体，以检测7-DHC氧合甾醇的生物活性。IM-MS是一种快速的二维分离技术，它根据迁移率、漂移时间和质荷比在微秒到毫秒内拆分生物分子离子。IM-MS技术在脂肪组学研究中的应用是创新的，因为这种方法需要最少的生物材料，并且在样品处理和数据采集方面效率很高。具体目标3侧重于通过抑制7-DHC氧化甾醇的形成来开发SLOS的治疗干预措施。由于体内7-DHC氧合甾醇的形成与自由基和酶促氧化有关，因此将在SLOS大鼠模型中探索抑制这两种途径的方法。将通过检测氧固醇水平、基因表达和脂质体来评估这些治疗的有效性。这项研究有望有助于阐明7-DHC衍生的氧化甾醇在SLOS的病理生理机制中的作用，最终通过检测患有IM-MS的SLOS患者的血脂脂体，最终导致一种快速而彻底的诊断方法，并为在补充胆固醇的同时抑制7-DHC氧合甾醇的形成而为综合治疗奠定基础。这项研究产生的关于基因表达和脂质体的知识以及治疗方法有望对其他与胆固醇生物合成或代谢异常有关的疾病产生重大影响，如X连锁显性点状软骨发育不良(CDPX2)、脑腱黄色瘤病(CTX)和自闭症。 与公共健康相关：拟议的研究有望丰富我们对7-脱氢胆固醇衍生的氧固醇在Smith-Lemli-Opitz综合征(SLOS)病理生理学中的作用的理解，SLOS是一种影响神经系统发育的胆固醇生物合成障碍，并通过抑制这些氧固醇的形成来开发治疗方法。该项目涉及NICHD了解智力和发育障碍，特别是影响大脑功能和发育的新陈代谢障碍的使命。

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.