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

候选人。我的博士后研究主要集中在脂质过氧化的速度和机制上 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、狩猎- 随着我的发现，7-脱氢胆固醇(7-DHC)是最容易氧化的脂质 到目前为止，我的研究重点转移到Smith-Lemli-Opitz综合征(SLOS)，因为明显的 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) 病人。随后发现了20个新的氧化甾醇作为7-DHC在溶液中的氧化产物， 在细胞内和体内。在溶液中形成的7-DHC氧化甾醇被发现具有细胞毒性和诱导有害基因 细胞中的表达发生变化。我在K99阶段的短期目标(具体目标1)是研究生物- 体内形成的7-DHC氧合甾醇对细胞基因表达和脂类(脂质体)的逻辑作用 SLOS的小叶模型，同时接受神经科学的细胞和分子生物学培训，以及切割 边缘离子迁移率质谱仪(IM-MS)。我在R00阶段的目标(具体目标2和3)是 在我现有的和新学到的技能基础上，将相同的研究扩展到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、机理、合成、 和分析，以了解与脂质相关的生物学过程，并开发翻译方法- 关注涉及脂类代谢异常的人类疾病。 环境问题。我的指导委员会由五位杰出的导师和合作伙伴组成- 在脂质过氧化、神经科学、质谱学、唇部- 免疫组学、SLOS、胆固醇代谢、基因表达等。 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研究所- 与GY(VICB)和范德比尔特肯尼迪中心密切相关的建议研究，全线核心 实验室和指定的职业发展办公室。VICB拥有一个强大的协作小组， 脂类研究，可供咨询和建立新的合作。总体而言，承诺 来自我的指导委员会和学院，以及范德比尔特丰富的学术环境，将 确保成功实施我的培训计划和拟议的研究。 这项研究。SLOS是一种常染色体隐性代谢性疾病，由先天错误引起 胆固醇的生物合成。SLOS表现出广泛的表型，包括多发性先天性畸形- 形体、神经缺陷、智力低下和行为问题。超过50%的SLOS儿童 表现出类似自闭症的行为。系统性红斑狼疮的传统治疗方法是补充胆固醇，但结果 是不一致的，也是有争议的。目前缺乏对7-DHC衍生代谢物的研究，这是 这项拟议的研究有望填补这一空白。该项目的中心假设是7-DHC衍生 氧化甾醇是SLOS潜在的分子和病理生理机制中的关键致病因素。在……里面 针对目标1和2，基因的表达将通过定量聚合酶链式反应进行分析，脂质体的分析将通过IM-MS进行。 建立SLOS细胞和/或动物模型，检测7-DHC氧合甾醇的生物活性。IM-MS是一种快速的 基于迁移率漂移时间和迁移率的生物分子离子的二维分离技术 质量-电荷比在微秒到毫秒内。IM-MS技术在脂质组学研究中的应用 创新是因为这种方法需要最少的生物材料，并且在SAM-2中是有效的 PLE处理和数据采集。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。因为自由基氧化和酶促氧化同时存在。 由于7-DHC氧合甾醇在体内的形成，抑制这两个途径的方法将在 SLOS大鼠模型。将通过检测氧固醇水平、基因表达和脂质体来评估其有效性-- 对这些疗法了如指掌。 这项拟议的研究有望有助于阐明7-DHC衍生的氧基-2，4-二羟基-4-羟基-4-羟基-4-羟基-4，4，4，4，4，6-二羟基-4，4，4，6-二羟基-4-羟基-4，4，4，4，4，4，4，4-二苯基-4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4， Terol在SLOS的病理生理学中的作用，最终导致一种快速而彻底的诊断方法 对系统性红斑狼疮患者进行IM-MS的血脂检测，为通过抑制-MS进行联合治疗奠定了基础。 在补充胆固醇的同时，促进7-DHC氧化甾醇的形成。关于基因表达的知识 这项研究产生的脂质体和治疗方法有望对人类健康产生重大影响。 对其他与胆固醇生物合成或代谢异常有关的疾病，如X-Lined 显性点状软骨发育不良(CDPX2)、脑腱黄瘤病(CTX)和自闭症。

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.