Oxysterols in SLOS Neurodevelopment: Pathological Role and Therapy

Smith-Lemli-Opitz综合征(SLOS)是一种神经发育障碍，由最常见的 胆固醇生物合成的先天错误在3-羟基类固醇-7-还原酶(β-β-Δ-7-Reductase，DHR7)的步骤中。这一缺陷 导致胆固醇水平下降和其前体7-脱氢胆固醇(7-DHC)在 受影响的个体。SLOS表型表现为多发性先天性畸形，神经缺陷， 以及自闭症行为。SLOS的传统治疗方法是补充胆固醇，加或不加辛伐他汀- 他丁，但这些方法并不能改善患者的神经缺陷。最新的发现表明，氧化剂- 7-DHC的代谢产物--氧甾醇是SLOS发病的重要因素，但其致病机制尚不清楚。 这些氧化甾醇在SLOS神经发育中的逻辑作用还没有得到系统的研究，这是 这一项目有望填补的空白。中心假设是7-DHC衍生的氧化甾醇是 系统性红斑狼疮患者神经发育缺陷的影响因素这个项目的长期目标是阐明 神经发育过程中胆固醇稳态紊乱的序列，并开发能够 改善神经功能缺陷。在目标1中，7-DHC衍生的氧化甾醇在神经原中的作用机制 Esis将使用来自WT和Dhcr7基因敲除(KO)小鼠的神经祖细胞(NPC)来阐明 以及来自WT和SLOS的人类诱导的多能干细胞(IPSCs)。Dhcr7KO对神经原损伤的影响 Esis将与7-DHC氧合甾醇的效果进行比较。7-DHC氧合甾醇的蛋白质靶点将被拉出 使用这些氧化类固醇的合成标记类似物。在目标2中，7-DHC氧化甾醇对 神经发育将使用WT和Dhcr7-KO小鼠模型在体内确定。Dhcr7的影响 KO和氧固醇在体内神经发生中的作用将通过免疫组织化学进行比较。时态 并将分析WT和Dhcr7-KO脑中甾醇、氧化甾醇和其他脂类的空间分布 通过质谱学(MS)技术，例如AIM 3中的高分辨率离子迁移率-MS和成像MS， 血脑屏障通透性小分子对SLOS患者神经功能缺陷的疗效将是 使用动物模型和来源于SLOS IPSCs的NPC进行评估。这里的假设是神经学的 SLOS的缺陷可以通过抗氧化剂抑制7-DHC衍生的氧化甾醇的形成来改善 和/或使用Hedgehog(HH)信号通路激动剂来抵消它们的影响，因为一些7-DHC 氧固醇拮抗HH信号转导。此外，血液和成纤维细胞中的类固醇、氧化类固醇和其他脂类 来自正在进行的抗氧化剂临床试验的SLOS患者的样本将被表征，旨在确定双 评估SLOS严重程度和治疗效果的征兆。这个项目代表了一个新的角度来 了解SLOS神经发生缺陷的分子机制并开发治疗方法 SLOS通过靶向7-DHC衍生的氧化甾醇。从这个项目中获得的知识可望从中受益 与胆固醇生物合成或代谢异常有关的其他疾病。

Smith-Lemli-Opitz syndrome (SLOS) is a neurodevelopmental disorder that is caused by the most common inborn error of cholesterol biosynthesis at the step of 3β-hydroxysterol-Δ7-reductase (DHCR7). This defect leads to decreased levels of cholesterol and accumulation of its precursor, 7-dehydrocholesterol (7-DHC), in affected individuals. SLOS phenotype manifests as multiple congenital malformations, neurological defects, and autistic behavior. Conventional therapy for SLOS is supplementation of cholesterol, with or without simvas- tatin, but these approaches do not improve neurological defects in patients. Recent findings suggested oxida- tive metabolites of 7-DHC, oxysterols, are important contributors to the pathogenesis of SLOS, but the patho- logical roles of these oxysterols in SLOS neurodevelopment have not been systematically studied, which is the gap that this project is expected to fill. The central hypothesis is that 7-DHC-derived oxysterols are causative factors for neurodevelopmental defects in SLOS. The long-term goals of this project are to elucidate the con- sequences of disrupted cholesterol homeostasis during neurodevelopment and to develop therapies that can ameliorate the neurological defects. In Aim 1, mechanisms of action of 7-DHC-derived oxysterols in neurogen- esis will be elucidated using neural progenitor cells (NPCs) derived from WT and Dhcr7-knock out (KO) mice and from WT and SLOS human induced pluripotent stem cells (iPSCs). The effects of Dhcr7 KO on neurogen- esis will be compared with the effects of 7-DHC oxysterols. Protein targets of 7-DHC oxysterols will be pulled down using synthetic tagged analogs of these oxysterols. In Aim 2, consequences of 7-DHC oxysterols on neural development will be determined in vivo using WT and Dhcr7-KO mouse models. The effects of Dhcr7 KO and those of oxysterols on neurogenesis in vivo will be compared using immunohistochemistry. Temporal and spatial distribution of sterols, oxysterols, and other lipids in both WT and Dhcr7-KO brains will be analyzed by mass spectrometry (MS) techniques, such as high-resolution ion mobility-MS and imaging MS. In Aim 3, effectiveness of blood-brain-barrier-permeable small molecules against neurological defects in SLOS will be evaluated using animal models and NPCs derived from SLOS iPSCs. The hypothesis here is that neurological defects in SLOS can be ameliorated by inhibiting the formation of 7-DHC-derived oxysterols with antioxidants and/or counteracting their effects using agonists of Hedgehog (Hh) signaling pathway because some 7-DHC oxysterols antagonize Hh signaling. In addition, sterols, oxysterols, and other lipids in blood and fibroblast samples of SLOS patients from an ongoing antioxidant clinical trial will be characterized, aiming to identify bi- omarkers for assessing SLOS severity and therapy effectiveness. This project represents a new angle to understand the molecular mechanisms underlying the neurogenesis defects in SLOS and develop therapies for SLOS by targeting 7-DHC-derived oxysterols. The knowledge obtained from this project is expected to benefit other diseases associated with abnormal cholesterol biosynthesis or metabolism.