Oxysterols in SLOS Neurodevelopment: Pathological Role and Therapy

Smith-Lemli-Opitz综合征（SLOS）是一种神经发育障碍， 胆固醇生物合成的先天性缺陷在3β-羟基甾醇-Δ7-还原酶（DHCR 7）步骤。这一缺陷 导致胆固醇水平降低及其前体7-脱氢胆固醇（7-DHC）的积累， 受影响的个人。SLOS表型表现为多种先天性畸形，神经缺陷， 和自闭症行为SLOS的常规治疗是补充胆固醇，有或没有辛伐他汀- 他汀，但这些方法不能改善患者的神经缺陷。最近的研究结果表明，氧化- 7-DHC的活性代谢产物氧化固醇是SLOS发病机制的重要贡献者，但病理性代谢产物氧化固醇是SLOS发病机制的重要贡献者。 这些氧固醇在SLOS神经发育中的逻辑作用尚未系统地研究，这是 这是该项目有望填补的空白。中心假设是7-DHC衍生的氧固醇是致病的 SLOS中神经发育缺陷的因素。该项目的长期目标是阐明该问题， 在神经发育过程中破坏胆固醇稳态的序列，并开发可以 改善神经缺陷。在目的1中，7-DHC-derived oxysterols在神经原中的作用机制， 将使用来自WT和Dhcr 7敲除（KO）小鼠的神经祖细胞（NPC）来阐明Esis 以及来自WT和SLOS人诱导多能干细胞（iPSC）。Dhcr 7基因敲除对神经元生长的影响 将与7-DHC氧化固醇的作用进行比较。7-DHC氧固醇的蛋白质目标将被取消 使用这些氧化固醇的合成标记类似物。在目标2中，7-DHC氧固醇对 使用WT和Dhcr 7-KO小鼠模型在体内测定神经发育。Dhcr 7的影响 KO和氧化固醇对体内神经发生的影响将使用免疫组织化学进行比较。时间 将分析WT和Dhcr 7-KO脑中甾醇、氧甾醇和其它脂质的空间分布 通过质谱（MS）技术，例如高分辨率离子迁移率-MS和成像MS。在Aim 3中， 血脑屏障渗透性小分子对SLOS中神经缺陷的有效性将是 使用动物模型和来源于SLOS iPSC的NPC进行评价。这里的假设是， SLOS的缺陷可以通过用抗氧化剂抑制7-DHC衍生的氧固醇的形成来改善 和/或使用Hedgehog（Hh）信号传导途径的激动剂抵消它们的作用，因为一些7-DHC 氧固醇拮抗Hh信号传导。此外，血液和成纤维细胞中的固醇、氧化固醇和其他脂质 来自正在进行的抗氧化剂临床试验的SLOS患者样本将被表征，旨在识别双- 用于评估SLOS严重程度和治疗有效性的标志物。这个项目代表了一个新的角度， 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衍生的氧固醇。从该项目中获得的知识预计将受益于 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.