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.

The proposed research is expected contribute to the understanding of the roles of 7-dehydrocholesterol- derived oxysterols in the pathogenesis of neurodevelopmental defects in Smith-Lemli-Opitz syndrome (SLOS) – the most common cholesterol biosynthesis disorder, and the development of new therapies that can ameliorate the detrimental effects of these oxysterols. This project relates to the mission of NICHD toward understanding intellectual and developmental disabilities, particularly disorders of the nervous system that affect brain function and development.