PROJECT SUMMARY The goal of the Center for Pediatric Research established at Sanford Research was to establish a multidisciplinary center to support translational research being performed by investigators with broad interests in pediatric disease. The proposed supplement will provide support for a new collaborative project encompassing the laboratories of three separate IDeA institution investigators, including one former project leader from our COBRE (Dr. Kevin Francis) and two investigators currently supported by a separate COBRE established at South Dakota State University (Dr. Natalie Thiex and Dr. Brandon Scott). The project itself is scientifically innovative, expanding upon a recent publication authored by two of the investigators’ laboratories, addressing a novel role for defects in endocytosis within pediatric disorders of cholesterol synthesis. The work proposed has the potential to provide significant contributions to these poorly characterized pediatric diseases. In addition, this project epitomizes a team science-based approach by combining overlapping interests with distinct expertise across each laboratory while utilizing COBRE-supported core facilities. While all three investigators have common interests in lipid regulation of cellular function, their complementary expertise and training will allow a thorough dissection and understanding of the impact of sterol biochemistry on endocytic trafficking with direct relevance to disorders of cholesterol synthesis. Eight known diseases are caused by genetic disruption of cholesterol synthesis enzymes, resulting in loss of cholesterol content and the aberrant expression of cholesterol precursor molecules. These disorders arise during childhood and create life-altering disabilities within affected individuals due to both developmental and functional deficits within affected children. Though these diseases share broad commonalities associated with developmental delay, biochemical deficits, and tissue malformations, disease pathogenesis varies greatly across individuals and thus therapeutic development for these children has been limited. While disease- associated deficits are likely due to combinatorial changes in cell signaling, membrane structure, and cell- specific processes, the precise effects of sterol biochemical changes and the mechanisms whereby cholesterol vs disease-associated sterols regulate membrane biology remain unresolved. Therefore, delineating how clinically relevant sterol biochemistry affects basic cellular functions with disease relevance constitutes an unmet scientific need for affected children. The synthesis of cholesterol from sterol precursors is a highly regulated pathway critical to cell structure and signaling across mammalian cells and tissues. Our recent work detailed a critical role for conserved sterol structural features and expression levels in the regulation of the critical cargo internalization mechanism clathrin-mediated endocytosis (CME). As the intracellular delivery of molecules is vital to maintenance of cell signaling, function, and health, fully defining the impact of disease-associated sterol biochemistry on membrane biology represents a novel biological mechanism which could help explain differences in the pathogenesis of cholesterol synthesis disorders and identify targetable, functional pathways to improve cell health. For this proposal, we will utilize disease-relevant cellular models to test the hypothesis that cellular membranes require highly specific sterol biochemistry to promote functional internalization of macromolecules and lipid ordering. In Aim 1, we will first define the role of clinically relevant sterols on functional internalization of macromolecules, differentiating between disease-specific impacts and defining cell signaling changes specific to immune cells of interest. In Aim 2, we will determine the mechanisms whereby disease-relevant sterol biochemical changes regulate membrane function through super resolution imaging of endocytic processes and recruitment of PtdIns species to endocytic sites. In Aim 3, we will utilize a CRISPR-based whole genome screen and follow-up validation to delineate genes, signaling pathways, and cellular processes that contribute to or compensate for endocytic defects resulting from cholesterol synthesis disruption. In summary, these studies constitute a novel proposal detailing how sterol metabolism regulates mammalian membrane biology with direct correlation to cell function and rare pediatric diseases. We anticipate completion of the proposed work will provide important insight into the regulation of endocytosis by sterol biochemistry with high relevance to this group of lethal pediatric disorders. Additionally, continued support will further foster this cross-institute collaboration and allow the supported investigators to extend these studies in the form of a multi-PI NIH application upon completion of the work proposed.

PROJECT NARRATIVE The Center for Pediatric Research was designed to support investigator-driven pediatric research with the potential for translational applications. This proposal will focus on diseases of cholesterol synthesis, a class of pediatric disorders in which affected individuals exhibit broad clinical phenotypes with limited treatment options due to poorly understood pathogenesis. Recent work and ongoing collaborative studies from the investigators involved have determined that defects in cholesterol synthesis inhibit the ability of cells to internalize macromolecules through endocytic mechanisms. This proposal will determine how sterol biochemistry regulates membrane internalization, will define the mechanisms involved, and will identify compensatory pathways which normalize cell health and function, using a team-science approach to address this complex class of disorders.