Zebrafish models for large-scale genetic screens in dyslipidemia and atherosclerosis Validation and application

Sammanfattning: Hundreds of loci have been robustly associated with circulating lipids, atherosclerosis and coronary artery disease; but for most loci the causal genes and mechanisms remain uncharacterized. The overall aim of my thesis is to develop and validate novel in vivo model systems that are suitable for high-throughput, image-based genetic screens in coronary artery disease and related traits, and use these model systems to systematically characterize positional candidate genes.In Study I, I developed an experimental pipeline to validate the suitability of zebrafish larvae as a model system for systematic, large-scale characterization of drugs and genes associated with dyslipidemia and atherosclerosis. Using this pipeline, I showed that five days of overfeeding and cholesterol supplementation have independent pro-atherogenic effects in zebrafish larvae, which could be diminished by concomitant treatment with atorvastatin and ezetimibe. CRISPR-Cas9-induced mutations in orthologues of proof-of-concept genes resulted in higher LDL cholesterol levels (apoea), and more early stage atherosclerosis (apobb.1). Finally, the pipeline helped me to identify putative causal genes for circulating lipids and early-stage atherosclerosis (LPAR2 and GATAD2A).In Study II, I characterized cardiometabolic traits in apoc2 mutant zebrafish larvae and found that, similar to humans, larvae with two non-functional apoc2 alleles have higher whole-body levels of triglycerides and total cholesterol, and more vascular lipid deposition than larvae without mutations in apoc2. Interestingly, apoc2 mutant larvae also had lower glucose levels after adjusting for triglyceride levels, suggesting that therapeutic stimulation of apoc2 to prevent hypertriglyceridemia may result in hyperglycemia. Still, zebrafish larvae with mutations in apoc2 can be a useful model to identify and characterize additional causal genes for triglyceride metabolism.In Study III, I examined the effects of mutations in pcsk9 on atherosclerosis and diabetes-related traits in nearly 5,000 zebrafish larvae. Similar to the loss-of-function mutations in PCSK9 in humans, larvae with mutations in pcsk9 had lower LDLc levels and were protected from early-stage atherosclerosis. Interestingly, mutations in pcsk9 also resulted in fewer pancreatic β-cells in 10 days old larvae, which suggests the higher risk of diabetes in humans with mutations in PCSK9 may result from a direct effect on the beta cell.Based on these large-scale proof-of-concept studies, my thesis confirms that zebrafish larvae can be used for large-scale, systematic genetic screens in dyslipidemia and early-stage atherosclerosis.