Defining mechanisms of lipoprotein turnover and their regulation by ASGR1

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High levels of plasma low-density lipoprotein (LDL) are correlated with an increased risk for cardiovascular disease (CVD). LDL is the smallest apolipoprotein-B containing lipoprotein (B-lp) and it accumulates modifications over time. These B-lp modifications may increase atherogenicity by increasing B-lp adherence to the vasculature and lowering their specificity to the LDL receptor (LDLR). However, the factors that control B-lp time in circulation, their turnover, remain to be fully defined. Current methods to study B-lp turnover rely on limited patient cohorts and require lipoprotein labeling, followed by complex mathematical modeling, that may skew the obtained data. A recent genome-wide association study (GWAS) underscores the importance to study LDL turnover. The GWAS reported lower risk for CVD, but only mildly reduced levels of LDL in individuals with a mutation in the asialoglycoprotein receptor 1 (ASGR1) gene. The reduction of LDL itself was not dramatic enough to account for the magnitude of the reduction in CVD risk. This proposal explores the hypothesis that ASGR1 modulates LDL turnover, a key understudied factor that may powerfully mediate CVD risk. I will use the optically clear zebrafish larva to obtain the first insight into general B-lp turnover in an in vivo, unperturbed context by developing multiple novel optical reporters. I generated and validated a tool to measure B-lp turnover by creating a zebrafish line that expresses the photoconvertible fluorescent protein Dendra2 fused to apolipoprotein B (ApoB). After photoconversion, Dendra2 fluoresces red and the subsequent loss of red fluorescence represents a readout of ApoB and thus B-lp turnover. I hypothesize that the general availability of lipids is a determinant of B-lp turnover and I will investigate this by genetic and dietary perturbations in zebrafish. To study the role of ASGR1 on B-lp metabolism, I identified the zebrafish ortholog of ASGR1 and created a mutant using CRISPR/Cas9. I found that the loss of ASGR1 in zebrafish does not change the total B-lp number or size. However, RNAseq analysis of ASGR1 mutants indicates that ASGR1 loss increases the expression of genes required for B-lp production and uptake. Together, these data are consistent with my hypothesis that the loss of ASGR1 increases B-lp turnover; I will directly test this by using the ApoB-Dendra2 reporter. Previous research suggests that ASGR1 binds LDLR and leads to endocytosis mediated degradation. Hence, I hypothesize that in the absence of ASGR1, LDLR escapes degradation and is more readily available. I will examine the interaction between ASGR1 and LDLR in the wild-type and ASGR1 mutants. The proposed experiments will not only generate a host of powerful new tools but will increase our understanding of B-lp regulation and provide me with exceptional training opportunities. While working on these studies, I will acquire hands-on experience with numerous ground-breaking techniques, while I expand my knowledge of lipid metabolism and CVD. Altogether, the synergy of world-renowned researchers and resources afforded by Johns Hopkins University and the Carnegie Institution create an outstanding environment to support the proposed studies and my Ph.D. training.