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Dolichol is a nonsterol isoprenoid derived from the mevalonate pathway. Dehydrodolichyl diphosphate synthase subunit (DHDDS) is a branch point enzyme that - in complex with Nogo-B receptor (NgBR) - catalyzes the first committed step of dolichol biosynthesis. Next, dolichol is phosphorylated, and used as carrier for oligosaccharides, which are transferred to nascent proteins in the N-linked glycosylation pathway. A growing body of evidence highlights the importance of N-linked glycosylation in regulating plasma lipid levels in humans. However, the underlying molecular mechanisms are largely unexplored. Therefore, the goal of this project is to define the role of dolichol biosynthesis in lipid metabolism in the liver, the major organ for the biosynthesis and clearance of lipoproteins. We used Adeno-Associated Virus (AAV)-mediated delivery of CRISPR/Cas9 (AAV-CRISPR) to delete Dhdds in mouse liver, to inhibit the hepatic biosynthesis of dolichol. Mice were injected with AAV-CRISPR or saline as control, and fed a high-fat diet for 4 to 12 weeks. Surprisingly, Dhdds liver-specific knockout (LSKO) mice gained less weight compared to control mice. Plasma total cholesterol and triglyceride levels were also reduced over time in Dhdds LSKO mice. Moreover, we observed less cholesterol content in the high-density lipoprotein (HDL) fractions as well as less triglycerides in very-low-density lipoproteins (VLDL). At the cellular level, Dhdds LSKO mice showed hepatic endoplasmic reticulum (ER) stress and decreased N-linked glycosylation. In addition, we observed significant downregulation of Sterol Regulatory Element-Binding Protein 1c ( Srebp-1c ) - a key regulator of de novo lipogenesis (DNL) - and downstream-regulated genes in the DNL pathway. Conversely, the expression of genes regulating cholesterol biosynthesis was unaffected. Taken together, our data show an important role for hepatic dolichol biosynthesis in regulating plasma lipids, potentially through modulation of Srebp-1c expression and DNL.

While the intestine is the critical interface between cholesterol absorption and excretion, surprisingly little is known about the role of de novo cholesterol synthesis in this organ and how it affects whole body cholesterol homeostasis. The mevalonate pathway is most well-known for the production of cholesterol, but it is also required for the production of essential non-sterol isoprenoids. 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway, is regulated by a three-protein complex made up of INSIG, SCAP, and SREBP2. Intestine specific knockouts of Scap and Srebp2 both result in severe enteropathy and reduced mouse survival. Here, we assessed the hypothesis that Hmgcr is required for enterocyte viability. Mice harboring floxed alleles for Hmgcr were bred with the Villin-Cre transgene to specifically knock out this enzyme in the villus and crypt epithelial cells of the small intestine (i-KO). The i-KO mice are viable through adulthood and fertile. Hmgcr is efficiently deleted based on analysis via drop digital PCR and qPCR. RNA sequencing shows reduction in all SREBP2 target genes throughout the mevalonate pathway in intestinal epithelial cells. Lipidomics confirms substantial reductions in abundance of all sterol and non-sterol isoprenoids, except 7-dehydrocholesterol and cholesterol. Cholesterol is likely maintained through reabsorption of biliary cholesterol or increased uptake from the circulation. Circulating cholesterol levels and cholesterol absorption are not altered in i-KO mice, but triglyceride absorption is increased through compensatory changes in bile acid composition and intestinal growth. At the cellular level, the intestine rapidly compensates for loss of Hmgcr via dramatic expansion of the stem cell compartment within the crypts. In conclusion, the mechanisms by which the intestine compensates for genetic loss of Hmgcr include altered triglyceride absorption, bile acid composition, increased absorptive surface area, and expansion of the resident stem cell compartment. Together these studies provide insight into the effects of HMGCR knockout in intestinal development and lipid metabolism.

Background: Current methods to study genetic contributions to atherosclerosis are time-consuming and expensive. The Ldlr and Apoe knockout (KO) models are currently the gold-standard, but require extensive backcrossing to homozygosity and congenic status on the C57BL6/J background. More rapid and efficient methods are needed to investigate the ever-growing list of candidate genes identified through human genetics.The C lustered R egularly- I nterspaced S hort P alindromic R epeats/Cas9 (CRISPR/Cas9) genome editing system is a powerful new tool for gene disruption. Hypothesis: Liver-directed somatic disruption of Ldlr using an all-in-one AAV-CRISPR vector can generate atherosclerotic lesions in adult mice, thereby avoiding the need to cross to Ldlr KO mice. Methods: An Adeno-Associated Viral (AAV) vector based on serotype 8 was used to deliver Staphylococcus aureus Cas9 (SaCas9) and small guide RNA (gRNA) targeting the Ldlr gene (AAV-CRISPR). This vector was compared head-to-head with AAV-mediated overexpression of a human gain-of-function variant of PCSK9 , a recent model that mimics Ldlr KO through promoting degradation of the Ldlr protein. Adult C57BL6/J mice received either: 1 ) saline, 2 ) AAV-CRISPR, or 3 ) AAV-hPCSK9, versus 4 ) germline Ldlr KO mice. Animals were placed on a Western diet for twenty weeks and followed for changes in plasma lipids, atherosclerotic lesion burden, and editing efficiency. Results and Conclusion: Disruption of Ldlr with AAV-CRISPR vector was robust, resulting in severe hypercholesterolemia and atherosclerosis on a standard western diet. AAV-hPCSK9 also produced atherosclerotic lesions as expected from previous reports. Lesion burden was slightly lower with AAV-CRISPR and AAV-hPCSK9 relative to germline Ldlr KO mice. Unexpected sexual dimorphism was also observed with AAV-CRISPR, with the greatest effects seen in male mice. In summary, our all-in-one AAV-CRISPR vector is a rapid and efficient alternative to the use of Ldlr KO mice to study atherosclerosis.

Hmg-CoA Reductase (Hmgcr) catalyzes the conversion of Hmg-CoA to mevalonate, which is the rate-limiting step in the cholesterol biosynthetic pathway. Hmgcr is the target of statins, which are the front line therapy for hypercholesterolemia. Statin-responsiveness varies greatly between individuals, and liver toxicity and transaminase elevations are observed in 1-3% of patients. In addition to cholesterol, Hmgcr activity is also required for the synthesis of heme A, dolichol, ubiquinone (Coenzyme Q), farnesylated and geranylgeranylated proteins, isopentenyl tRNA, and vitamin K2. Surprisingly, very little is known about the effects of statins on these non-sterol isoprenoids in vivo , which may be essential for hepatocyte viability and function. The goal of this project is to investigate the effects of Hmgcr inhibition in the liver, in order to better understand the physiological regulation and function of the mevalonate pathway. We have developed a new inducible genetic model of Hmgcr deletion, which enables us acutely inhibit the mevalonate pathway in the liver, thus avoiding well-known compensatory responses to statins in rodents. Hmgcr floxed mice were injected with liver-specific AAV-Cre recombinase (AAV-Cre) and followed for 1, 2, 4 and 8 weeks. We observed massive apoptosis followed by a burst of hepatocyte proliferation at 2 weeks post-injection. We also observed compensatory upregulation of genes in the mevalonate pathway, likely a response to sterol depletion. Hepatocytes from AAV-Cre mice had expanded and swollen endoplasmic reticulum (ER), and upregulation of Chop and Bax, suggesting ER stress-induced apoptosis. Genetic analysis of AAV-Cre treated livers showed progressive loss of the null allele over time, indicating a selective advantage for hepatocytes that escaped complete Hmgcr deletion. The injection of a five-fold higher dose of AAV-Cre was acutely lethal in less than 2 weeks, confirming that liver Hmgcr activity is essential for survival. This new model will be used to identify the essential mevalonate-derived products in the liver, and the molecular pathways underlying statin hepatotoxicity and responsiveness.