ATVB presentation 5-10-2019.pptx

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. In addition to cholesterol, Hmgcr activity is also required for the synthesis of non-sterol isoprenoids, such as heme A, dolichol, ubiquinone, farnesylated and geranylgeranylated proteins. The goal of this project is to investigate the effects of Hmgcr inhibition on non-sterol isoprenoids in the liver, in order to better understand the physiological regulation and function of the mevalonate pathway. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using viral delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and liver regeneration. We observed compensatory upregulation of genes in the mevalonate pathway, likely a response to sterol depletion. At the biochemical level, we observed decreased levels of sterol intermediates and cholesterol. In parallel, we also detected decreased production of the non-sterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr null hepatocytes showed expanded and swollen ER and upregulation of Chop, suggestive of ER stress. Given the important role for N-linked glycosylation in protein folding in the ER, we hypothesized that depletion of dolichol, a scaffold for synthesis of N-linked glycans, could be responsible for the hepatocyte ER stress. To test this, we used AAV-CRISPR to somatically disrupt Dehydrodolichyl diphosphate synthase subunit (Dhdds), a branch point enzyme required for the synthesis of dolichol. Dhdds null livers showed severe hepatocyte apoptosis and regeneration, along with significant induction of Chop, partially mimicking the phenotype observed in Hmgcr null livers. Taken together, our data show a critical role for mevalonate-derived dolichol in the liver, and suggest that dolichol depletion is at least partially responsible for ER-stress induced apoptosis upon potent Hmgcr inhibition.