Project Summary Nonalcoholic fatty liver disease (NAFLD) is an alarming public health concern and now considered the most common liver disease in the Western world. Patients with NAFLD may develop nonalcoholic steatohepatitis (NASH) of which may develop hepatic injury that may progress to liver cirrhosis. We have shown that activation of the alpha-calcitonin gene-related peptide (CGRP)/Calcitonin receptor-like receptor (CRLR) axis plays a key role in cholangiocyte proliferation induced by biliary damage during cholestasis in animal models. Recent evidence and our novel preliminary data indicate that cholangiocytes play a key role in the pathogenesis of NAFLD/NASH through activation of biliary damage, ductular reaction (DR), senescence, and subsequent liver fibrosis. Our preliminary data showing that the CGRP/CRLR axis is upregulated in cholangiocytes in animal models of NAFLD/NASH (high-fat diet (HFD) and methionine and choline-deficient (MCD) hepatoxic diet) and human liver samples with NAFLD and NASH support the concept that the CGRP/CRLR axis plays a key role in the progression of NAFLD and NASH phenotypes. Based upon these findings, we propose the central hypothesis that the CGRP/CRLR axis signaling is critical for mediating DR and activated profibrogenic biliary phenotype that triggers HSC activation, as well as steatosis in hepatocytes contributing to the progression of hepatic fibrosis during NAFLD/NASH. To test our hypothesis, two Specific Aims are proposed: (i) HFD and MCD hepatoxic diet-induced activation of the CGRP/CRLR axis stimulates DR, biliary senescence triggering the subsequent steatosis and fibrosis during NAFLD/NASH; and (ii) Therapeutic inhibition of the CGRP/CRLR axis and downstream pathways attenuates the activated neuroendocrine/profibrogenic biliary phenotype hepatic steatosis and fibrosis during the progression of NAFLD/NASH. The proposed studies will be performed in cell specific CRLR knockout mice models as well as in vitro studies in human NAFLD/NASH cholangiocytes cell lines and 3D organoids. The activation of the CGRP/CRLR axis and downstream pathways will be correlated in human samples of NAFLD/NASH. Successful completion of the proposed studies will provide a translational mechanism of how activation of the CGRP/CLR axis mediates DR and hepatobiliary fibrosis during the progression of NAFLD/NASH. Our study will also provide insight for novel therapeutic approaches for NAFLD/NASH and other liver diseases characterized by ductular reaction and hepatobiliary fibrosis.