PROJECT SUMMARY/ABSTRACT In humans, mutations in the ABCB11 gene impair bile salt export pump (BSEP) function and cause progressive familial intrahepatic cholestasis type II (PFIC2), which is a fast-progressing and often-fatal liver disease in infancy. Liver transplantation remains as the only curable treatment for many PFIC2 patients. The main challenge for developing alternative therapy is that there is no known mechanism allowing restoration of bile excretion in the absence of BSEP. Our long-term goal is to discover molecular targets to treat cholestatic liver diseases. The overall objective for this application is to identify mechanisms that can be activated to restore bile excretion in the context of BSEP deficiency. Based on the preliminary data generated using the abcb11b mutant zebrafish, the central hypothesis is that alternative transporters can be prompted to excrete bile acids in BSEP-deficient hepatocytes. The rationale for the proposed research is that, finding the alternative bile salt transporter and delineating the molecular mechanisms triggering its bile excretion function will facilitate the designing of targeted therapies to treat cholestasis caused by BSEP deficiency. Identifying novel strategies to modulate bile secretion will also benefit the patients with other cholestatic liver diseases. The central hypothesis will be tested by the experiments proposed in three complementary specific aims: 1) Determine the alternative transporter that can be prompted to excrete bile acids in BSEP-deficient hepatocytes; 2) Define the cellular and molecular mechanisms that can be activated to restore bile excretion in BSEP-deficient hepatocytes; and 3) Identify genetic modifiers that restore bile excretion in BSEP-deficient hepatocytes. The first aim will test the hypothesis that recovering the localization of another ABC transporter MDR1 to the bile canaliculus allows it to assume bile excretion function in BSEP-deficient hepatocytes. The second aim will investigate whether augmentation of autophagy represents a novel mechanism to restore bile excretion in hepatocytes with BSEP deficiency. The third aim will utilize an unbiased whole-genome sequencing approach to identify genetic modifiers of BSEP-deficiency phenotype severity. The research proposed in this application is highly innovative, because it uses the zebrafish model that offers unique advantages for studying bile excretion, and abcb11b mutant zebrafish is the first animal model in which loss of BSEP results in severe perturbation of bile excretion as seen in patients with PFIC2. The proposed research is significant, because it is expected to reveal strategies to restore bile excretion in BSEP-deficient hepatocytes and uncover new therapeutic targets for treating cholestatic liver diseases.

PROJECT NARRATIVE The proposed research is relevant to public health because the discovery of mechanisms that can be activated to restore bile excretion in the context of BSEP deficiency is expected to inform new therapies for neonatal cholestasis. Thus, the proposed research is relevant to the part of NIH's mission that pertains to seeking fundamental knowledge that will enhance health, lengthen life, and reduce illness and disability. !