Impaired Redox and Protein Homeostasis as Risk Factors and Therapeutic Targets in Toxin-Induced Biliary Atresia.

BACKGROUND & AIMS: Extrahepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA. METHODS: We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screening of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model, with subsequent validation. RESULTS: Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors and other cyclic guanosine monophosphate signaling activators worked synergistically with the glutathione precursor N-acetylcysteine in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. Phosphodiesterase-5 inhibitors enhanced proteasomal degradation and required intact HSP90 chaperone. CONCLUSION: Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone and may account for the reported association between BA transplant-free survival and glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with N-acetylcysteine and cyclic guanosine monophosphate signaling enhancers warrants further investigation as therapy for BA.

The cardiovascular manifestations of Alagille syndrome and JAGI mutations.

Alagille syndrome is an autosomal-dominant disorder characterized by hepatic, cardiac, ocular, skeletal, and facial abnormalities. The disease gene, Jaggedl (JAG]), was identified by molecular analyses of chromosomal alterations involving chromosome 20p. Total gene deletions (3-7%) and intragenic mutations (70%) of JAG] have been identified in Alagille patients. Identifying JAG] mutations is challenging, given its size of 26 exons. Methods to identify both whole-gene deletions and intragenic mutations of JAG] are described in detail, including fluorescence in situ hybridization (FISH), conformation-sensitive gel electrophoresis (CSGE), and complementary DNA (cDNA) sequencing.