BACKGROUND and AIMS: Extra-hepatic 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 screen 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 (PDE5i) and other cGMP signaling activators worked synergistically with the glutathione precursor Nacetylcysteine (NAC) in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. PDE5i enhanced proteasomal degradation and required intact HSP90 chaperone. CONCLUSION: Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone, and mirrors recently reported BA risk stratification linked to glutathione metabolism gene expression. Human BA can be causatively linked to genetic
Extra-hepatic biliary atresia (BA) is an important pediatric liver disease of unknown etiology. The identification of biliatresone, a plant electrophile with selective toxicity for extra-hepatic cholangiocytes (EHC) and linked to outbreaks of epidemic BA in livestock, demonstrated the feasibility of an environmental trigger for this enigmatic condition. Here, we show that susceptibility of zebrafish EHC to biliatresone arises from their inability to restore stress-induced depletion of glutathione, and that heterozygous mutations in glutathione metabolism genes differentially sensitize EHC and the normally resistant intra-hepatic cholangiocytes (IHC) to biliatresone-mediated injury. Collectively these findings argue that genetic variants in stressresponse genes could be risk factors for human BA. Supporting this idea, we identified de novo loss-of-function mutations in the STIP1 and REV1 gene, both part of the HSP90 pathway, in two children with BA, respectively, and showed that modulation of their expression sensitized zebrafish larvae and human cholangiocytes to biliatresone. Using an in vivo drug screening assay, we identified activators of cGMP signaling as potent enhancers of the anti-oxidant Nacetylcysteine (NAC) in biliatresone-treated larvae and human cholangiocytes that act independently of glutathione and upstream of chaperone-mediated protein quality control (PQC) pathways. Collectively, these data highlighted novel aspects of cholangiocyte injury responses, identified new genetic risk factors for BA, and suggest combined treatment with NAC and cGMP signaling modulators as a rational therapeutic strategy for BA. University of Pennsylvania School of Medicine (Philadelphia, PA) zebrafish facility in accord with protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee.Generation of targeted mutations in zebrafish using CRISPR/Cas9 system. The target sequences for gclm, abcc2, stip1 were identified using the CHOPCHOP website: http://chopchop.cbu.uib. no/. The single-guide RNA (sgRNA) was generated as described(70). Cas9 protein were obtained from PNABIO. Approximately 60 to 120 pg of sgRNA and 180 pg of Cas9 protein were coinjected into WT embryos at the one-cell stage. Genomic DNAs was extracted from pools of 20 one-day old embryos, and the targeted areas were amplified and sequenced using primers listed in Supplementary Table 1 to confirm somatic mutations. Injected embryos were raised to adulthood (F0) and screened for the germline-transmitting founders. The F0 founders were subsequently outcrossed with WT fish to obtain the heterozygous F1 and F2 offspring. All experiments were performed on the animals of F3 or subsequent generations. The gclm, abcc2 mutants were genotyped using primers listed in Table S1. The gsr and stip1 mutant fish were genotyped using the KASP genotyping assays (KBioscience).Redox-Sensitive GFP Redox Mapping Double transgenic (Tg(ef1a:Grx-roGFP; tp1:mCherry)) larvae in gclm, abcc2, gsr genetic background were used for the redox mapping of hep...
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