Impaired redox and protein homeostasis as risk factors and therapeutic targets in toxin-induced biliary atresia

Abstract

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...

Exome Sequencing in Individuals with Isolated Biliary Atresia

“…Based on the de novo variant found in the human exome data, the identification of the heatshock response pathway as differentially expressed in zebrafish, our collaborators performed a CRISPR/Cas9 based experiment to introduce a frameshift mutation in exon 1 of the zebrafish stip1 gene to create heterozygous mutant fish. When treated with toxin, stip1 heterozygous fish were highly sensitive to a low dose of biliatresone compared to the wild-type 49 . REV1 encodes a protein similar to the S. cerevisiae mutagenesis protein Rev1 and is known to be involved in DNA repair.…”

Exome Sequencing in Individuals with Isolated Biliary Atresia

“…Based on the de novo variant found in the human exome data, along with evidence that the heatshock response pathway is differentially expressed in zebrafish treated with the plant toxin biliatresone, our collaborators used CRISPR/Cas9 methods to introduce a frameshift mutation in the zebrafish stip1 gene. When treated with biliatresone toxin, stip1 heterozygous fish were highly sensitive to a low dose that is normally inactive in wild-type fish 52 . Similar sensitization was detected in human cholangiocytes following siRNA knockdown of the STIP1 isoform targeted by the BA variant 52 .…”

“…When treated with biliatresone toxin, stip1 heterozygous fish were highly sensitive to a low dose that is normally inactive in wild-type fish 52 . Similar sensitization was detected in human cholangiocytes following siRNA knockdown of the STIP1 isoform targeted by the BA variant 52 . Heterozygous rev1 mutation in zebrafish and siRNA knockdown of REV1 in human cholangiocytes were sensitized to low dose biliatresone, similar to mutation and knockdown of stip1/STIP1 52 .…”