A precursor‐inducible zebrafish model of acute protoporphyria with hepatic protein aggregation and multiorganelle stress

Abstract

Protoporphyria is a metabolic disease that causes excess production of protoporphyrin IX (PP-IX), the final biosynthetic precursor to heme. Hepatic PP-IX accumulation may lead to end-stage liver disease. We tested the hypothesis that systemic administration of porphyrin precursors to zebrafish larvae results in protoporphyrin accumulation and a reproducible nongenetic porphyria model. Retro-orbital infusion of PP-IX or the iron chelator deferoxamine mesylate (DFO), with the first committed heme precursor α-aminolevulinic acid (ALA), generates high levels of PP-IX in zebrafish larvae. Exogenously infused or endogenously produced PP-IX accumulates preferentially in the liver of zebrafish larvae and peaks 1 to 3 d after infusion. Similar to patients with protoporphyria, PP-IX is excreted through the biliary system. Porphyrin accumulation in zebrafish liver causes multiorganelle protein aggregation as determined by mass spectrometry and immunoblotting. Endoplasmic reticulum stress and induction of autophagy were noted in zebrafish larvae and corroborated in 2 mouse models of protoporphyria. Furthermore, electron microscopy of zebrafish livers from larvae administered ALA + DFO showed hepatocyte autophagosomes, nuclear membrane ruffling, and porphyrin-containing vacuoles with endoplasmic reticulum distortion. In conclusion, systemic administration of the heme precursors PP-IX or ALA + DFO into zebrafish larvae provides a new model of acute protoporphyria with consequent hepatocyte protein aggregation and proteotoxic multiorganelle alterations and stress.-Elenbaas, J. S., Maitra, D., Liu, Y., Lentz, S. I., Nelson, B., Hoenerhoff, M. J., Shavit, J. A., Omary, M. B. A precursor-inducible zebrafish model of acute protoporphyria with hepatic protein aggregation and multiorganelle stress.

Loss of hepatocyte β-catenin protects mice from experimental porphyria-associated liver injury

“…Cytosolic (K8/K18) and nuclear (Lamin A/C, Lamin B1) IF proteins have been reported to be highly susceptible to porphyrin-mediated aggregation. 11,12,24,25 Indeed, DDC caused upregulation of both K8 and K18 in both control+DDC and β-catenin DsiRNA+DDC livers as previously described 4 (Fig. 6A, 6B, upper panel, and Fig.…”

“…Given that recent studies have shown the unique role of porphyrins in causing protein aggregation, 11,12,24 we next analyzed the extent of protein aggregation after DDC. SDS-PAGE and Coomassie blue staining show changes in the protein banding pattern as a function of DDC (Fig.…”

“…Previous studies showed that porphyrins cause ER stress through a non-canonical pathway involving ER protein aggregation. 11,12 Since IRE1a is essential in activating the unfolded protein response during ER stress, we analyzed its expression before and after DDC. Under basal conditions, we saw increased IRE1a expression in β-catenin DsiRNA-treated livers (Fig.…”

“…10 Protein aggregation hinders clearance, causing proteasomal inhibition and induction of autophagy. 11,12 Because of crystallized porphyrin biliary plugs, DDC also results in ductular reaction, pericholangitis and periductal fibrosis, similar to that seen in patients with primary sclerosing cholangitis. 13…”

Molecular Characterization, 3D Modeling of Grass Carp Autophagy Related 16 Protein Like 1 (ATG16L1)

“…Most sequences in NCBI database were predicted based on bioinformatic analysis. In bony fishes, only the sequence of zebrafish (danio rerio) [11] was cloned. Interestingly, similar to yeast homology, zebrafish ATG16L1 is lack of WD40-repeat domain but predicted ATG16L1 sequence of other bony fishes such as common carp (cyprinus carpio) not.…”