Activation of autophagy, observed in liver tissues from patients with Wilson disease and from Atp7b-deficient animals, protects hepatocytes from copper-induced apoptosis

Elena V Polishchuk, Assunta Merolla, Josef Lichtmannegger, Alessia Romano, Alessia Indrieri, Ekaterina Y Ilyechova, Mafalda Concilli, Rossella De Cegli, Roberta Crispino, Marta Mariniello, Raffaella Petruzzelli, Giusy Ranucci, Raffaele Iorio, Federico Pietrocola, Claudia Einer, Sabine Borchard, Andree Zibert, Hartmut H Schmidt, Elia Di Schiavi, Ludmila V PuchkovaBrunella Franco, Guido Kroemer, Hans Zischka, Roman S Polishchuk

Research output: Contribution to journalArticle

Abstract

BACKGROUND AND AIMS: Wilson disease is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in liver and brain. It is caused by mutations in the ATPase copper transporting beta gene (ATP7B), which encodes a protein that transports copper out of heaptocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to reduce copper toxicity in patients with Wilson disease.

METHODS: We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b-/- and Atp7b+/- (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with Wilson disease in Italy and liver tissues from patients without Wilson disease (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, and apoptosis assays, as well as by electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy.

RESULTS: Following exposure to copper, ATP7B-knockout cells had significant increases in expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3), compared with wild-type cells. Electron and confocal microscopy revealed more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells following copper exposure. Hepatocytes in liver tissues from patients with Wilson disease, as well as Atp7b-/- mice and rats (but not controls), had multiple autophagosomes. In ATP7B-knockout cells, mTOR had reduced activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB (TFEB) to the nucleus, and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated death of ATP7B-knockout HepG2 cells, compared to wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death.

CONCLUSION: ATP7B-deficient hepatocytes, such as in those in patients with Wilson disease, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might reduce copper toxicity in patients with Wilson disease.

Original languageEnglish
JournalGastroenterology
DOIs
Publication statusE-pub ahead of print - Nov 16 2018

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    Polishchuk, E. V., Merolla, A., Lichtmannegger, J., Romano, A., Indrieri, A., Ilyechova, E. Y., Concilli, M., De Cegli, R., Crispino, R., Mariniello, M., Petruzzelli, R., Ranucci, G., Iorio, R., Pietrocola, F., Einer, C., Borchard, S., Zibert, A., Schmidt, H. H., Di Schiavi, E., ... Polishchuk, R. S. (2018). Activation of autophagy, observed in liver tissues from patients with Wilson disease and from Atp7b-deficient animals, protects hepatocytes from copper-induced apoptosis. Gastroenterology. https://doi.org/10.1053/j.gastro.2018.11.032