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Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis.
Gastroenterology. 2019 03; 156(4):1173-1189.e5.G

Abstract

BACKGROUND & AIMS

Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase copper transporting β gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD.

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 WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and 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

After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b-/- mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB 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 the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death.

CONCLUSION

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

Authors+Show Affiliations

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy; ITMO University, St. Petersburg, Russia; Institute of Biosciences and Bioresources CNR, Italy.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy.Institute of Molecular Toxicology and Pharmacology, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy; Department of Translational Medical Science, "Federico II" University of Naples, Naples, Italy.ITMO University, St. Petersburg, Russia; Department of Molecular Genetics, Institute of Experimental Medicine, St. Petersburg, Russia.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy.Division of Metabolism, IRCCS Bambino Gesù Children's Hospital, Rome, Italy.Department of Translational Medical Science, "Federico II" University of Naples, Naples, Italy.Equipe 11 labellisée Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Institut National de la Santé et de la Recherche Médicale, UMR1138, Equipe labellisée Ligue Nationale Contre le Cancer, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie, Paris, France; Cell Biology and Metabolomics Platforms, Gustave Roussy Cancer Campus, Villejuif, France.Institute of Molecular Toxicology and Pharmacology, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany.Institute of Molecular Toxicology and Pharmacology, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany.Klinik für Transplantationsmedizin, Universitätsklinikum Münster, Münster, Germany.Klinik für Transplantationsmedizin, Universitätsklinikum Münster, Münster, Germany.Institute of Biosciences and Bioresources CNR, Italy.ITMO University, St. Petersburg, Russia.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy; Department of Translational Medical Science, "Federico II" University of Naples, Naples, Italy.Equipe 11 labellisée Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Institut National de la Santé et de la Recherche Médicale, UMR1138, Equipe labellisée Ligue Nationale Contre le Cancer, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie, Paris, France; Cell Biology and Metabolomics Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France; Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden.Institute of Molecular Toxicology and Pharmacology, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany; Institute of Toxicology and Environmental Hygiene, Technical University Munich, Munich, Germany.Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy. Electronic address: mpolish@tigem.it.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30452922

Citation

Polishchuk, Elena V., et al. "Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis." Gastroenterology, vol. 156, no. 4, 2019, pp. 1173-1189.e5.
Polishchuk EV, Merolla A, Lichtmannegger J, et al. Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis. Gastroenterology. 2019;156(4):1173-1189.e5.
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. (2019). Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis. Gastroenterology, 156(4), 1173-e5. https://doi.org/10.1053/j.gastro.2018.11.032
Polishchuk EV, et al. Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis. Gastroenterology. 2019;156(4):1173-1189.e5. PubMed PMID: 30452922.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis. AU - Polishchuk,Elena V, AU - Merolla,Assunta, AU - Lichtmannegger,Josef, AU - Romano,Alessia, AU - Indrieri,Alessia, AU - Ilyechova,Ekaterina Y, AU - Concilli,Mafalda, AU - De Cegli,Rossella, AU - Crispino,Roberta, AU - Mariniello,Marta, AU - Petruzzelli,Raffaella, AU - Ranucci,Giusy, AU - Iorio,Raffaele, AU - Pietrocola,Federico, AU - Einer,Claudia, AU - Borchard,Sabine, AU - Zibert,Andree, AU - Schmidt,Hartmut H, AU - Di Schiavi,Elia, AU - Puchkova,Ludmila V, AU - Franco,Brunella, AU - Kroemer,Guido, AU - Zischka,Hans, AU - Polishchuk,Roman S, Y1 - 2018/11/17/ PY - 2018/03/14/received PY - 2018/10/23/revised PY - 2018/11/10/accepted PY - 2018/11/20/pubmed PY - 2019/4/4/medline PY - 2018/11/20/entrez KW - Copper KW - Copper Homeostasis KW - Metal Toxicity KW - Mitophagy SP - 1173 EP - 1189.e5 JF - Gastroenterology JO - Gastroenterology VL - 156 IS - 4 N2 - BACKGROUND & AIMS: Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase copper transporting β gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD. 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 WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and 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: After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b-/- mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB 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 the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death. CONCLUSION: ATP7B-deficient hepatocytes, such as in those in patients with WD, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might decrease copper toxicity in patients with WD. SN - 1528-0012 UR - https://www.unboundmedicine.com/medline/citation/30452922/Activation_of_Autophagy_Observed_in_Liver_Tissues_From_Patients_With_Wilson_Disease_and_From_ATP7B_Deficient_Animals_Protects_Hepatocytes_From_Copper_Induced_Apoptosis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0016-5085(18)35280-6 DB - PRIME DP - Unbound Medicine ER -