Elena V Polishchuk1, Assunta Merolla2, Josef Lichtmannegger3, Alessia Romano2, Alessia Indrieri4, Ekaterina Y Ilyechova5, Mafalda Concilli2, Rossella De Cegli2, Roberta Crispino2, Marta Mariniello2, Raffaella Petruzzelli2, Giusy Ranucci6, Raffaele Iorio7, Federico Pietrocola8, Claudia Einer3, Sabine Borchard3, Andree Zibert9, Hartmut H Schmidt9, Elia Di Schiavi10, Ludmila V Puchkova11, Brunella Franco4, Guido Kroemer12, Hans Zischka13, Roman S Polishchuk14. 1. Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy; ITMO University, St. Petersburg, Russia; Institute of Biosciences and Bioresources CNR, Italy. 2. Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy. 3. Institute of Molecular Toxicology and Pharmacology, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany. 4. Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy; Department of Translational Medical Science, "Federico II" University of Naples, Naples, Italy. 5. ITMO University, St. Petersburg, Russia; Department of Molecular Genetics, Institute of Experimental Medicine, St. Petersburg, Russia. 6. Division of Metabolism, IRCCS Bambino Gesù Children's Hospital, Rome, Italy. 7. Department of Translational Medical Science, "Federico II" University of Naples, Naples, Italy. 8. 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. 9. Klinik für Transplantationsmedizin, Universitätsklinikum Münster, Münster, Germany. 10. Institute of Biosciences and Bioresources CNR, Italy. 11. ITMO University, St. Petersburg, Russia. 12. 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. 13. 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. 14. Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy. Electronic address: mpolish@tigem.it.
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.
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: Cortnie Hartwig; Gretchen Macías Méndez; Shatabdi Bhattacharjee; Alysia D Vrailas-Mortimer; Stephanie A Zlatic; Amanda A H Freeman; Avanti Gokhale; Mafalda Concilli; Erica Werner; Christie Sapp Savas; Samantha Rudin-Rush; Laura Palmer; Nicole Shearing; Lindsey Margewich; Jacob McArthy; Savanah Taylor; Blaine Roberts; Vladimir Lupashin; Roman S Polishchuk; Daniel N Cox; Ramon A Jorquera; Victor Faundez Journal: J Neurosci Date: 2020-11-18 Impact factor: 6.167
Authors: Daniel J Klionsky; Giulia Petroni; Ravi K Amaravadi; Eric H Baehrecke; Andrea Ballabio; Patricia Boya; José Manuel Bravo-San Pedro; Ken Cadwell; Francesco Cecconi; Augustine M K Choi; Mary E Choi; Charleen T Chu; Patrice Codogno; Maria Isabel Colombo; Ana Maria Cuervo; Vojo Deretic; Ivan Dikic; Zvulun Elazar; Eeva-Liisa Eskelinen; Gian Maria Fimia; David A Gewirtz; Douglas R Green; Malene Hansen; Marja Jäättelä; Terje Johansen; Gábor Juhász; Vassiliki Karantza; Claudine Kraft; Guido Kroemer; Nicholas T Ktistakis; Sharad Kumar; Carlos Lopez-Otin; Kay F Macleod; Frank Madeo; Jennifer Martinez; Alicia Meléndez; Noboru Mizushima; Christian Münz; Josef M Penninger; Rushika M Perera; Mauro Piacentini; Fulvio Reggiori; David C Rubinsztein; Kevin M Ryan; Junichi Sadoshima; Laura Santambrogio; Luca Scorrano; Hans-Uwe Simon; Anna Katharina Simon; Anne Simonsen; Alexandra Stolz; Nektarios Tavernarakis; Sharon A Tooze; Tamotsu Yoshimori; Junying Yuan; Zhenyu Yue; Qing Zhong; Lorenzo Galluzzi; Federico Pietrocola Journal: EMBO J Date: 2021-08-30 Impact factor: 14.012
Authors: Antonella Capuozzo; Sandro Montefusco; Vincenzo Cacace; Martina Sofia; Alessandra Esposito; Gennaro Napolitano; Eduardo Nusco; Elena Polishchuk; Maria Teresa Pizzo; Maria De Risi; Elvira De Leonibus; Nicolina Cristina Sorrentino; Diego Luis Medina Journal: Mol Ther Date: 2022-02-02 Impact factor: 12.910
Authors: Eva J Ge; Ashley I Bush; Angela Casini; Paul A Cobine; Justin R Cross; Gina M DeNicola; Q Ping Dou; Katherine J Franz; Vishal M Gohil; Sanjeev Gupta; Stephen G Kaler; Svetlana Lutsenko; Vivek Mittal; Michael J Petris; Roman Polishchuk; Martina Ralle; Michael L Schilsky; Nicholas K Tonks; Linda T Vahdat; Linda Van Aelst; Dan Xi; Peng Yuan; Donita C Brady; Christopher J Chang Journal: Nat Rev Cancer Date: 2021-11-11 Impact factor: 69.800
Authors: Tiffany Tsang; Xingxing Gu; Caroline I Davis; Jessica M Posimo; Zoey A Miller; Donita C Brady Journal: Mol Cancer Res Date: 2022-07-06 Impact factor: 6.333