title: Effects of copper overload in P19 neurons: impairment of glutathione redox homeostasis and crosstalk between caspase and calpain protease systems in ROS-induced apoptosis.
abstract: Copper, a transition metal with essential biological functions, exerts neurotoxic effects when present in excess. The aim of the present study was to better elucidate cellular and molecular mechanisms of CuSO4 toxicity in differentiated P19 neurons. Exposure to 0.5 mM CuSO4 for 24 h provoked moderate decrease in viability, accompanied with barely increased generation of reactive oxygen species (ROS) and caspase-3/7 activity. Glutathione (GSH) and ATP contents were depleted, lactate dehydrogenase inactivated, and glyceraldehyde-3-phosphate dehydrogenase overexpressed. In severely damaged neurons exposed to only two times higher concentration, classical caspase-dependent apoptosis was triggered as evidenced by marked caspase-3/7 activation and chromatin condensation. Multifold increase in ROS, together with very pronounced ATP and GSH loss, strongly suggests impairment of redox homeostasis. At higher copper concentration protease calpains were also activated, and neuronal injury was prevented in the presence of calpain inhibitor leupeptin through the mechanism that affects caspase activation. MK-801 and nifedipine, inhibitors of calcium entry, and H-89 and UO126, inhibitors of PKA and ERK signaling respectively, exacerbated neuronal death only in severely damaged neurons, while ROS-scavenger quercetin and calcium chelator BAPTA attenuated toxicity only at lower concentration. In a dose-dependent manner copper also provoked transcriptional changes of genes involved in intracellular signaling and induction of apoptosis (p53, c-fos, Bcl-2 and Bax). The obtained results emphasize differences in triggered neuronal-death processes in a very narrow range of concentrations and give further insight into the molecular mechanisms of copper toxicity with the potential to improve current therapeutic approaches in curing copper-related neurodegenerative diseases.
authors.affiliation: Laboratory for Molecular Neuropharmacology, Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, HR-10 000, Zagreb, Croatia, Maja.Jazvinscak.Jembrek@irb.hr.
authors.forename: Maja
authors.initials: M
authors.lastname: Jazvinšćak Jembrek
authors.forename: Josipa
authors.initials: J
authors.lastname: Vlainić
authors.forename: Vedrana
authors.initials: V
authors.lastname: Radovanović
authors.forename: Julija
authors.initials: J
authors.lastname: Erhardt
authors.forename: Nada
authors.initials: N
authors.lastname: Oršolić
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics; D002154:Calpain / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000494:pharmacology; D002843:Chromatin / Q000187:drug effects / Q000648:ultrastructure; D019327:Copper Sulfate / Q000633:toxicity*; D016291:Dizocilpine Maleate / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000096:biosynthesis / Q000235:genetics; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects*; D009543:Nifedipine / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D011480:Protease Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D018243:Teratocarcinoma / Q000473:pathology
chemical_list: D051017:Apoptosis Regulatory Proteins; D002614:Chelating Agents; D002843:Chromatin; D007976:Leupeptins; D009363:Neoplasm Proteins; D011480:Protease Inhibitors; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016291:Dizocilpine Maleate; D000255:Adenosine Triphosphate; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D002154:Calpain; D020169:Caspases; D005978:Glutathione; D009543:Nifedipine; C032854:leupeptin; D019327:Copper Sulfate