Copper dopamine complex induces mitochondrial autophagy preceding caspase-independent apoptotic cell death.
Parkinsonism is one of the major neurological symptoms in Wilson disease, and young workers who worked in the copper smelting industry also developed Parkinsonism. We have reported the specific neurotoxic action of copper dopamine complex in neurons with dopamine uptake. Copper dopamine complex (100 microm) induces cell death in RCSN-3 cells by disrupting the cellular redox state, as demonstrated by a 1.9-fold increase in oxidized glutathione levels and a 56% cell death inhibition in the presence of 500 microm ascorbic acid; disruption of mitochondrial membrane potential with a spherical shape and well preserved morphology determined by transmission electron microscopy; inhibition (72%, p < 0.001) of phosphatidylserine externalization with 5 microm cyclosporine A; lack of caspase-3 activation; formation of autophagic vacuoles containing mitochondria after 2 h; transfection of cells with green fluorescent protein-light chain 3 plasmid showing that 68% of cells presented autophagosome vacuoles; colocalization of positive staining for green fluorescent protein-light chain 3 and Rhod-2AM, a selective indicator of mitochondrial calcium; and DNA laddering after 12-h incubation. These results suggest that the copper dopamine complex induces mitochondrial autophagy followed by caspase-3-independent apoptotic cell death. However, a different cell death mechanism was observed when 100 microm copper dopamine complex was incubated in the presence of 100 microm dicoumarol, an inhibitor of NAD(P)H quinone:oxidoreductase (EC 1.6.99.2, also known as DT-diaphorase and NQ01), because a more extensive and rapid cell death was observed. In addition, cyclosporine A had no effect on phosphatidylserine externalization, significant portions of compact chromatin were observed within a vacuolated nuclear membrane, DNA laddering was less pronounced, the mitochondria morphology was more affected, and the number of cells with autophagic vacuoles was a near 4-fold less.
Paris
Irmgard
I
Programme of Molecular and Clinical Pharmacology, Faculty of Medicine, Casilla 70000, Santiago 7, Chile; Department of Basic Sciences, Universidad Santo Tomás, 2561780 Viña del Mar, Chile; Department of Biological Sciences, Universidad Andrés Bello, 2561156 Viña del Mar, Chile.
Perez-Pastene
Carolina
C
Programme of Molecular and Clinical Pharmacology, Faculty of Medicine, Casilla 70000, Santiago 7, Chile.
Couve
Eduardo
E
Department of Biology, University of Valparaiso, Casilla 5030, Valparaiso, Chile.
Caviedes
Pablo
P
Programme of Molecular and Clinical Pharmacology, Faculty of Medicine, Casilla 70000, Santiago 7, Chile.
LeDoux
Susan
S
Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, Alabama 36608.
Segura-Aguilar
Juan
J
Programme of Molecular and Clinical Pharmacology, Faculty of Medicine, Casilla 70000, Santiago 7, Chile. Electronic address: jsegura@med.uchile.cl.
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D003300:Copper / Q000009:adverse effects* / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D001728:Dicumarol / Q000494:pharmacology; D004298:Dopamine / Q000009:adverse effects* / Q000494:pharmacology; D015259:Dopamine Agents / Q000009:adverse effects* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors; D016273:Occupational Exposure / Q000009:adverse effects; D010302:Parkinson Disease, Secondary / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D051381:Rats; D014617:Vacuoles / Q000378:metabolism / Q000473:pathology
D015259:Dopamine Agents; D004791:Enzyme Inhibitors; D003300:Copper; D001728:Dicumarol; D016572:Cyclosporine; D016660:NAD(P)H Dehydrogenase (Quinone); D020169:Caspases; D004298:Dopamine