Effects of dopamine on LC3-II activation as a marker of autophagy in a neuroblastoma cell model.
Dopamine at 100-500 microM has toxic effects on human SH-SY5Y neuroblastoma cells, manifested as apoptotic cell loss and strong autophagy. The molecular mechanisms and types of dopamine-induced cell death are not yet well known. Their identification is important in the study of neurodegenerative diseases that specifically involve dopaminergic neurons. We looked for changes in expression and content of proteins involved in apoptosis and autophagy after dopamine treatment. All the changes found were prevented by avoiding dopamine oxidation with N-acetylcysteine, indicating a key role for the products of dopamine oxidation in dopamine toxicity. As early as 1-2h after treatment we found an increase in hypoxia-inducible factor-1alpha (HIF-1alpha) and an accumulation of ubiquitinated proteins. Proteins regulated by HIF-1alpha and involved in apoptosis and/or autophagy, such as p53, Puma and Bnip3, were subsequently increased. However, apoptotic parameters (caspase-3, caspase-7, PARP) were only activated after 12h of 500muM dopamine treatment. Autophagy, monitored by the LC3-II increase after LC3-I linkage to autophagic vacuoles, was evident after 6h of treatment with both 100 and 500 microM dopamine. The mTOR pathway was inhibited by dopamine, probably due to the intracellular redox changes and energy depletion leading to AMPK activation. However, this mechanism is not sufficient to explain the high LC3-II activation caused by dopamine: the LC3-II increase was not reversed by IGF-1, which prevented this effect when caused by the mTOR inhibitor rapamycin. Our results suggest that the aggregation of ubiquitinated non-degraded proteins may be the main cause of LC3-II activation and autophagy. As we have reported previously, cytosolic dopamine may cause damage by autophagy in neuroblastoma cells (and presumably in dopaminergic neurons), which develops to apoptosis and leads to cell degeneration.
Giménez-Xavier
Pol
P
Unitat de Bioquímica, Dep. Ciències Fisiològiques II, Facultat de Medicina, Campus Universitari de Bellvitge - IDIBELL, Universitat de Barcelona, Barcelona, Spain.
Francisco
Roser
R
Santidrián
Antonio F
AF
Gil
Joan
J
Ambrosio
Santiago
S
D000704:Analysis of Variance; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004298:Dopamine / Q000494:pharmacology*; D015259:Dopamine Agents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000473:pathology / Q000648:ultrastructure; D051058:Oncogene Protein v-akt / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases
D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D015853:Cysteine Proteinase Inhibitors; D004298:Dopamine; D015259:Dopamine Agents; D005452:Fluoresceins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007976:Leupeptins; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D051058:Oncogene Protein v-akt; D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D058570:TOR Serine-Threonine Kinases; C438378:BBC3 protein, human; C477012:MAP1LC3A protein, human; C498702:PMAIP1 protein, human; C102215:brain injury-derived neurotrophic peptide; C063261:acetylleucyl-leucyl-norleucinal; C029569:diacetyldichlorofluorescein; C546842:MTOR protein, human