Абстрактный

Melatonin precludes cytoskeletal collapse caused by hydrogen peroxide: participation of protein kinase C

Gloria Ben�?­tez-King, Leonardo Ortiz-L�?³pez and Graciela Jim�?©nez-Rubio

Background: Oxidative stress is a hallmark of aging and most neurodegenerative diseases such as Alzheimers, Parkinsons, and diffuse Lewy body diseases. Hydrogen peroxide (H2O2) causes free radical generation and apoptosis similar to that present in neurogenerative diseases. It has been thought that H2O2 disorganizes the cortical actin cytoskeleton and, in brain cell extracts, inhibits actin polymerization. Moreover, microtubule network disruption has also been observed in cortical neurons exposed to high levels of free radicals. Melatonin, the main product secreted by the pineal gland, protects the neuronal cells by its action as a free radical scavenger. In addition, this antioxidant inhibits apoptosis and induces microtubule and microfilament reorganization as well as neurite formation in neuronal cultured cells. Objective: In this work, we characterized the effects of H2O2 on neurite structure and studied the effects of melatonin on cytoskeletal organization in N1E- 115 cells damaged by H2O2. Methods: Melatonin treatment and H2O2 effects on neurite cytoskeletal arrangements were evaluated by measuring both the major axis cell length or by simultaneous immunofluorescence staining of microtubules and microfilaments. Free radical levels and cell death were also quantified by measuring lipid peroxides and the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) method, respectively. Results: The results demonstrated that H2O2 causes loss of neurites and a cytoskeletal retraction toward the perinuclear region. Melatonin precluded microfilament and microtubule collapse in N1E-115 cells, as well as the increased lipid peroxidation and apoptosis caused by H2O2 and restores neurite formation, microtubule enlargement and microfilament organization in microspikes and growth cones in cells damaged with H2O2. The results presented here support the idea that an intracellular melatonin mechanism, not mediated by melatonin membrane receptors participates in the mechanisms by which melatonin precludes cytoskeletal damage caused by free radicals. Data show that protein kinase C participates in this mechanism. The protein kinase C agonist, phorbol 12-myristate 13-acetate, caused cytoskeletal reorganization in the presence of H2O2, while the protein kinase C inhibitor, bisindolylmaleimide, blocked neurite formation and microfilament reorganization elicited by melatonin. In addition, the calmodulin antagonist, ophiobolin was not capable of protecting cells against the damage caused by H2O2. However, phorbol 12-myristate 13-acetate and ophiobolin resembled the melatonin effects in cells treated with H2O2. A cytoskeleton organized in neurites and a network all over the cytoplasm was observed. In contrast, the melatonin receptor antagonist did not abolish the protective effects of melatonin against the damage caused by H2O2. Conclusions: The results presented here support the fact that melatonin may be useful in the treatment of neurodegenerative diseases by restoring microspikes, lamellipodia, growth cones and microtubule enlargement to form neurites that will eventually re-establish synaptic connectivity and an improvement of cognition.