[P198]: Melatonin stimulates RHO/ROCK activity downstream the PKC pathway and increases microfilament organization in growth cones in N1E‐115 cells

Abstract

Introduction: Melatonin the main product secreted by the pineal gland has been shown to increase neurite formation in N1E-115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) stimulation. Besides microtubule and intermediate filament reorganization, neurite formation involves microfilament rearrangements in microspikes, lamellipodia and filopodia to form growth cones in the neurite growing tips. In this work, we studied the effect of melatonin on growth cone formation and the possible participation of Rho associated kinase (ROCK) a downstream kinase in the PKC signaling pathway.Methods: N1E-115 cells were incubated for 6 h with either the vehicle, 1 nM melatonin, 2 nM of the PKC activator phorbol-12-myristate-13acetate (PMA), or with melatonin or PMA in the presence of the PKC Inhibitor, bisindolylmaleimide (5 M), the Rho inhibitor C3 (100 ng/ml) or the ROCK inhibitor, Y27632 (10 M) and melatonin or PMA. Microfilaments organized in growth cones were stained with rhodamine-phalloidin and observed by fluorescence microscopy. ROCK activity was determined in cells cultured and in whole cell extracts with the mentioned compounds by using the ROCK substrate long S6 peptide.Results: The results showed that melatonin caused an increase in the number of cells with growth cones. Similar results were obtained with the PKC activator PMA. While, the PKC inhibitor bisindolylmaleimide, Rho inhibitor C3, or the ROCK inhibitorY27632, abolished the microfilament reorganizations elicited by either melatonin or PMA. Both ROCK and PKC activity measured in whole cell extracts and in N1E-115 cells were increased in the presence of melatonin and PMA.Discussion: The results indicate that ROCK activity is stimulated by melatonin downstream the PKC pathway in N1E-115 cells and in whole cell extracts. Also, data suggest that melatonin induces activation of PKC which in turn activates RHO/ROCK pathway increasing growth cone formation that precludes neurite enlargement elicited by melatonin. Acknowledgement

Melatonin potentiates running wheel‐induced neurogenesis in the dentate gyrus of adult C3H/HeN mice hippocampus

“…In our cell survival protocol, melatonin was given in the first 12 days of the experiment, and it is unlikely to affect the subsequent 28‐day survival stage directly through its free‐radical scavenging metabolic pathway. Second, melatonin enhances dendrite maturation and complexity of hippocampal newborn neuron to facilitate the incorporation of newly generated neuron in the hippocampal circuitry, which is important to the neuron survival process . Third, melatonin treatment increases BDNF, an important factor in regulating neurotrophic mechanism in the brain .…”