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Melatonin stimulates dendrite formation and complexity in the hilar zone of the rat hippocampus: participation of the Ca++/Calmodulin complex.

Domínguez-Alonso A, Valdés-Tovar M, Solís-Chagoyán H, Benítez-King G - Int J Mol Sci (2015)

Bottom Line: We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices.Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction.Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Neurofarmacología, Subdirección de Investigaciones Clínicas, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Calzada México-Xochimilco No. 101, Col. San Lorenzo-Huipulco, CP 14370 Tlalpan, DF, Mexico. aline.dmgzalonso@gmail.com.

ABSTRACT
Melatonin (MEL), the main product synthesized by the pineal gland, stimulates early and late stages of neurodevelopment in the adult brain. MEL increases dendrite length, thickness and complexity in the hilar and mossy neurons of hippocampus. Dendrite formation involves activation of Ca2+/Calmodulin (CaM)-dependent kinase II (CaMKII) by CaM. Previous work showed that MEL increased the synthesis and translocation of CaM, suggesting that MEL activates CaM-dependent enzymes by this pathway. In this work we investigated whether MEL stimulates dendrite formation by CaMKII activation in organotypic cultures from adult rat hippocampus. We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices. Also, PKC inhibition abolished dendritogenesis, while luzindole, an antagonist of MEL receptors (MT1/2), partially blocked the effects of MEL. Moreover, autophosphorylation of CaMKII and PKC was increased in presence of MEL, as well as phosphorylation of ERK1/2. Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction. Dendritogenesis elicited by MEL also required PKC activation, and signaling through MT1/2 receptors was partially involved. Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII.

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Relative content of phosphorylated Ca2+/CaM Kinase II, Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2 in hippocampal slices incubated with Melatonin. Hippocampal slices were incubated for 3 h with either the vehicle (VEH) or 100 nM melatonin (MEL). CaMKII and Thr286-phospho-CaMKII (P-CaMKII), PKC and phospho-PKC (P-PKC) as well as ERK1/2 and phospho-ERK1/2 (P-ERK1/2) were determined by Western blot. Upper panels show immunodetection of GAPDH as load control. Representative fluorograms of total and phosphorylated enzymes revealed by ECL are shown below. Histograms correspond to densitometric analysis of bands depicted immediately above. Results are expressed as the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisks indicate significant differences (p < 0.05).
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ijms-16-01907-f004: Relative content of phosphorylated Ca2+/CaM Kinase II, Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2 in hippocampal slices incubated with Melatonin. Hippocampal slices were incubated for 3 h with either the vehicle (VEH) or 100 nM melatonin (MEL). CaMKII and Thr286-phospho-CaMKII (P-CaMKII), PKC and phospho-PKC (P-PKC) as well as ERK1/2 and phospho-ERK1/2 (P-ERK1/2) were determined by Western blot. Upper panels show immunodetection of GAPDH as load control. Representative fluorograms of total and phosphorylated enzymes revealed by ECL are shown below. Histograms correspond to densitometric analysis of bands depicted immediately above. Results are expressed as the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisks indicate significant differences (p < 0.05).

Mentions: Upon activation, PKC autophosphorylates in two different sites [51]. Similarly, Ca2+/CaM activation of CaMKII is followed by its intramolecular autophosphorylation in the threonine 286 residue [31,32] and ERK1/2 is phosphorylated after PKC activation in the hippocampus [52]. Therefore, to demonstrate that these enzymes are involved in dendritogenesis elicited by MEL, the relative levels of Thr286-phospho-CaMKII, phospho-PKC, and phospho-ERK1/2 were determined by Western blot. As shown in Figure 4, an increase of 32% in the phospho-kinase/total kinase ratio of CaMKII was evident in hippocampal slices incubated with 100 nM MEL. Also in this condition, the corresponding ratios of PKC and ERK1/2 were increased by 60% and 27% respectively. No increase in the amount of total CaMKII, PKC, ERK1/2 or GAPDH was evident (Figure 4 upper panels). CaMKII participation in the mechanism of action by which MEL increases dendritogenesis was demonstrated by blocking the enzyme activity with KN-62. However, because KN-62 may block the activity of various CaM kinases [32], autophosphorylation of CaMKII was determined to corroborate its activation by MEL treatment. Data clearly showed that the amount of autophosphorylated CaMKII was increased, supporting that MEL activates this enzyme. Similarly, an increase in both autophosphorylated PKC and phosphoERK1/2 was observed in hippocampal slices incubated with MEL, indicating that both enzymes are activated in the process of dendritogenesis elicited by this indoleamine.


Melatonin stimulates dendrite formation and complexity in the hilar zone of the rat hippocampus: participation of the Ca++/Calmodulin complex.

Domínguez-Alonso A, Valdés-Tovar M, Solís-Chagoyán H, Benítez-King G - Int J Mol Sci (2015)

Relative content of phosphorylated Ca2+/CaM Kinase II, Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2 in hippocampal slices incubated with Melatonin. Hippocampal slices were incubated for 3 h with either the vehicle (VEH) or 100 nM melatonin (MEL). CaMKII and Thr286-phospho-CaMKII (P-CaMKII), PKC and phospho-PKC (P-PKC) as well as ERK1/2 and phospho-ERK1/2 (P-ERK1/2) were determined by Western blot. Upper panels show immunodetection of GAPDH as load control. Representative fluorograms of total and phosphorylated enzymes revealed by ECL are shown below. Histograms correspond to densitometric analysis of bands depicted immediately above. Results are expressed as the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisks indicate significant differences (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307341&req=5

ijms-16-01907-f004: Relative content of phosphorylated Ca2+/CaM Kinase II, Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2 in hippocampal slices incubated with Melatonin. Hippocampal slices were incubated for 3 h with either the vehicle (VEH) or 100 nM melatonin (MEL). CaMKII and Thr286-phospho-CaMKII (P-CaMKII), PKC and phospho-PKC (P-PKC) as well as ERK1/2 and phospho-ERK1/2 (P-ERK1/2) were determined by Western blot. Upper panels show immunodetection of GAPDH as load control. Representative fluorograms of total and phosphorylated enzymes revealed by ECL are shown below. Histograms correspond to densitometric analysis of bands depicted immediately above. Results are expressed as the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisks indicate significant differences (p < 0.05).
Mentions: Upon activation, PKC autophosphorylates in two different sites [51]. Similarly, Ca2+/CaM activation of CaMKII is followed by its intramolecular autophosphorylation in the threonine 286 residue [31,32] and ERK1/2 is phosphorylated after PKC activation in the hippocampus [52]. Therefore, to demonstrate that these enzymes are involved in dendritogenesis elicited by MEL, the relative levels of Thr286-phospho-CaMKII, phospho-PKC, and phospho-ERK1/2 were determined by Western blot. As shown in Figure 4, an increase of 32% in the phospho-kinase/total kinase ratio of CaMKII was evident in hippocampal slices incubated with 100 nM MEL. Also in this condition, the corresponding ratios of PKC and ERK1/2 were increased by 60% and 27% respectively. No increase in the amount of total CaMKII, PKC, ERK1/2 or GAPDH was evident (Figure 4 upper panels). CaMKII participation in the mechanism of action by which MEL increases dendritogenesis was demonstrated by blocking the enzyme activity with KN-62. However, because KN-62 may block the activity of various CaM kinases [32], autophosphorylation of CaMKII was determined to corroborate its activation by MEL treatment. Data clearly showed that the amount of autophosphorylated CaMKII was increased, supporting that MEL activates this enzyme. Similarly, an increase in both autophosphorylated PKC and phosphoERK1/2 was observed in hippocampal slices incubated with MEL, indicating that both enzymes are activated in the process of dendritogenesis elicited by this indoleamine.

Bottom Line: We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices.Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction.Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Neurofarmacología, Subdirección de Investigaciones Clínicas, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Calzada México-Xochimilco No. 101, Col. San Lorenzo-Huipulco, CP 14370 Tlalpan, DF, Mexico. aline.dmgzalonso@gmail.com.

ABSTRACT
Melatonin (MEL), the main product synthesized by the pineal gland, stimulates early and late stages of neurodevelopment in the adult brain. MEL increases dendrite length, thickness and complexity in the hilar and mossy neurons of hippocampus. Dendrite formation involves activation of Ca2+/Calmodulin (CaM)-dependent kinase II (CaMKII) by CaM. Previous work showed that MEL increased the synthesis and translocation of CaM, suggesting that MEL activates CaM-dependent enzymes by this pathway. In this work we investigated whether MEL stimulates dendrite formation by CaMKII activation in organotypic cultures from adult rat hippocampus. We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices. Also, PKC inhibition abolished dendritogenesis, while luzindole, an antagonist of MEL receptors (MT1/2), partially blocked the effects of MEL. Moreover, autophosphorylation of CaMKII and PKC was increased in presence of MEL, as well as phosphorylation of ERK1/2. Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction. Dendritogenesis elicited by MEL also required PKC activation, and signaling through MT1/2 receptors was partially involved. Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII.

Show MeSH
Related in: MedlinePlus