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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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Calmodulin content in hippocampal slices treated with Melatonin. Hippocampal slices were incubated during 6 h with either the vehicle (VEH) or melatonin (MEL). Calmodulin (CaM) in the homogenates (A); and in the soluble (SOL) and cytoskeletal fractions (CSK) separated by centrifugation (B); was determined by Western blot. Upper panels show Carbonic Anhydrase (CA) used as external load control and stained with Coomassie blue. Representative fluorograms of CaM are shown immediately below. First lane of both gel and fluorogram from panel A was loaded with pure CA (5 µg) and CaM (1 µg), respectively. CaM was recognized with a specific CaM antibody and ECL. Histograms correspond to densitometric analysis of the bands shown in the upper panels. Results are the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisk indicates significant differences (p < 0.05).
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ijms-16-01907-f005: Calmodulin content in hippocampal slices treated with Melatonin. Hippocampal slices were incubated during 6 h with either the vehicle (VEH) or melatonin (MEL). Calmodulin (CaM) in the homogenates (A); and in the soluble (SOL) and cytoskeletal fractions (CSK) separated by centrifugation (B); was determined by Western blot. Upper panels show Carbonic Anhydrase (CA) used as external load control and stained with Coomassie blue. Representative fluorograms of CaM are shown immediately below. First lane of both gel and fluorogram from panel A was loaded with pure CA (5 µg) and CaM (1 µg), respectively. CaM was recognized with a specific CaM antibody and ECL. Histograms correspond to densitometric analysis of the bands shown in the upper panels. Results are the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisk indicates significant differences (p < 0.05).

Mentions: As mentioned before, one of the crucial conditions for CaMKII activation is the availability of Ca2+/CaM for binding and activating it. Therefore, as a first step to disclose the mechanism by which MEL stimulates the activity of CaMKII we determined CaM levels in total homogenates and in the soluble and cytoskeletal fractions of hippocampal slices incubated with either the vehicle or 100 nM MEL for 6 h. As shown in Figure 5A, a slight increase of 8% in total CaM content was observed after a 6 h-incubation with 100 nM MEL. No changes were observed in the carbonic anhydrase used as external load control (Figure 4, upper panel). Figure 5B shows the subcellular distribution of CaM in the soluble and cytoskeletal fractions of hippocampal slices. In the presence of MEL, a 43% increase in CaM was observed in the soluble fraction with a slight decrease in the cytoskeletal fraction. Data suggest that both an increase in synthesis and translocation might occur in presence of MEL, in agreement with previous observations made in epithelial MDCK cells where the indoleamine induced an increase in both parameters [36]. Moreover, in vitro assays have shown that MEL directly stimulates PKC alpha and phosphorylates CaM. In this regard, it is known that phosphorylation of proteins induces a conformational change that increases the affinity constant for specific protein binding and therefore, they are targeted to specific subcellular compartments [22]. Although our experimental strategy does not allow us to elucidate the mechanisms by which CaM is enriched in the soluble fraction, our data indicates that MEL might increase availability of CaM to activate CaMKII and therefore trigger dendrite formation.


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)

Calmodulin content in hippocampal slices treated with Melatonin. Hippocampal slices were incubated during 6 h with either the vehicle (VEH) or melatonin (MEL). Calmodulin (CaM) in the homogenates (A); and in the soluble (SOL) and cytoskeletal fractions (CSK) separated by centrifugation (B); was determined by Western blot. Upper panels show Carbonic Anhydrase (CA) used as external load control and stained with Coomassie blue. Representative fluorograms of CaM are shown immediately below. First lane of both gel and fluorogram from panel A was loaded with pure CA (5 µg) and CaM (1 µg), respectively. CaM was recognized with a specific CaM antibody and ECL. Histograms correspond to densitometric analysis of the bands shown in the upper panels. Results are the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisk indicates significant differences (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-01907-f005: Calmodulin content in hippocampal slices treated with Melatonin. Hippocampal slices were incubated during 6 h with either the vehicle (VEH) or melatonin (MEL). Calmodulin (CaM) in the homogenates (A); and in the soluble (SOL) and cytoskeletal fractions (CSK) separated by centrifugation (B); was determined by Western blot. Upper panels show Carbonic Anhydrase (CA) used as external load control and stained with Coomassie blue. Representative fluorograms of CaM are shown immediately below. First lane of both gel and fluorogram from panel A was loaded with pure CA (5 µg) and CaM (1 µg), respectively. CaM was recognized with a specific CaM antibody and ECL. Histograms correspond to densitometric analysis of the bands shown in the upper panels. Results are the mean ± SEM of four densitometric scannings obtained from two independent experiments. Asterisk indicates significant differences (p < 0.05).
Mentions: As mentioned before, one of the crucial conditions for CaMKII activation is the availability of Ca2+/CaM for binding and activating it. Therefore, as a first step to disclose the mechanism by which MEL stimulates the activity of CaMKII we determined CaM levels in total homogenates and in the soluble and cytoskeletal fractions of hippocampal slices incubated with either the vehicle or 100 nM MEL for 6 h. As shown in Figure 5A, a slight increase of 8% in total CaM content was observed after a 6 h-incubation with 100 nM MEL. No changes were observed in the carbonic anhydrase used as external load control (Figure 4, upper panel). Figure 5B shows the subcellular distribution of CaM in the soluble and cytoskeletal fractions of hippocampal slices. In the presence of MEL, a 43% increase in CaM was observed in the soluble fraction with a slight decrease in the cytoskeletal fraction. Data suggest that both an increase in synthesis and translocation might occur in presence of MEL, in agreement with previous observations made in epithelial MDCK cells where the indoleamine induced an increase in both parameters [36]. Moreover, in vitro assays have shown that MEL directly stimulates PKC alpha and phosphorylates CaM. In this regard, it is known that phosphorylation of proteins induces a conformational change that increases the affinity constant for specific protein binding and therefore, they are targeted to specific subcellular compartments [22]. Although our experimental strategy does not allow us to elucidate the mechanisms by which CaM is enriched in the soluble fraction, our data indicates that MEL might increase availability of CaM to activate CaMKII and therefore trigger dendrite formation.

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