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Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics.

Speranza L, Giuliano T, Volpicelli F, De Stefano ME, Lombardi L, Chambery A, Lacivita E, Leopoldo M, Bellenchi GC, di Porzio U, Crispino M, Perrone-Capano C - Front Behav Neurosci (2015)

Bottom Line: In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics.Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation.Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Naples Federico II Naples, Italy ; Institute of Genetics and Biophysics "Adriano Buzzati Traverso", CNR Naples, Italy.

ABSTRACT
Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

No MeSH data available.


Stimulation of 5-HT7R enhances axonal outgrowth in cultured hippocampal neurons. (A) Schematic representation of microfluidic chambers. (B) Co-immunolabeling of neurons cultured in microfluidic chambers with the neuronal marker Tuj1 (green) and either the axonal marker Tau (top row, red) or the dendritic marker Map2 (bottom row, red). Dendritic immunolabeling is confined in the soma compartment and only axons cross the micro-channels to reach the other compartment. (C) The number of axons crossing the micro-channels is significantly higher in LP-211 treated cultures (LP, red line), respect to control (DMSO, blue line), after 3, 4, and 5 DIV. Asterisk (*): values significantly different from DMSO by One Way ANOVA followed by Dunnett post-hoc test (p < 0.05). (D) Representative hippocampal cultures grown in the presence of DMSO or LP and immunolabeled with the Tuj1 antibody (green).
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Figure 8: Stimulation of 5-HT7R enhances axonal outgrowth in cultured hippocampal neurons. (A) Schematic representation of microfluidic chambers. (B) Co-immunolabeling of neurons cultured in microfluidic chambers with the neuronal marker Tuj1 (green) and either the axonal marker Tau (top row, red) or the dendritic marker Map2 (bottom row, red). Dendritic immunolabeling is confined in the soma compartment and only axons cross the micro-channels to reach the other compartment. (C) The number of axons crossing the micro-channels is significantly higher in LP-211 treated cultures (LP, red line), respect to control (DMSO, blue line), after 3, 4, and 5 DIV. Asterisk (*): values significantly different from DMSO by One Way ANOVA followed by Dunnett post-hoc test (p < 0.05). (D) Representative hippocampal cultures grown in the presence of DMSO or LP and immunolabeled with the Tuj1 antibody (green).

Mentions: Then, we cultured HIPP neurons in microfluidic chambers (Park et al., 2006), implementing culture medium with 100 nM of the agonist. Neurons were plated on one side of the culture chamber (soma compartment) and axons, but not dendrites, grew into the other compartment, passing through interconnecting micro-channels (450 μm long) (Figure 8A). We first verified whether in our system the only fibers crossing the channels were axons, by co-immunolabeling hippocampal neurons with Tuj1 (neuronal marker) and either anti-Tau (axon specific marker) or anti-Map2 (dendrite specific marker). As expected, the anti-Tau antibody labeled axons in both compartments, while dendritic labeling with Map2 was confined to the soma compartment (Figure 8B). Therefore, we plated HIPP neurons in the presence of either LP-211 or vehicle (CTRL), added to both compartments. Cultures were maintained for 6 days and axons reaching the appropriate compartment were counted daily, from 1DIV to 6DIV. In LP-211-treated cultures we observed a significant increase in the number of axons crossing the channels by 3 to 5DIV, clearly indicating that stimulation of the 5-HT7R promotes axonal growth, at least at early stages (Figure 8C). This difference disappeared at 6DIV, suggesting receptor desensitization, although we have also considered the hypothesis that the number of axons would eventually reach a plateau, since the number of channels to cross is limited. Figure 8D shows two typical cultures, grown in the presence of either DMSO or LP-211, indicating that, before plateau is reached, the number of axons reaching the side compartment is higher in the presence of 5-HT7R agonist.


Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics.

Speranza L, Giuliano T, Volpicelli F, De Stefano ME, Lombardi L, Chambery A, Lacivita E, Leopoldo M, Bellenchi GC, di Porzio U, Crispino M, Perrone-Capano C - Front Behav Neurosci (2015)

Stimulation of 5-HT7R enhances axonal outgrowth in cultured hippocampal neurons. (A) Schematic representation of microfluidic chambers. (B) Co-immunolabeling of neurons cultured in microfluidic chambers with the neuronal marker Tuj1 (green) and either the axonal marker Tau (top row, red) or the dendritic marker Map2 (bottom row, red). Dendritic immunolabeling is confined in the soma compartment and only axons cross the micro-channels to reach the other compartment. (C) The number of axons crossing the micro-channels is significantly higher in LP-211 treated cultures (LP, red line), respect to control (DMSO, blue line), after 3, 4, and 5 DIV. Asterisk (*): values significantly different from DMSO by One Way ANOVA followed by Dunnett post-hoc test (p < 0.05). (D) Representative hippocampal cultures grown in the presence of DMSO or LP and immunolabeled with the Tuj1 antibody (green).
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Figure 8: Stimulation of 5-HT7R enhances axonal outgrowth in cultured hippocampal neurons. (A) Schematic representation of microfluidic chambers. (B) Co-immunolabeling of neurons cultured in microfluidic chambers with the neuronal marker Tuj1 (green) and either the axonal marker Tau (top row, red) or the dendritic marker Map2 (bottom row, red). Dendritic immunolabeling is confined in the soma compartment and only axons cross the micro-channels to reach the other compartment. (C) The number of axons crossing the micro-channels is significantly higher in LP-211 treated cultures (LP, red line), respect to control (DMSO, blue line), after 3, 4, and 5 DIV. Asterisk (*): values significantly different from DMSO by One Way ANOVA followed by Dunnett post-hoc test (p < 0.05). (D) Representative hippocampal cultures grown in the presence of DMSO or LP and immunolabeled with the Tuj1 antibody (green).
Mentions: Then, we cultured HIPP neurons in microfluidic chambers (Park et al., 2006), implementing culture medium with 100 nM of the agonist. Neurons were plated on one side of the culture chamber (soma compartment) and axons, but not dendrites, grew into the other compartment, passing through interconnecting micro-channels (450 μm long) (Figure 8A). We first verified whether in our system the only fibers crossing the channels were axons, by co-immunolabeling hippocampal neurons with Tuj1 (neuronal marker) and either anti-Tau (axon specific marker) or anti-Map2 (dendrite specific marker). As expected, the anti-Tau antibody labeled axons in both compartments, while dendritic labeling with Map2 was confined to the soma compartment (Figure 8B). Therefore, we plated HIPP neurons in the presence of either LP-211 or vehicle (CTRL), added to both compartments. Cultures were maintained for 6 days and axons reaching the appropriate compartment were counted daily, from 1DIV to 6DIV. In LP-211-treated cultures we observed a significant increase in the number of axons crossing the channels by 3 to 5DIV, clearly indicating that stimulation of the 5-HT7R promotes axonal growth, at least at early stages (Figure 8C). This difference disappeared at 6DIV, suggesting receptor desensitization, although we have also considered the hypothesis that the number of axons would eventually reach a plateau, since the number of channels to cross is limited. Figure 8D shows two typical cultures, grown in the presence of either DMSO or LP-211, indicating that, before plateau is reached, the number of axons reaching the side compartment is higher in the presence of 5-HT7R agonist.

Bottom Line: In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics.Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation.Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Naples Federico II Naples, Italy ; Institute of Genetics and Biophysics "Adriano Buzzati Traverso", CNR Naples, Italy.

ABSTRACT
Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

No MeSH data available.