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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.


Related in: MedlinePlus

Stimulation of 5-HT7R enhances neurite outgrowth in hippocampal primary cultures. Hippocampal cells were treated for 2 h with the 5-HT7R selective agonist LP-211 (LP, 100 nM), alone or in combination with the selective 5-HT7R antagonist SB269970 (SB, 100 nM). Neurite length was measured on cells stained with anti-Tuj1 antibody, and expressed as percentage of values measured in the corresponding vehicle-treated cultures (CTRL, set to 100%). The bars represent means ± SEM from randomly selected fields for each cell culture conditions (n = 10). Asterisk (*): values significantly different from CTRL by One Way ANOVA followed by Dunnett post-hoc test (p < 0.05). The panels below each bar display representative images of hippocampal neurons immunostained with the neuronal marker Tuj1 (red) and counterstained with the nuclear marker DAPI (blue; magnification 20x).
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Figure 7: Stimulation of 5-HT7R enhances neurite outgrowth in hippocampal primary cultures. Hippocampal cells were treated for 2 h with the 5-HT7R selective agonist LP-211 (LP, 100 nM), alone or in combination with the selective 5-HT7R antagonist SB269970 (SB, 100 nM). Neurite length was measured on cells stained with anti-Tuj1 antibody, and expressed as percentage of values measured in the corresponding vehicle-treated cultures (CTRL, set to 100%). The bars represent means ± SEM from randomly selected fields for each cell culture conditions (n = 10). Asterisk (*): values significantly different from CTRL by One Way ANOVA followed by Dunnett post-hoc test (p < 0.05). The panels below each bar display representative images of hippocampal neurons immunostained with the neuronal marker Tuj1 (red) and counterstained with the nuclear marker DAPI (blue; magnification 20x).

Mentions: Although neurite outgrowth is studied in cultured neurons before the occurrence of neuronal polarization, it is generally considered the equivalent of axonal extension (Conti et al., 1997). To determine whether 5-HT7R could indeed modulate axonal growth, we used microfluidic culture platforms that allow physical separation between soma and axons of cultured neurons (Park et al., 2006). Microfluidic chambers have been mostly used with HIPP neurons for their ability to develop long axons. Therefore, we first tested the effect of 2 h stimulation with LP-211 on neurite elongation of E18 mouse HIPP neurons cultured in traditional chamber slides. These cultures were immunostained with TuJ1 antibodies and counterstained with the nuclear marker DAPI, indicating that most of cultured cells were neurons (see panels in Figure 7). Consistent with previous results obtained with other 5-HT7R agonists (Kvachnina et al., 2005), we observed that the length of neurites significantly increased in LP-211-treated cultures compared to CTRL; the co-treatment of neurons with LP-211 and SB-269970 completely abolished neurite elongation, while addition of the antagonist alone did not affect neurite outgrowth (Figure 7).


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 neurite outgrowth in hippocampal primary cultures. Hippocampal cells were treated for 2 h with the 5-HT7R selective agonist LP-211 (LP, 100 nM), alone or in combination with the selective 5-HT7R antagonist SB269970 (SB, 100 nM). Neurite length was measured on cells stained with anti-Tuj1 antibody, and expressed as percentage of values measured in the corresponding vehicle-treated cultures (CTRL, set to 100%). The bars represent means ± SEM from randomly selected fields for each cell culture conditions (n = 10). Asterisk (*): values significantly different from CTRL by One Way ANOVA followed by Dunnett post-hoc test (p < 0.05). The panels below each bar display representative images of hippocampal neurons immunostained with the neuronal marker Tuj1 (red) and counterstained with the nuclear marker DAPI (blue; magnification 20x).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356071&req=5

Figure 7: Stimulation of 5-HT7R enhances neurite outgrowth in hippocampal primary cultures. Hippocampal cells were treated for 2 h with the 5-HT7R selective agonist LP-211 (LP, 100 nM), alone or in combination with the selective 5-HT7R antagonist SB269970 (SB, 100 nM). Neurite length was measured on cells stained with anti-Tuj1 antibody, and expressed as percentage of values measured in the corresponding vehicle-treated cultures (CTRL, set to 100%). The bars represent means ± SEM from randomly selected fields for each cell culture conditions (n = 10). Asterisk (*): values significantly different from CTRL by One Way ANOVA followed by Dunnett post-hoc test (p < 0.05). The panels below each bar display representative images of hippocampal neurons immunostained with the neuronal marker Tuj1 (red) and counterstained with the nuclear marker DAPI (blue; magnification 20x).
Mentions: Although neurite outgrowth is studied in cultured neurons before the occurrence of neuronal polarization, it is generally considered the equivalent of axonal extension (Conti et al., 1997). To determine whether 5-HT7R could indeed modulate axonal growth, we used microfluidic culture platforms that allow physical separation between soma and axons of cultured neurons (Park et al., 2006). Microfluidic chambers have been mostly used with HIPP neurons for their ability to develop long axons. Therefore, we first tested the effect of 2 h stimulation with LP-211 on neurite elongation of E18 mouse HIPP neurons cultured in traditional chamber slides. These cultures were immunostained with TuJ1 antibodies and counterstained with the nuclear marker DAPI, indicating that most of cultured cells were neurons (see panels in Figure 7). Consistent with previous results obtained with other 5-HT7R agonists (Kvachnina et al., 2005), we observed that the length of neurites significantly increased in LP-211-treated cultures compared to CTRL; the co-treatment of neurons with LP-211 and SB-269970 completely abolished neurite elongation, while addition of the antagonist alone did not affect neurite outgrowth (Figure 7).

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.


Related in: MedlinePlus