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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

Characterization of striatal and cortical primary cultures. (A) Photomicrographs of the cells in striatal and cortical cultures immunostained with specific antibodies against neuronal marker Tuj1 (red), and 5-HT7R (green), as indicated in each panel. Cell bodies were counterstained with the nuclear marker DAPI (blue). (B) Expression levels of 5-HT7R mRNAs, determined by real time RT-PCR, in neuronal cultures. The bars represent the 5-HT7R mRNA levels normalized with those of the housekeeping gene HPRT (means ± SEM; n = 3). (C) Quantitation of 5-HT7R protein in neuronal cultures. The bars represent the densitometric values of 5-HT7R Western blot signals normalized with those of β-actin signals in the same samples (mean ± SEM; n = 3). STR: cultures from the striatal complex of E15 mouse embryos; CTX: cultures from the cortex of E15 mouse embryos.
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Figure 1: Characterization of striatal and cortical primary cultures. (A) Photomicrographs of the cells in striatal and cortical cultures immunostained with specific antibodies against neuronal marker Tuj1 (red), and 5-HT7R (green), as indicated in each panel. Cell bodies were counterstained with the nuclear marker DAPI (blue). (B) Expression levels of 5-HT7R mRNAs, determined by real time RT-PCR, in neuronal cultures. The bars represent the 5-HT7R mRNA levels normalized with those of the housekeeping gene HPRT (means ± SEM; n = 3). (C) Quantitation of 5-HT7R protein in neuronal cultures. The bars represent the densitometric values of 5-HT7R Western blot signals normalized with those of β-actin signals in the same samples (mean ± SEM; n = 3). STR: cultures from the striatal complex of E15 mouse embryos; CTX: cultures from the cortex of E15 mouse embryos.

Mentions: As expected from previous data on rat primary cultures (Speranza et al., 2013), the phenotype of cells obtained from the CTX and STR of mouse brains at E15 showed that almost 95% of STR and CTX cells in culture were neurons, as judged by co-localization of Tuj1 antibodies with the nuclear DAPI staining. In addition, about 90% of striatal and cortical neurons were positive to 5-HT7R (Figure 1A).


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)

Characterization of striatal and cortical primary cultures. (A) Photomicrographs of the cells in striatal and cortical cultures immunostained with specific antibodies against neuronal marker Tuj1 (red), and 5-HT7R (green), as indicated in each panel. Cell bodies were counterstained with the nuclear marker DAPI (blue). (B) Expression levels of 5-HT7R mRNAs, determined by real time RT-PCR, in neuronal cultures. The bars represent the 5-HT7R mRNA levels normalized with those of the housekeeping gene HPRT (means ± SEM; n = 3). (C) Quantitation of 5-HT7R protein in neuronal cultures. The bars represent the densitometric values of 5-HT7R Western blot signals normalized with those of β-actin signals in the same samples (mean ± SEM; n = 3). STR: cultures from the striatal complex of E15 mouse embryos; CTX: cultures from the cortex of E15 mouse embryos.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Characterization of striatal and cortical primary cultures. (A) Photomicrographs of the cells in striatal and cortical cultures immunostained with specific antibodies against neuronal marker Tuj1 (red), and 5-HT7R (green), as indicated in each panel. Cell bodies were counterstained with the nuclear marker DAPI (blue). (B) Expression levels of 5-HT7R mRNAs, determined by real time RT-PCR, in neuronal cultures. The bars represent the 5-HT7R mRNA levels normalized with those of the housekeeping gene HPRT (means ± SEM; n = 3). (C) Quantitation of 5-HT7R protein in neuronal cultures. The bars represent the densitometric values of 5-HT7R Western blot signals normalized with those of β-actin signals in the same samples (mean ± SEM; n = 3). STR: cultures from the striatal complex of E15 mouse embryos; CTX: cultures from the cortex of E15 mouse embryos.
Mentions: As expected from previous data on rat primary cultures (Speranza et al., 2013), the phenotype of cells obtained from the CTX and STR of mouse brains at E15 showed that almost 95% of STR and CTX cells in culture were neurons, as judged by co-localization of Tuj1 antibodies with the nuclear DAPI staining. In addition, about 90% of striatal and cortical neurons were positive to 5-HT7R (Figure 1A).

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