Effects of fluoxetine on Rho- and BDNF-signaling in the mouse brain

Research output: ThesisMaster's thesisTheses

Abstract

The antidepressant drug fluoxetine has been shown to increase neuronal plasticity in the adult central nervous system (CNS), which promotes the reorganization of neuronal connections. The neuroplastic effects of fluoxetine are at least partly mediated by the brain derived neurotrophic factor BDNF and its receptor TrkB that mediates both the formation and differentiation of new neurons and the growth and potentiation of neuronal connections. In addition to these “anabolic” or neurotrofic changes, the reorganization of neuronal connections also requires the removal of unnecessary connections, but the effects of fluoxetine on this process have not been thoroughly studied. Because the precursor of BDNF, proBDNF, has been shown to decrease the growth of neuronal connections and the stability of neuronal branches by activating the p75-neurotrophin receptor (p75NTR) mediated RhoA signaling in cell cultures, the primary objective of this Master’s thesis was to investigate the connections between the signaling pathways of TrkB and RhoA in vitro and the effects of short and long term fluoxetine treatment on Rho signaling in vivo.

Three week fluoxetine treatments with investigated dosages (0.08/0.16 mg/ml ad libitum in drinking water) did not alter the levels of RhoA protein levels in the frontal cortex and hippocampus of adult mice. Apart from the 0.16 mg/ml fluoxetine treatment, which caused an increase in the hippocampal BDNF-protein levels, no changes were detected in any of the other selected plasticity markers (NOGO, NR2A, CaMKII). The in vitro experiments consisted of transfections with different Rho plasmids, the wildtype RhoA, a dominant negative RhoA and a constitutively active RhoA. The transfected cells were treated with three concentrations of BDNF (0, 5, 20 ng/ml) and the levels of phosphorylated proteins related to TrkB signaling were measured. The most significant results were obtained with the 20 ng/ml BDNF-concentration, which showed the RhoA-T19N-transfection to induce increased TrkB phosphorylation compared to the cells transfected with GFP, RhoA-WT and RhoA-Q63L. The constitutively active RhoA-Q63L-transfected cells also showed a decrease in the phosphorylation levels of AKT- and GSK3β –proteins. The acute treatment with fluoxetine (30 mg/kg, i.p. 30 min) did not affect the activity of Rho-GTPases in hippocampal samples, then again the phosphorylation of the TrkB receptor was also unaffected in contrast to previous findings. An inhibitor of RhoA signaling, the ROCK-inhibitor Y27632 (10 mg/kg, i.p.) was also without effects on the phosphorylation of TrkB or the essential signaling molecules related to it.

According to the results Rho-signaling might interact with TrkB-signaling, but the nature of these interactions should be carefully researched in a more relevant context. The detection of plasticity related changes in vivo with the methods used in this study might also require different experimental settings that activate the laboratory animals besides the drug treatments.
Translated title of the contributionEffects of fluoxetine on Rho- and BDNF-signaling in the mouse brain
Original languageFinnish
Place of Publication[Helsinki]
Publication statusPublished - 2016
MoE publication typeG2 Master's thesis, polytechnic Master's thesis

Fields of Science

  • 1182 Biochemistry, cell and molecular biology
  • fluoxetine
  • RHO
  • TRKB
  • BDNF
  • Neuroplasticity
  • reorganization of neuronal networks
  • 317 Pharmacy

Cite this

@phdthesis{f62365d752674108a2575ed92e748dfd,
title = "Fluoksetiinin vaikutus Rho- ja BDNF-signalointiin hiiren aivoissa",
abstract = "The antidepressant drug fluoxetine has been shown to increase neuronal plasticity in the adult central nervous system (CNS), which promotes the reorganization of neuronal connections. The neuroplastic effects of fluoxetine are at least partly mediated by the brain derived neurotrophic factor BDNF and its receptor TrkB that mediates both the formation and differentiation of new neurons and the growth and potentiation of neuronal connections. In addition to these “anabolic” or neurotrofic changes, the reorganization of neuronal connections also requires the removal of unnecessary connections, but the effects of fluoxetine on this process have not been thoroughly studied. Because the precursor of BDNF, proBDNF, has been shown to decrease the growth of neuronal connections and the stability of neuronal branches by activating the p75-neurotrophin receptor (p75NTR) mediated RhoA signaling in cell cultures, the primary objective of this Master’s thesis was to investigate the connections between the signaling pathways of TrkB and RhoA in vitro and the effects of short and long term fluoxetine treatment on Rho signaling in vivo.Three week fluoxetine treatments with investigated dosages (0.08/0.16 mg/ml ad libitum in drinking water) did not alter the levels of RhoA protein levels in the frontal cortex and hippocampus of adult mice. Apart from the 0.16 mg/ml fluoxetine treatment, which caused an increase in the hippocampal BDNF-protein levels, no changes were detected in any of the other selected plasticity markers (NOGO, NR2A, CaMKII). The in vitro experiments consisted of transfections with different Rho plasmids, the wildtype RhoA, a dominant negative RhoA and a constitutively active RhoA. The transfected cells were treated with three concentrations of BDNF (0, 5, 20 ng/ml) and the levels of phosphorylated proteins related to TrkB signaling were measured. The most significant results were obtained with the 20 ng/ml BDNF-concentration, which showed the RhoA-T19N-transfection to induce increased TrkB phosphorylation compared to the cells transfected with GFP, RhoA-WT and RhoA-Q63L. The constitutively active RhoA-Q63L-transfected cells also showed a decrease in the phosphorylation levels of AKT- and GSK3β –proteins. The acute treatment with fluoxetine (30 mg/kg, i.p. 30 min) did not affect the activity of Rho-GTPases in hippocampal samples, then again the phosphorylation of the TrkB receptor was also unaffected in contrast to previous findings. An inhibitor of RhoA signaling, the ROCK-inhibitor Y27632 (10 mg/kg, i.p.) was also without effects on the phosphorylation of TrkB or the essential signaling molecules related to it. According to the results Rho-signaling might interact with TrkB-signaling, but the nature of these interactions should be carefully researched in a more relevant context. The detection of plasticity related changes in vivo with the methods used in this study might also require different experimental settings that activate the laboratory animals besides the drug treatments.",
keywords = "1182 Biokemia, solu- ja molekyylibiologia, fluoxetine, RHO, TRKB, BDNF, Neuroplasticity, reorganization of neuronal networks, 317 Farmasia",
author = "Kohtala, {Henrik Samuel}",
year = "2016",
language = "suomi",

}

Fluoksetiinin vaikutus Rho- ja BDNF-signalointiin hiiren aivoissa. / Kohtala, Henrik Samuel.

[Helsinki], 2016. 77 p.

Research output: ThesisMaster's thesisTheses

TY - THES

T1 - Fluoksetiinin vaikutus Rho- ja BDNF-signalointiin hiiren aivoissa

AU - Kohtala, Henrik Samuel

PY - 2016

Y1 - 2016

N2 - The antidepressant drug fluoxetine has been shown to increase neuronal plasticity in the adult central nervous system (CNS), which promotes the reorganization of neuronal connections. The neuroplastic effects of fluoxetine are at least partly mediated by the brain derived neurotrophic factor BDNF and its receptor TrkB that mediates both the formation and differentiation of new neurons and the growth and potentiation of neuronal connections. In addition to these “anabolic” or neurotrofic changes, the reorganization of neuronal connections also requires the removal of unnecessary connections, but the effects of fluoxetine on this process have not been thoroughly studied. Because the precursor of BDNF, proBDNF, has been shown to decrease the growth of neuronal connections and the stability of neuronal branches by activating the p75-neurotrophin receptor (p75NTR) mediated RhoA signaling in cell cultures, the primary objective of this Master’s thesis was to investigate the connections between the signaling pathways of TrkB and RhoA in vitro and the effects of short and long term fluoxetine treatment on Rho signaling in vivo.Three week fluoxetine treatments with investigated dosages (0.08/0.16 mg/ml ad libitum in drinking water) did not alter the levels of RhoA protein levels in the frontal cortex and hippocampus of adult mice. Apart from the 0.16 mg/ml fluoxetine treatment, which caused an increase in the hippocampal BDNF-protein levels, no changes were detected in any of the other selected plasticity markers (NOGO, NR2A, CaMKII). The in vitro experiments consisted of transfections with different Rho plasmids, the wildtype RhoA, a dominant negative RhoA and a constitutively active RhoA. The transfected cells were treated with three concentrations of BDNF (0, 5, 20 ng/ml) and the levels of phosphorylated proteins related to TrkB signaling were measured. The most significant results were obtained with the 20 ng/ml BDNF-concentration, which showed the RhoA-T19N-transfection to induce increased TrkB phosphorylation compared to the cells transfected with GFP, RhoA-WT and RhoA-Q63L. The constitutively active RhoA-Q63L-transfected cells also showed a decrease in the phosphorylation levels of AKT- and GSK3β –proteins. The acute treatment with fluoxetine (30 mg/kg, i.p. 30 min) did not affect the activity of Rho-GTPases in hippocampal samples, then again the phosphorylation of the TrkB receptor was also unaffected in contrast to previous findings. An inhibitor of RhoA signaling, the ROCK-inhibitor Y27632 (10 mg/kg, i.p.) was also without effects on the phosphorylation of TrkB or the essential signaling molecules related to it. According to the results Rho-signaling might interact with TrkB-signaling, but the nature of these interactions should be carefully researched in a more relevant context. The detection of plasticity related changes in vivo with the methods used in this study might also require different experimental settings that activate the laboratory animals besides the drug treatments.

AB - The antidepressant drug fluoxetine has been shown to increase neuronal plasticity in the adult central nervous system (CNS), which promotes the reorganization of neuronal connections. The neuroplastic effects of fluoxetine are at least partly mediated by the brain derived neurotrophic factor BDNF and its receptor TrkB that mediates both the formation and differentiation of new neurons and the growth and potentiation of neuronal connections. In addition to these “anabolic” or neurotrofic changes, the reorganization of neuronal connections also requires the removal of unnecessary connections, but the effects of fluoxetine on this process have not been thoroughly studied. Because the precursor of BDNF, proBDNF, has been shown to decrease the growth of neuronal connections and the stability of neuronal branches by activating the p75-neurotrophin receptor (p75NTR) mediated RhoA signaling in cell cultures, the primary objective of this Master’s thesis was to investigate the connections between the signaling pathways of TrkB and RhoA in vitro and the effects of short and long term fluoxetine treatment on Rho signaling in vivo.Three week fluoxetine treatments with investigated dosages (0.08/0.16 mg/ml ad libitum in drinking water) did not alter the levels of RhoA protein levels in the frontal cortex and hippocampus of adult mice. Apart from the 0.16 mg/ml fluoxetine treatment, which caused an increase in the hippocampal BDNF-protein levels, no changes were detected in any of the other selected plasticity markers (NOGO, NR2A, CaMKII). The in vitro experiments consisted of transfections with different Rho plasmids, the wildtype RhoA, a dominant negative RhoA and a constitutively active RhoA. The transfected cells were treated with three concentrations of BDNF (0, 5, 20 ng/ml) and the levels of phosphorylated proteins related to TrkB signaling were measured. The most significant results were obtained with the 20 ng/ml BDNF-concentration, which showed the RhoA-T19N-transfection to induce increased TrkB phosphorylation compared to the cells transfected with GFP, RhoA-WT and RhoA-Q63L. The constitutively active RhoA-Q63L-transfected cells also showed a decrease in the phosphorylation levels of AKT- and GSK3β –proteins. The acute treatment with fluoxetine (30 mg/kg, i.p. 30 min) did not affect the activity of Rho-GTPases in hippocampal samples, then again the phosphorylation of the TrkB receptor was also unaffected in contrast to previous findings. An inhibitor of RhoA signaling, the ROCK-inhibitor Y27632 (10 mg/kg, i.p.) was also without effects on the phosphorylation of TrkB or the essential signaling molecules related to it. According to the results Rho-signaling might interact with TrkB-signaling, but the nature of these interactions should be carefully researched in a more relevant context. The detection of plasticity related changes in vivo with the methods used in this study might also require different experimental settings that activate the laboratory animals besides the drug treatments.

KW - 1182 Biokemia, solu- ja molekyylibiologia

KW - fluoxetine

KW - RHO

KW - TRKB

KW - BDNF

KW - Neuroplasticity

KW - reorganization of neuronal networks

KW - 317 Farmasia

M3 - Pro gradu

CY - [Helsinki]

ER -