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Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.

Cheung ZH, Chin WH, Chen Y, Ng YP, Ip NY - PLoS Biol. (2007)

Bottom Line: Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)-triggered dendritic growth in primary hippocampal neurons.In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth.Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

ABSTRACT
Neurotrophins are key regulators of neuronal survival and differentiation during development. Activation of their cognate receptors, Trk receptors, a family of receptor tyrosine kinases (RTKs), is pivotal for mediating the downstream functions of neurotrophins. Recent studies reveal that cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase, may modulate RTK signaling through phosphorylation of the receptor. Given the abundant expression of both Cdk5 and Trk receptors in the nervous system, and their mutual involvement in the regulation of neuronal architecture and synaptic functions, it is of interest to investigate if Cdk5 may also modulate Trk signaling. In the current study, we report the identification of TrkB as a Cdk5 substrate. Cdk5 phosphorylates TrkB at Ser478 at the intracellular juxtamembrane region of TrkB. Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)-triggered dendritic growth in primary hippocampal neurons. In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth. Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.

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Cdk5-Mediated Phosphorylation of TrkB Affected BDNF-Induced Dendritic Growth through Attenuation of Cdc42 Activity(A) Hippocampal neurons were transfected with the WT or DN form of Rac1, RhoA, or Cdc42. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of DN Cdc42 markedly reduced BDNF-induced increase in primary dendrites compared to overexpression of WT Cdc42, indicating that Cdc42 may contribute to the BDNF-dependent induction of dendritic growth in hippocampal neurons.(B) Cortical neurons were pretreated with Cdk5 selective inhibitor Ros or vehicle (DMSO) for 30 min prior to treatment with BDNF for different time intervals. Ros pretreatment markedly reduced BDNF-induced increase in Cdc42 activity following 15 and 30 min of BDNF treatment, indicating that Cdk5 activity was involved in BDNF-triggered Cdc42 activation. Quantification of the changes in Cdc42 activity following BDNF stimulation with or without Ros pretreatment was normalized to the value obtained for the DMSO-treated group at time 0 and is shown in the histogram. *, p < 0.05.(C) TrkB WT or TrkB M1 mutant were co-transfected with WT or CA Cdc42 in hippocampal neurons. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of CA Cdc42 reversed the abrogation of BDNF-induced increase in primary dendrites following overexpression of TrkB M1. *, p < 0.05.(D) Hippocampal neurons isolated from cdk5+/+ and cdk5−/− brains were transfected with WT or CA Cdc42. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of CA Cdc42 rescued the lack of dendritic growth following BDNF treatment in Cdk5−/− hippocampal neurons. *, p < 0.05.
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pbio-0050063-g006: Cdk5-Mediated Phosphorylation of TrkB Affected BDNF-Induced Dendritic Growth through Attenuation of Cdc42 Activity(A) Hippocampal neurons were transfected with the WT or DN form of Rac1, RhoA, or Cdc42. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of DN Cdc42 markedly reduced BDNF-induced increase in primary dendrites compared to overexpression of WT Cdc42, indicating that Cdc42 may contribute to the BDNF-dependent induction of dendritic growth in hippocampal neurons.(B) Cortical neurons were pretreated with Cdk5 selective inhibitor Ros or vehicle (DMSO) for 30 min prior to treatment with BDNF for different time intervals. Ros pretreatment markedly reduced BDNF-induced increase in Cdc42 activity following 15 and 30 min of BDNF treatment, indicating that Cdk5 activity was involved in BDNF-triggered Cdc42 activation. Quantification of the changes in Cdc42 activity following BDNF stimulation with or without Ros pretreatment was normalized to the value obtained for the DMSO-treated group at time 0 and is shown in the histogram. *, p < 0.05.(C) TrkB WT or TrkB M1 mutant were co-transfected with WT or CA Cdc42 in hippocampal neurons. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of CA Cdc42 reversed the abrogation of BDNF-induced increase in primary dendrites following overexpression of TrkB M1. *, p < 0.05.(D) Hippocampal neurons isolated from cdk5+/+ and cdk5−/− brains were transfected with WT or CA Cdc42. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of CA Cdc42 rescued the lack of dendritic growth following BDNF treatment in Cdk5−/− hippocampal neurons. *, p < 0.05.

Mentions: Rho GTPases, including RhoA, Rac1, and Cdc42, are key regulators of actin cytoskeleton dynamics. Since BDNF stimulation has been observed to activate Rac1 and Cdc42 in neurons [15], we were interested to delineate if Rho GTPases contribute to BDNF-stimulated dendritic growth. To investigate if Rho GTPases are involved, and to identify the Rho GTPase(s) implicated, hippocampal neurons were transfected with WT or DN Rac1, Cdc42, or RhoA. We found that while overexpression of WT and DN Rac1 increased the basal number of dendrites in the absence of BDNF treatment, overexpression of both forms of Rac1 abolished BDNF-stimulated dendritic growth. On the other hand, while overexpression of DN RhoA slightly enhanced primary dendrites irrespective of BDNF stimulation, overexpression of both WT and DN forms of RhoA inhibited BDNF-stimulated dendritic growth. Remarkably, in contrast to the inhibition of BDNF-stimulated dendritic growth in cells overexpressing WT Rac1 and RhoA, BDNF stimulation of hippocampal neurons overexpressing WT Cdc42 resulted in an increase in primary dendrites, which was nearly abolished by overexpression of DN Cdc42 (Figure 6A). Our observations therefore suggest that while Rac1 and RhoA may also modulate BDNF-stimulated dendritic growth, it is the activation of Cdc42 following BDNF stimulation that most likely mediates the increase in primary dendrites by BDNF.


Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.

Cheung ZH, Chin WH, Chen Y, Ng YP, Ip NY - PLoS Biol. (2007)

Cdk5-Mediated Phosphorylation of TrkB Affected BDNF-Induced Dendritic Growth through Attenuation of Cdc42 Activity(A) Hippocampal neurons were transfected with the WT or DN form of Rac1, RhoA, or Cdc42. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of DN Cdc42 markedly reduced BDNF-induced increase in primary dendrites compared to overexpression of WT Cdc42, indicating that Cdc42 may contribute to the BDNF-dependent induction of dendritic growth in hippocampal neurons.(B) Cortical neurons were pretreated with Cdk5 selective inhibitor Ros or vehicle (DMSO) for 30 min prior to treatment with BDNF for different time intervals. Ros pretreatment markedly reduced BDNF-induced increase in Cdc42 activity following 15 and 30 min of BDNF treatment, indicating that Cdk5 activity was involved in BDNF-triggered Cdc42 activation. Quantification of the changes in Cdc42 activity following BDNF stimulation with or without Ros pretreatment was normalized to the value obtained for the DMSO-treated group at time 0 and is shown in the histogram. *, p < 0.05.(C) TrkB WT or TrkB M1 mutant were co-transfected with WT or CA Cdc42 in hippocampal neurons. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of CA Cdc42 reversed the abrogation of BDNF-induced increase in primary dendrites following overexpression of TrkB M1. *, p < 0.05.(D) Hippocampal neurons isolated from cdk5+/+ and cdk5−/− brains were transfected with WT or CA Cdc42. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of CA Cdc42 rescued the lack of dendritic growth following BDNF treatment in Cdk5−/− hippocampal neurons. *, p < 0.05.
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pbio-0050063-g006: Cdk5-Mediated Phosphorylation of TrkB Affected BDNF-Induced Dendritic Growth through Attenuation of Cdc42 Activity(A) Hippocampal neurons were transfected with the WT or DN form of Rac1, RhoA, or Cdc42. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of DN Cdc42 markedly reduced BDNF-induced increase in primary dendrites compared to overexpression of WT Cdc42, indicating that Cdc42 may contribute to the BDNF-dependent induction of dendritic growth in hippocampal neurons.(B) Cortical neurons were pretreated with Cdk5 selective inhibitor Ros or vehicle (DMSO) for 30 min prior to treatment with BDNF for different time intervals. Ros pretreatment markedly reduced BDNF-induced increase in Cdc42 activity following 15 and 30 min of BDNF treatment, indicating that Cdk5 activity was involved in BDNF-triggered Cdc42 activation. Quantification of the changes in Cdc42 activity following BDNF stimulation with or without Ros pretreatment was normalized to the value obtained for the DMSO-treated group at time 0 and is shown in the histogram. *, p < 0.05.(C) TrkB WT or TrkB M1 mutant were co-transfected with WT or CA Cdc42 in hippocampal neurons. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of CA Cdc42 reversed the abrogation of BDNF-induced increase in primary dendrites following overexpression of TrkB M1. *, p < 0.05.(D) Hippocampal neurons isolated from cdk5+/+ and cdk5−/− brains were transfected with WT or CA Cdc42. Twenty-four hours after transfection, cells were exposed to BDNF for 3 d. Overexpression of CA Cdc42 rescued the lack of dendritic growth following BDNF treatment in Cdk5−/− hippocampal neurons. *, p < 0.05.
Mentions: Rho GTPases, including RhoA, Rac1, and Cdc42, are key regulators of actin cytoskeleton dynamics. Since BDNF stimulation has been observed to activate Rac1 and Cdc42 in neurons [15], we were interested to delineate if Rho GTPases contribute to BDNF-stimulated dendritic growth. To investigate if Rho GTPases are involved, and to identify the Rho GTPase(s) implicated, hippocampal neurons were transfected with WT or DN Rac1, Cdc42, or RhoA. We found that while overexpression of WT and DN Rac1 increased the basal number of dendrites in the absence of BDNF treatment, overexpression of both forms of Rac1 abolished BDNF-stimulated dendritic growth. On the other hand, while overexpression of DN RhoA slightly enhanced primary dendrites irrespective of BDNF stimulation, overexpression of both WT and DN forms of RhoA inhibited BDNF-stimulated dendritic growth. Remarkably, in contrast to the inhibition of BDNF-stimulated dendritic growth in cells overexpressing WT Rac1 and RhoA, BDNF stimulation of hippocampal neurons overexpressing WT Cdc42 resulted in an increase in primary dendrites, which was nearly abolished by overexpression of DN Cdc42 (Figure 6A). Our observations therefore suggest that while Rac1 and RhoA may also modulate BDNF-stimulated dendritic growth, it is the activation of Cdc42 following BDNF stimulation that most likely mediates the increase in primary dendrites by BDNF.

Bottom Line: Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)-triggered dendritic growth in primary hippocampal neurons.In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth.Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

ABSTRACT
Neurotrophins are key regulators of neuronal survival and differentiation during development. Activation of their cognate receptors, Trk receptors, a family of receptor tyrosine kinases (RTKs), is pivotal for mediating the downstream functions of neurotrophins. Recent studies reveal that cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase, may modulate RTK signaling through phosphorylation of the receptor. Given the abundant expression of both Cdk5 and Trk receptors in the nervous system, and their mutual involvement in the regulation of neuronal architecture and synaptic functions, it is of interest to investigate if Cdk5 may also modulate Trk signaling. In the current study, we report the identification of TrkB as a Cdk5 substrate. Cdk5 phosphorylates TrkB at Ser478 at the intracellular juxtamembrane region of TrkB. Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)-triggered dendritic growth in primary hippocampal neurons. In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth. Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.

Show MeSH