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GABAB receptor upregulates fragile X mental retardation protein expression in neurons.

Zhang W, Xu C, Tu H, Wang Y, Sun Q, Hu P, Hu Y, Rondard P, Liu J - Sci Rep (2015)

Bottom Line: Fragile X mental retardation protein (FMRP) is an RNA-binding protein important for the control of translation and synaptic function.In addition, a positive allosteric modulator of the GABAB receptor, CGP7930, stimulated Fmrp expression in neurons.These results suggest a role for GABAB receptor in Fmrp regulation and a potential interest of GABAB receptor signaling in FXS improvement.

View Article: PubMed Central - PubMed

Affiliation: Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan, Hubei, China.

ABSTRACT
Fragile X mental retardation protein (FMRP) is an RNA-binding protein important for the control of translation and synaptic function. The mutation or silencing of FMRP causes Fragile X syndrome (FXS), which leads to intellectual disability and social impairment. γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the mammalian central nervous system, and its metabotropic GABAB receptor has been implicated in various mental disorders. The GABAB receptor agonist baclofen has been shown to improve FXS symptoms in a mouse model and in human patients, but the signaling events linking the GABAB receptor and FMRP are unknown. In this study, we found that GABAB receptor activation upregulated cAMP response element binding protein-dependent Fmrp expression in cultured mouse cerebellar granule neurons via two distinct mechanisms: the transactivation of insulin-like growth factor-1 receptor and activation of protein kinase C. In addition, a positive allosteric modulator of the GABAB receptor, CGP7930, stimulated Fmrp expression in neurons. These results suggest a role for GABAB receptor in Fmrp regulation and a potential interest of GABAB receptor signaling in FXS improvement.

No MeSH data available.


Related in: MedlinePlus

PKC activated by GABAB receptor acts downstream of FAK and is independent of IGF-1R signaling.(A) Time course of phosphorylation of the PKC substrate MARCKS induced by baclofen in CGNs. (B) Effect of the FAK inhibitor PF573228 on MARCKS phosphorylation in CGNs. pMARCKS ratio was defined as the ratio between the density of each band and the sum of the densities of all the bands in a given blot. Values represent the mean ± SEM from three independent experiments. *P < 0.05 vs. basal level. ##P < 0.01 vs. baclofen-treated group. (C,D) Effect of siRNA-mediated knockdown of FAK or IGF-1R on baclofen-induced phosphorylation of MARCKS, ERK1/2, Akt, and CREB in MEFs co-transfected with GABAB1 and GABAB2. Full-size blots are shown in Supplementary Figure S8 and the band of interest is indicated by a red box.
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f5: PKC activated by GABAB receptor acts downstream of FAK and is independent of IGF-1R signaling.(A) Time course of phosphorylation of the PKC substrate MARCKS induced by baclofen in CGNs. (B) Effect of the FAK inhibitor PF573228 on MARCKS phosphorylation in CGNs. pMARCKS ratio was defined as the ratio between the density of each band and the sum of the densities of all the bands in a given blot. Values represent the mean ± SEM from three independent experiments. *P < 0.05 vs. basal level. ##P < 0.01 vs. baclofen-treated group. (C,D) Effect of siRNA-mediated knockdown of FAK or IGF-1R on baclofen-induced phosphorylation of MARCKS, ERK1/2, Akt, and CREB in MEFs co-transfected with GABAB1 and GABAB2. Full-size blots are shown in Supplementary Figure S8 and the band of interest is indicated by a red box.

Mentions: PKC was previously shown to be activated by baclofen treatment15. Phosphorylation of the PKC substrate MARCKS was increased in a time-dependent manner by baclofen treatment (Fig. 5A, Figure S8A). Phospho-MARCKS level was reduced by application of the FAK inhibitor PF573228 or by siRNA-mediated knockdown of FAK (Fig. 5B, C, Figure S8B, C), but not by IGF-1R knockdown (Fig. 5D, Figure S8D), suggesting that PKC acts downstream of FAK but independently of IGF-1R. Three PKC inhibitors (GF109203x, Gö-6983, and Gö-6976) were used to analyse the effect of PKC on CREB activation; GF109203x and Gö-6983 inhibit all PKC isozymes2526, whereas Gö-6976 is selective for Ca2+ -sensitive PKC isotypes26. All three inhibitors blocked baclofen-induced CREB phosphorylation but had no effect on the phosphorylation of ERK1/2 and Akt (Fig. 6A, B, Figure S9A, B and Figure S10A). In addition, siRNA-mediated knockdown of PKCα or PKCβ in MEFs co-expressing GABAB1 and GABAB2 subunits of the GABAB receptor decreased baclofen-mediated CREB phosphorylation, whereas no changes in IGF-1R transactivation or ERK1/2 and Akt phosphorylation were observed (Fig. 6C, Figure S9C and Figure S10B). These results indicate that Ca2+ -sensitive PKCs are required for GABAB-induced CREB activation, but that this effect is independent of IGF-1R transactivation.


GABAB receptor upregulates fragile X mental retardation protein expression in neurons.

Zhang W, Xu C, Tu H, Wang Y, Sun Q, Hu P, Hu Y, Rondard P, Liu J - Sci Rep (2015)

PKC activated by GABAB receptor acts downstream of FAK and is independent of IGF-1R signaling.(A) Time course of phosphorylation of the PKC substrate MARCKS induced by baclofen in CGNs. (B) Effect of the FAK inhibitor PF573228 on MARCKS phosphorylation in CGNs. pMARCKS ratio was defined as the ratio between the density of each band and the sum of the densities of all the bands in a given blot. Values represent the mean ± SEM from three independent experiments. *P < 0.05 vs. basal level. ##P < 0.01 vs. baclofen-treated group. (C,D) Effect of siRNA-mediated knockdown of FAK or IGF-1R on baclofen-induced phosphorylation of MARCKS, ERK1/2, Akt, and CREB in MEFs co-transfected with GABAB1 and GABAB2. Full-size blots are shown in Supplementary Figure S8 and the band of interest is indicated by a red box.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4447080&req=5

f5: PKC activated by GABAB receptor acts downstream of FAK and is independent of IGF-1R signaling.(A) Time course of phosphorylation of the PKC substrate MARCKS induced by baclofen in CGNs. (B) Effect of the FAK inhibitor PF573228 on MARCKS phosphorylation in CGNs. pMARCKS ratio was defined as the ratio between the density of each band and the sum of the densities of all the bands in a given blot. Values represent the mean ± SEM from three independent experiments. *P < 0.05 vs. basal level. ##P < 0.01 vs. baclofen-treated group. (C,D) Effect of siRNA-mediated knockdown of FAK or IGF-1R on baclofen-induced phosphorylation of MARCKS, ERK1/2, Akt, and CREB in MEFs co-transfected with GABAB1 and GABAB2. Full-size blots are shown in Supplementary Figure S8 and the band of interest is indicated by a red box.
Mentions: PKC was previously shown to be activated by baclofen treatment15. Phosphorylation of the PKC substrate MARCKS was increased in a time-dependent manner by baclofen treatment (Fig. 5A, Figure S8A). Phospho-MARCKS level was reduced by application of the FAK inhibitor PF573228 or by siRNA-mediated knockdown of FAK (Fig. 5B, C, Figure S8B, C), but not by IGF-1R knockdown (Fig. 5D, Figure S8D), suggesting that PKC acts downstream of FAK but independently of IGF-1R. Three PKC inhibitors (GF109203x, Gö-6983, and Gö-6976) were used to analyse the effect of PKC on CREB activation; GF109203x and Gö-6983 inhibit all PKC isozymes2526, whereas Gö-6976 is selective for Ca2+ -sensitive PKC isotypes26. All three inhibitors blocked baclofen-induced CREB phosphorylation but had no effect on the phosphorylation of ERK1/2 and Akt (Fig. 6A, B, Figure S9A, B and Figure S10A). In addition, siRNA-mediated knockdown of PKCα or PKCβ in MEFs co-expressing GABAB1 and GABAB2 subunits of the GABAB receptor decreased baclofen-mediated CREB phosphorylation, whereas no changes in IGF-1R transactivation or ERK1/2 and Akt phosphorylation were observed (Fig. 6C, Figure S9C and Figure S10B). These results indicate that Ca2+ -sensitive PKCs are required for GABAB-induced CREB activation, but that this effect is independent of IGF-1R transactivation.

Bottom Line: Fragile X mental retardation protein (FMRP) is an RNA-binding protein important for the control of translation and synaptic function.In addition, a positive allosteric modulator of the GABAB receptor, CGP7930, stimulated Fmrp expression in neurons.These results suggest a role for GABAB receptor in Fmrp regulation and a potential interest of GABAB receptor signaling in FXS improvement.

View Article: PubMed Central - PubMed

Affiliation: Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology and the Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan, Hubei, China.

ABSTRACT
Fragile X mental retardation protein (FMRP) is an RNA-binding protein important for the control of translation and synaptic function. The mutation or silencing of FMRP causes Fragile X syndrome (FXS), which leads to intellectual disability and social impairment. γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the mammalian central nervous system, and its metabotropic GABAB receptor has been implicated in various mental disorders. The GABAB receptor agonist baclofen has been shown to improve FXS symptoms in a mouse model and in human patients, but the signaling events linking the GABAB receptor and FMRP are unknown. In this study, we found that GABAB receptor activation upregulated cAMP response element binding protein-dependent Fmrp expression in cultured mouse cerebellar granule neurons via two distinct mechanisms: the transactivation of insulin-like growth factor-1 receptor and activation of protein kinase C. In addition, a positive allosteric modulator of the GABAB receptor, CGP7930, stimulated Fmrp expression in neurons. These results suggest a role for GABAB receptor in Fmrp regulation and a potential interest of GABAB receptor signaling in FXS improvement.

No MeSH data available.


Related in: MedlinePlus