Roles of CREB in the regulation of FMRP by group I metabotropic glutamate receptors in cingulate cortex

Abstract

Background: Fragile X syndrome is caused by lack of fragile X mental retardation protein (FMRP) due to silencing of the FMR1 gene. The metabotropic glutamate receptors (mGluRs) in the central nervous system contribute to higher brain functions including learning/memory, mental disorders and persistent pain. The transcription factor cyclic AMP-responsive element binding protein (CREB) is involved in important neuronal functions, such as synaptic plasticity and neuronal survival. Our recent study has shown that stimulation of Group I mGluRs upregulated FMRP and activated CREB in anterior cingulate cortex (ACC), a key region for brain cognitive and executive functions, suggesting that activation of Group I mGluRs may upregulate FMRP through CREB signaling pathway.

Results: In this study, we demonstrate that CREB contributes to the regulation of FMRP by Group I mGluRs. In ACC neurons of adult mice overexpressing dominant active CREB mutant, the upregulation of FMRP by stimulating Group I mGluR is enhanced compared to wild-type mice. However, the regulation of FMRP by Group I mGluRs is not altered by overexpression of Ca2+-insensitive mutant form of downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor involved in synaptic transmission and plasticity.

Conclusion: Our study has provided further evidence for CREB involvement in regulation of FMRP by Group I mGluRs in ACC neurons, and may help to elucidate the pathogenesis of fragile X syndrome.

Figure 1

Upregulation of FMRP by Group I mGluRs was enhanced in ACC from CREB mutant mice, whereas it was not affected in transgenic mice overexpressing a Ca²⁺-insensitive DREAM mutant (TgDREAM). A, The basal levels of FMRP in ACC slices of CREB mutant mice were not affected. B, The increase of FMRP after treatment with Group I mGluR agonist DHPG (100 μM) for 30 min, was enhanced in ACC slices from CREB mutant mice, as compared to wild-type (WT) mice. Representative Western blot (top) and quantification data (bottom) of FMRP are shown for the corresponding treatments. C, The basal levels of FMRP in ACC slices of TgDREAM mice were not affected. D, The increase of FMRP after treatment with Group I mGluR agonist DHPG (100 μM) for 30 min, was not changed in ACC slices from TgDREAM mutant mice, as compared to wild-type (WT) mice. Representative Western blot (top) and quantification data (bottom) of FMRP are shown for the corresponding treatments. Data were normalized by WT control values. ** P < 0.01, compared to control mice; # P < 0.05, compared to WT DHPG treatment. n = 5 mice for each group.

Figure 2

Putative CREs in the FMR1 promoter. A, Conserved regions among multiple placental mammalian species were identified by UCSC Genome browser. Two putative CREs are indicated with yellow. TSS; transcription start site. B, Two putative CREs are highly conserved across species (mouse, rat, human, cow, opossum). Conserved CRE sequences are highlighted in yellow.

Figure 3

The signaling pathway for CREB in the regulation of FMRP by Group I mGluRs in ACC neurons. Stimulation of mGluR1/5 triggers the Ca²⁺ release from intracellular calcium stores by IP3 and Ca²⁺ influx from L-VDCCs through membrane depolarization. The increase of Ca²⁺ leads to activation of Ca²⁺-calmodulin (CaM) dependent pathways, including Ca²⁺ and CaM stimulated AC1-cAMP dependent protein kinase (PKA) and CaMKIV. PKA and CaMKIV then phosphorylates CREB. Phosphorylated CREB (pCREB) initiates the CREB-dependent transcription of Fmr1 gene and upregulates FMRP in the cytoplasm. The mutant CREB (Y134F) contributes to transcription of Fmr1 gene, whereas DREAM may not be involved in Fmr1 gene expression. FMRP may interact with its interactors and modulate neuronal functions in ACC.