Preliminary Study On The CREB Signaling Pathway Induced By GABA_B Receptor In Cerebella Granule Neurons

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate CNS (central nervous system). The biological effects of GABA are mediated by GABA receptors. There are two kinds of GABA receptors in CNS, one of them is GABAB receptor, which belongs to metabotropic G-protein coupled receptors (GPCR) and mediates slow and prolonged synaptic inhibition. The GABAB receptor is a heterodimer that is composed of two distinct subunits, GB1 and GB2, which is broadly distributed in neurons and involves in many physiological procedures. Recent studies suggest GABAB receptor can promote neuronal survival involving CREB(cAMP response element-binding protein) activation, but the molecular mechanism underlying this neuroprotection is poorly characterized.In this study, I mainly study on the CREB activation induced by GABAB receptor in cerebellar granule neurons (CGN). I incubate the CGN cells with baclofen which is the agonist of GABAB receptor. I found that activation of GABAB receptor could rapidly stimulate CREB peaked at 10 min,and then decreased. I incubate the CGN cells with CGP54626 which is the specific inhibitor of GABAB receptor , it repressed the activation of CREB . I also found that CGP7930 which is the agonist of GB2 could rapidly stimulate the CREB activation. The activation of CREB incubated by baclofen follows the activation of the Gi/o protein, and it can be blocked by the pretreatment with the inhibitor of Gi/o protein PTX. I pretreat CGN with the PLC inhibitor U73122 and the Calcium chelator BAPTA in independent experiments, then incubating with baclofen and the activation of CREB are blocked. RNAi-mediated knockdown experiments of IGF-R blocked baclofen-induced CREB phosphorylation in MEF (mouse embryonic fibroblast) cells coexpressing GABAB1 and GABAB2 .I pretreat CGN with the Src inhibitor PP2 and the PI3K inhibitor LY294002 in independent experiments, then incubating with baclofen and the activation of CREB are blocked.Taken together, this research revealed that the activation of GABAB receptor can activate CREB through a mechanism that involves transactivation of the IGF-1 receptor (IGF-1R). Further work demonstrated that this cross talk was dependent on Gi/o-protein, PLC and cytosolic Ca2+, while IGF-1R-induced signaling involved Src kinase, PI3 kinase, and CREB activation.This research made a foundation for the further investigation in the mechanism of the CREB signaling pathway and for the investigation of the physiological functions of this pathway.