Study On The Mechanism Of GABA_BR-Mediated Protective Effect On ER Stress-induced Apoptosis In Neurons

Gamma-aminobutyric acid (GABA) type B receptor is a type of metabotropicreceptors and belongs to the G-protein coupled receptors (GPCRs) family C. GABA_BR iswidely distributed in the mammalian central neural system where it mediates slow andprolonged inhibition of synaptic transmission. However, in recent years, GABA_Breceptorhas been found exist in various tissues besides the central neural system. Studies haveshown that GABA_Breceptor activation can trans-activates IGF-1R which mediates thePI3K/AKT downstream signaling pathways such as PI3K/AKT, ERK activation,PI3K/AKT, ERK signals can regulate cell survival.The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells andresponsible for the synthesis, processing and transfer of nascent protein. In addition, ER isan important place for lipid synthesis and calcium storage. Various stimulations includinghypoxia, hunger can lead to unfolded or misfolded proteins increasing and calcium flowdisorders in ER, causing ER stress response. Extreme or prolonged external stimulationsbeyond the ER stress response can cause cell apoptosis.The crosstalk between ER stress and GABA_Breceptor-mediated signaling has notbeen reported although they are involved in many important physiological andpathological processes. In this study, the effect of GABA_Breceptor activation-mediatedsignaling on ER stress response was investigated in cerebellar granule neurons (CGNs).CGNs were pretreated with GABA_Breceptor agonist baclofen, MTT and PI staining assayshowed that GABA_Breceptor activation inhibited thapsigargin (TG, one ER stressinducer)-induced apoptosis. The effect of baclofen on ER stress-induced apoptosis signalwas detected by Western Blot. Baclofen pretreatment down-regulated the level ofCaspase-3activation and CHOP expression induced by TG. These results indicated thatGABA_Breceptor activation protect CGNs form ER stress-induced apoptosis. To study theimpact of the GABA_Breceptor activation on ER stress signaling, ER stress-related threesignaling pathways were detected. Results showed that pretreatment of baclofen had noeffect on phosphorylation of IRE1, XBP1mRNA splicing, and Bip expression, but significantly weaked the PERK/p-eIF2α/ATF4signaling. Co-immunoprecipitationexperiment was carried out to detect potential interaction between AKT and PERK. It wasinteresting to find that PERK interacted with AKT constitutively in CGNs, and eitherbaclofen or TG treatment had no effect on this interaction. We presumed that activatedAKT induced by baclofen downregulates PERK activity by interacting with PERK. Toconfirm the effect of PI3K/AKT pathway on ER stress-induced PERK signaling, PI3Kinhibitor Wortmannin was used to pretreat CGNs, followed by detection of caspase-3cleavage after both baclofen and TG treatment. Data showed that wortmannin obviouslyabrogated baclofen-mediated inhibitory effect on TG-induced caspase-3avtivation, assuggested that AKT pathway mediated the protective effect of baclofen on ER-stressedneurons.In summary, this study confirmed that the GABA_Breceptor activation can protectCGNs from ER stress-induced apoptosis. GABA_Breceptor negatively regulated PERKpathway and down-regulated CHOP expression and caspase-3cleavage induced by ERstress via activation of PI3K/AKT pathway, thereby enhancing the survival of CGNssubjected to ER stress. Our results here will contribute to the exploration of signalingnetwork responsible for GABA_Breceptor-mediated long-term effects, and also will throwlight on related mechanism underlying either GABA_Breceptor or ER stress-involvedphysiological and pathological processes.