Research On The Role Of GSK3β In ER Stress Response Of Cerebellar Granule Neurons

Endoplasmic Reticulum is an important organelle in eukaryotic cells, which charges in protein synthesis, folding, modification and protein translocation and so on. Disequilibrium between this ER load and folding capacity is referred to as ER stress, which can activate UPR (Unfolded Protein Response) to improve the ability to process proteins of ER. Prolonged stress leads to apoptosis and may thus be an important factor in the pathogenesis of many diseases, including some neurodegenerative diseases. Glycogen synthase kinase 3β(GSK3β), a serine-threonine kinase with constitutive activation, is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. Recently it has been identified that activation of GSK3βplays a role in ER stress-induced apoptosis. However, the mechanisms mediating ER stress-induced activation of GSK3βand cell apoptosis remain incompletely elucidated.To further study the role of GSK3βin ER stress in cerebellar neurons (CGNs), two ER stress inducers, Tunicamycin (TM), an inhibitor of N-linked glycosylation, and 1,4–Dithiothreitol (DTT), were used in the study. CGNs displayed different sensitivity to TM and DTT treatment, with DTT generating a more serious apoptotic response. TM showed a lower toxicity towards CGNs than DTT since more than 60% CGNs survived after 24 hours treatment even when TM was used at a concentration of 50μg/ml, while only 30% CGNs survived after 24 hours in the presence of DTT used at 1 mM. Phosphorylation at the N terminal ser9 of GSK3βhas an inhibitory effect on GSK3βactivity and plays an important role in regulation of GSK3βfunction. Western blot analysis showed that the phosphorylation of ser-9-GSK3βincreased in TM-treated CGNs, whereas DTT treatment resulted in dephosphorylation (activation) of GSK3β. Previous studies have indicated that several kinases, such as Akt and JNK, are involved in regulating GSK3βactivation. Therefore, kinetics of Akt and JNK activation were detected in CGNs after TM or DTT treatment. Our data showed that TM induced transient JNK activation with the peak value appearing at five minutes, and pretreatment with JNK inhibitor, SP600125, led to both GSK3βand caspase-3 activation. While DTT induced prolonged JNK activation lasting about 6 hours with no implication in regulation of GSK3βactivation when JNK was inhibited. We further investigated the role of GSK3βin ER stress-induced apoptosis. Pretreatment with GSK3βinhibitor, LiCl, decreased the TM-induced CHOP expression and caspase-3 activation, but showed less effect on caspase-3 activation induced by DTT, which suggested that transient JNK activation is a protective event in CGNs via phosphorylation of GSK3βat ser-9. In addition, we detected the protein expression of protein phosphatase 2A (PP2A). Upon TM and DTT treatment, the levels of PP2A protein expression were differentially altered. TM treatment resulted in an obvious increase of PP2A expression, which was consistent with TM-induced transient JNK activation.In a word, TM-induced transient JNK activation contributes to ser9-GSK3βphosphorylation, which influence the survival of CGNs. PP2A may play a role in ER stress-induced cell apoptosis.