Role Of O-GlcNAcylation In Neurological Disorders

Part I Diverse Regulation of the Insulin-PI3K-AKT Signaling Pathway by O-GlcNAcylation in Various Types of CellsObjective:To investigate how O-GlcNAcylation regulates insulin-PI3K-AKT signaling in various types of cells.Methods:HEK293FT, HepG2, mouse neuroblastoma N2a cells, and mouse primary hippocampal neuronal cells were used for this study. Alteration of O-GlcNAcylation level was achieved by various means, including over-expression and knock-down of O-GlcNAc trans ferase and treatments of cells with selective inhibitors of the key enzymes regulating protein O-GlcNAcylation. O-GlcNAcylated proteins were analyzed by immunoprecipitation and Western blots with monoclonal antibodies RL2and CTD110.6. Akt and GSK-3β phosphorylation and their substrates phosphorylation were determined by Western blots.Results:By employing immunoprecipitation and Western blots, we found protein kinase B (AKT) and glycogen synthase kinase-3β(GSK-3β) to be the major O-GlcNAc-modified components of the insulin-PI3K-AKT signaling pathway in HEK-293FT cells (human embryonic kidney cells transformed with the SV40large T antigen). By using both molecular and pharmacological approaches to modify cellular O-GlcNAcylation in various types of cells, we observed that O-GlcNAcylation regulated phosphorylation of AKT and GSK-3β in different manners in HEK-293FT cells, but did not affect these two kinases in human hepatocellular carcinoma HepG2cells. In neuroblastoma N2a cells and primary mouse hippocampal neuronal cells, O-GlcNAcylation regulated phosphorylation of AKT negatively, but had no effect on GSK-3β phosphorylation.Conclusions:These results demonstrate protein-specific and cell type-specific regulation of AKT and GSK-3β by O-GlcNAcylation. Our findings suggest that O-GlcNAcylation may regulate insulin-PI3K-AKT signaling in a tissue-and cell type-specific manner.Part Ⅱ Involvement of O-GlcNAcylation in neuronal apoptosis of cerebral ischemiaObjective:To investigate the involvement of O-GlcNAcylation in the apoptosis induced by cerebral ischemiaMethods:O-GlcNAcylation level and apoptotic markers in mouse hippocampus of middle cerebral artery occlusion (MCAO) model were measured by using Western blots or immunohistochemical staining. To determinethe role of O-GlcNAcylation in apoptotosis, we altered O-GlcNAcylation level molecularly in HEK-293FT cells and detected apoptotic. markers with different methods. To investigate the molecular mechanism of O-GlcNAcylation in apoptosis, we analyzed AKT phosphorylation and activity, and levels of phosphorylated Bad, cleaved Caspase3and PARP with Western blots.Results:We found an elevation of O-GlcNAcylation level and apoptosis in hippocampi of mouse with MCAO for2or4h. Overexpression of OGT in HEK-293FT cells led to decreased cell viability, cell morphological changes such as condensed and/or fragmented nuclei, increased apoptotic cell number as determined by flow cytometry, and enhanced caspase3activity. Up-regulation of O-GlcNAcylation inhibitedphosphorylation of AKT at Thr308and Ser473sites and its activity, resulting in activation of caspase3and cell apoptosis. By co-immunoprecipitation and mutagenesis, we found that AKT was O-GlcNAcylated at both Thr308and Ser473. Overexpression of AKT attenuated OGT-induced apoptotic events, but not mutated AKT. In ischemic models, phosphorylation of AKT is correlated to the O-GlcNAcylation negatively.Conclusions:Our results suggest that cerebral ischemia-induced elevation of O-GlcNAcylation down regulates AKT activity by inhibition of its phosphorylation which leads activation of caspase3and neuronal apoptosis.Part Ⅲ Decreased O-GlcNAcylation in Alzheimer disease brain affects FoxO1-mediated autophagy and tau pathogenesisObjective:To investigate the mechanism by which O-GlcNAcylaion regulates FoxO1function and the relationship of FoxO1-mediated autophagy with the accumulation of tau in Alzheimer disease brainMethods:The levels of FoxO1protein and its phosphorylation were measured by Western blot, and the level of mRNA was measured by Q-PCR. Different inhibitors of transcription, translation, autophagy-lysosome pathway, and ubiquitin-proteasome pathway were used to treat cells to study by which O-GlcNAcylation regulates FoxO1protein levels. O-GlcNAcylated proteins, ubiquitinated proteins and FoxO1were immunoprecipitated to study how O-GlcNAcylation affected FoxO1degradation. FoxO1regulated target gene expressions and autophagy were measured with Q-PCR and Western blot. The levels of FoxO1, AKT, and their phosphorylation and autophagy functions were measured in the forebrain-specific knock out OGT mice. One-trail object recognition, Morris water maze, reversal Morris water maze were used to study the learning and memory fuction in OGT KO mice. The levels of O-GlcNAcylation, FoxO1, autophagy functions and tau were studied by Western blot in AD brains.Results:The alteration of O-GlcNAcylation in HEK-293FT cells not only affected FoxO1phosphorylation by the change of AKT kinase activity, but also regulated FoxO1protein level. O-GlcNAcylation of FoxO1suppressed its ubiquitination and degradation by the ubiquitin-proteasome pathway, resulting in an increased FoxO1. Up-regulation of O-GlcNAcylation enhanced FoxO1transcriptional activity and function of autophagy, but did not affect its subcellular distribution. Down-regulation of O-GlcNAcylation by knock-out of OGT in mice forebrain neurons led to decreased AKT kinase activity and FoxO1protein levels, as well as autophagy functions. Short-term memory and episodic-like memory were deficit in these KO mice. Both O-GlcNAcylation level and foxO1in AD brains were decreased, related to impairment of autophagy identified by increased p62protein levels and decreased LC3protein levels. Decreased FoxO1in AD brains was negatively correlated with the level of total tau and aggregated tau, suggesting impaired foxO1-autophagy may contribute to the accumulation of tau in AD brain.Conclusions:Our results suggest that FoxO1is O-GlcNAcylated and O-GlcNAcylation of FoxOl prevented its degradation by proteasome. Dowe-regulation of O-GlcNAcylation in AD brains promotes FoxO1degradation and suppresses autophagy functions, leading to impairment in clearance of aggregated tau and accumulation of tau.