TLRs Mediated The Changes Of GLUTs And MCTs Expression In Neurons And Its Mechanism

Glucose is the main fuel for neuronal activity. Neural activities cause the increase ofenergy demand, which is met by oxidative metabolism of glucose. In most mammaliancells, glucose is transported by glucose transporters (GLUTs) family. In the mammalianbrain, GLUT1and GLUT3play the main role to transport glucose. Brain neurons expressGLUT3, GLUT6and GLUT8. Insulin, nerve growth factors or H2O2can regulate theGLUT3expression and the glucose uptake by the PI3K-AKT, MAPKs, cAMP or NF-kBsignal transduction pathways. In recent years, the astrocyte-neuron lactate shuttlehypothesis (ANLSH) consider that during the neural activities, neurons intake lactic acidwhich is released from glial cells as its main substrate for energy metabolism. Lactic acidfrom glycolysis is transported out of the glial cells through the cell membranemonocarboxylate transporters (MCTs), and is easily taken in by neuron through its ownMCT. MCTs transport substances including lactic acid, pyruvic acid and acetoacetate,β-hydroxybutyric acid, etc. The central nervous system (CNS) expresses MCT1, MCT2andMCT4, while MCT2is expressed mainly in neurons. The study found that norepinephrineincreased MCT2expression through the PI3K-Akt and mTOR/S6K pathways. There havebeen reported, when the neurons in the environment of glucose and lactic acid-rich, theywill first use lactic acid as the main oxidation substrate. Even in the case of normal bloodglucose, lactic acid can also be in preference to glucose metabolism as the fuel of the brain.Toll-like receptors (TLRs) are the important pattern recognition receptors (PRRs),which are able to recognize pathogen-associated molecular patterns (PAMPs), andconstitute the first line of the body defense against pathogen invasion. The availableliterature shows that, TLRs express in the CNS of the human and mouse, and indicates thatthey play an important role in a series of infectious and non-infectious diseases of brain. Inrecent years, more and more evidence show that TLRs are not only expressed on glial cells,but also on neurons. Among the several members of the TLR family, TLR3and TLR8werefirst reported in human neuronal cell lines and primary cultured mouse cortical neurons,respectively. Mouse neurons express TLR1-8and TLR10-13mRNA and the proteins ofTLR2and TLR6. After the neurons were infected by virus, the expression of TLR2, TLR4,TLR6, TLR7and TLR8was induced. It is reported that the stimulation of TLR9reduceenergy substrates and increase the AMP/ATP ratio, subsequently activating AMP-activatedkinase (AMPK), leading to increased stress tolerance against hypoxia in cardiomyocyteswithout inducing the canonical inflammatory response. In bone marrow derived adipocytes, GLUT4expression was downregulated after stimulated with the TLR4or TLR3specificligand, LPS or poly (I:C), respectively, and resulted to decreased insulin-induced glucoseuptake and increased lipolysis. Another report showed that TLR2and TLR4expressionwas increased in cerebral cortical neurons in response to energy lack, which lead to neurondeath. These results suggest that TLRs play a very important role in the energy metabolismof cells. In the virus infection of the neurons, ssRNA and dsRNA were synthesized in thereplication and transcription processes of the virus, and could be be recongnized by TLR3,TLR7and TLR8as pathogen associated molecular patterns, and then initiated immuneresponse. In this project, we used poly (I:C), a synthetic dsRNA, and R848, a ligand forTLR7and TLR8, as dsRNA and ssRNA to stimulate the neurons cultured in vitro, andstudied whether dsRNA interacted with TLR3or ssRNA interacted with TLR7/8wouldaffect the expression of neuronal GLUTs and MCTs, the uptake of glucose and lactic acidby neurons, and the survival of neurons in order to investigate the mechanism of virusdsRNA and ssRNA cause nerve injury through TLRs, and provide a new evidence forexploring the prevention and treatment measures of infectious diseases in nervous system.PartⅠEffect of TLR3or TLR7/8activation on the survival of the mouseembryonic cortical neurons in vitroObjective: To observe the effects of TLR3pathway activated by poly(I:C) andTLR7/8pathway activated by R848on the survival of the cultured mouse embryoniccortical neurons in vitro. Methods: The wild-type and TLR3-/-C57BL/6mouses with16-18days of pregnancy were chosed and sacrificed. The cerebral cortex of the embryonicmouse was removed into a dish, cut into pieces, digested by trypsin and made into singlecell suspension. At last, the cell suspension was seeded in6/12-well plates pretreated withpoly-L-lysine and cultured in Neurobasal medium (containing2%B27,1%fetal bovineserum and1%glutamine) in vitro. Half the amount of medium was replaced with freshneurobasal medium every three days, and the cells could be used for the experiment8daysafter the culture. The neuron purity was detected by immunofluorescence assay, in whichMAP2was used as a specific marker of neuronal cells, and the nuclei of all cells werestained by DAPI. Under a fluorescence microscope, the number of cells expressing MAP2and the number of DAPI stained cell nucleus were counted, and calculated the percentageof neurons according to the formula, the number of cells expressing MAP2/the number ofcell nucleus. The neurons cultured for8days were stimulated by poly (I:C) or R848for different time period, and the cell activity of the wild-type and TLR3-/-neurons weredetected by MTT assay and flow cytometry. Results: The purity of the mouse embryoniccortical neurons was more than90%, and the cultured neurons could be used for otherexperiment. After24hours of poly(I:C) or R848stimulation, the cell activity of thewild-type mouse embryonic cortical neurons decreased significantly, compared with thecontrol group, while the cell activity of the TLR3-/-neurons had no significant differencebetween poly(I:C) stimulated group and control group. Flow cytometry analysis showedthat the early and late apoptosis of the wild-type mouse embryonic cortical neuronstimulated by poly(I:C) for24hours increased significantly, compared with control group,but no significant difference between the poly(I:C) stimulated group and control group inthe TLR3-/-mouse embryonic cortical neurons. Conclusion: The activation of TLR3orTLR7/8in the mouse embryonic cortical neurons decreases the neuronal survival.Part Ⅱ TLR3or TLR7/8mediated changes of the expression of GLUTsand MCTs in mouse embryonic cortical neuronsObjective: To detect the effects of poly(I:C) activated TLR3pathway and R848activated TLR7/8pathway on the expression of GLUTs and MCTs in mouse embryoniccortical neurons. Methods: The wild-type and TLR3-/-mouse embryonic cortical neuronswere cultured in vitro for8days and made sure the purity of the neurons was more than90%. The cells were divided into the control group, the groups stimulated by20μg/ml finalconcentration of poly(I:C) for different time periods (1,4,10or5hours), the groupsstimulated by different concentrations (10,20or40μg/ml) of poly(I:C) for10hours, thegroups stimulated by30μg/ml final concentration of R848for different time periods (1,4,10or5hours) and the groups stimulated by different concentrations (5,30and60μg/ml)of R848for12hours. Total RNA was extracted from the neuronal cells in the differentgroups, and the reverse transcript was performed, then mRNA expression of GLUTs andMCTs was detected by Real-time qPCR. The mouse embryonic cortical neurons culturedfor8days were divided into the control group, the groups stimulated with20μg/ml finalconcentration of poly(I:C) for different time periods (12,24or36hours), the groupsstimulated with different concentrations (10,20or40μg/ml) of poly(I:C) for24hours, thegroups stimulated with30μg/ml final concentration of R848for different time periods (12,24and36hours) and the groups stimulated with different concentrations (5,30or60μg/ml) of R848for24hours. The protein expression of GLUTs and MCTs of the neuronal cells from different groups was examined by Western Blotting. Results: After thewild-type neurons were stimulated by poly(I:C) for10hours, the mRNA expression levelsof GLUT3, GLUT8and MCT2in stimulated group were significant lower than controlgroup, and this result was not observed in TLR3-/-mouse embryonic cortical neurons. Theexpression of GLUT3, GLUT8and MCT2proteins decreased significantly after thewild-type neurons were stimulated by poly(I:C) for24hours compared with control group,while the mRNA and protein expression of GLUT3and MCT2increased after the neuronswere stimulated by R848. Conclusion: The expression of GLUT3, GLUT8and MCT2decreased after the mouse embryonic cortical neurons were stimulated by poly(I:C), whilethe expression of GLUT3and MCT2increased after stimulated by R848.Part Ⅲ Mechanisms of the effects of TLR3and TLR7/8pathways onGLUTs and MCTs expression in mouse embryonic cortical neuronsObjective: To study the mechanisms of the effects of TLR3and TLR7/8pathways onGLUTs and MCTs expression in mouse embryonic cortical neurons. Method: Thewild-type and TLR3-/-mouse embryonic cortical neurons were cultured in vitro for8days,and made sure the purity of the neurons was more than90%. The neurons were dividedinto control group,20μg/ml final concentration of poly(I:C) stimulated group,30μg/mlfinal concentration of R848stimulated group,20μg/ml poly(I:C)+PDTC or bx-795stimulated group, PDTC or bx-795stimulated group. The mRNAs and proteins of IFN-α,IFN-β, TNF-α and IL-6were detected by Real-time qPCR and ELISA, respectively. Thephosphorylations of TBK1, IRF3, IRF7, p65, p38, ERK and JNK were detected by WesternBlotting. The neurons were pretreated with PDTC, a NF-κB inhibitor, or bx-795, a TBK1inhibitor, or without pretreatment, and then unstimulated or stimulated by poly(I:C). TheReal-time qPCR and Western Blotting were used to detect the mRNA and protein levels ofGLUT3、GLUT8and MCT2. Results: The mRNA expression of IFN-α, IFN-β, TNF-α andIL-6were significantly increased in wild-type mouse embryonic cortical neuronsstimulated by poly(I:C) for10hours or by R848for12hours. The levels of IL-6and IFN-βwere significantly increased in poly(I:C) stimulated group, while only IFN-β protein wassignificantly increased in R848stimulated group. The phosphorylations of TBK1, IRF3,IRF7and p65of poly(I:C) stimulated group were higher than control group, while thephosphorylations of p38, ERK and JNK were not changed significantly. After thepretreatments of PDTC and bx-795to inhibit NF-κB and TBK1activation in neurons, respectively, the results showed that it was PDTC, but not bx-795, inhibited thedown-regulation of GLUT3, GLUT8and MCT2in neurons stimulated by poly(I:C).Conclusion: The suppressive effects of poly(I:C) on the expression of GLUT3, GULT8and MCT2may be through TLR3-mediated NF-κB signal transduction pathway，and causethe energy metabolism disorder，which affects the survival of the neurons and may berelated to the cytokines induced by TLR3or TLR7/8activation.