Effects Of TLR7/8 On The Expression Of GLUTs And MCTs In Neurons And Its Mechanisms

Population aging phenomenon becomes more and more serious in modern society. The incidence of diseases such as senile dementia, Parkinson’s disease (PD) and cerebral apoplexy was increased. Besides that, the infectious diseases of the brain, such as human immunodeficiency virus (HIV) infection caused dementia, also seriously endanger human life.There is a close relationship between the energy metabolism of brain and the brain diseases. The research on the relationship between energy metabolism and brain diseases using advanced technology has become one of the hotspots in the brain disease research.Glucose is one of the main sources of the brain energy supply, and also the necessary energy substrate in the brain. Glucose into the brain parenchyma provides energy to the neurons and other kinds of cells through the circulation. The energy metabolism activity of the brain is mainly done by the glucose oxidation metabolism. The glucose transport depends on the glucose transporter (GLUT) on the cell membrane. Lactic acid produced by glycolysis also can be used to supply the energy to the brain. The transport of lactic acid depends on the monocarboxylate transporter (MCT) on the cell membrane. Studies have found that while plasma glucose in normal, lactic acid was first used to supply energy to the brain compared with glucose and as the priority fuel for cerebral energy metabolism, but its specific action mechanism is not yet clear. Various transporters can be detected in the neurons, including GLUT3, GLUT6, GLUT8 and MCT2, and regulate the intake of glucose and lactic acid respectively by different signal transduction pathways.Toll-like receptors (TLRs) play an important role in the innate immune responses and are highly conservative in evolution. In mammals, TLRs can recognize conservative composition of viruses or bacteria, that is, pathogen associated molecular patterns (PAMPs). After recognizing the ligands, TLRs cause the activation of related signal pathways and promot the release of inflammatory cytokines, finally clearing the invading pathogens. Although TLRs mainly play a role in the immune system in mammals, but there are also some TLRs in neurons. These TLRs have other important roles. The members of TLR family expressed in the brain are mainly TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9. In drosophila embryogenesis and embryonic development, such as synaptogenesis, the formation of neural axon and axons (output) direction, TLRs play an important role. In the mammalian central nervous system (CNS), TLRs expresse in microglia and astrocytes, and a high expression of TLRs is in the infection and tissue damage. TLRs can activate inflammatory cells in the neural degeneration.In rat embryonic development process, TLR3 inhibits the neural progenitor cell proliferation in the telencephalon and mediates the axon growth. TLR2 and TLR4 play an important role in adult hippocampus neurogenesis. TLR7 is also expressed in the mouse brain development, which is mainly in the early embryo. In the development process of mouse, a dynamic expression of TLR8 mainly focuses on the neurons and axons. The stimulation of TLR8 ligand can inhibit the growth of axons and mediate apoptosis. The activation of TLR9 affects the AMP/ATP value within the neurons and the tolerance to hypoxia. TLR3 or TLR4 activation in fat cells can regulate the expression of GLUT4 and affect the glucose uptake and the adipose decompose.The previous studies in our laboratory have found that TLR3, mainly recognizes virus double-stranded RNA (dsRNA), play an important role in energy metabolism in neurons. TLR7 and TLR8 are the receptors mainly recognizing virus single-stranded RNA (ssRNA). Whether TLR7/8 activation is involved in the energy metabolism of the nerve cells and its specific signaling pathway has not been reported. Therefore, in this study, we investigated the effects of TLR7/8 activation on the expression of GLUTs and MCTs, which play a key role in energy metabolism of neurons, and probed the related mechanism in order to explore the roles of TLR7/8 expression in the brain diseases from a new visual angle.Ⅰ. Effects of TLR7/8 activation on neuronal activityTo investigate the effects of TLR7 or TLR8 activation on the neuron survival, the original generation of mouse fetal cerebral cortex neurons in vitro cultivation system was established in this part of the experiments. The cultured neurons were stimulated by Loxoribine, a specific ligand for TLR7, or CL075 (3M-002), a ligand for TLR7/8 but priority for TLR8, and the cell activity and apoptosis were observed at different time points with CCK8 test method and flow cytometry (FCM) analysis, respectively. The results showed that the purity of neurons cultured in vitro was more than 90%, and the cells can be used in subsequent experiments. Using CCK8 test method, a significantly decreased neuron activity was observed after the stimulation of Loxoribine or CL075 (3M-002). The results of FCM analysis showed that the neuron apoptosis in the groups treated by Loxoribine or CL075 (3M-002) increased in comparison with control group. We synthesized TLR7 interference small RNA (siRNA) further, and then the cortical neurons treated by siRNA were stimulated with two kinds of ligands, respectively. The results showed that the obvious change of the cell activity did not take place, and the significant increase of neuron apoptosis did not appeared, compared with the control. Therefore, the first part of the experimental results indicats that TLR7/8 activation can reduce the neuron activity and cause neuron apoptosis.Ⅱ. Effects of TLR7/8 on the expression of GLUTs and MCTs in neuronal cellsMany factors are able to cause neuron activity decrease or apoptosis, such as mitochondrial pathway and caspase pathway. This study mainly explored the roles of TLR signaling pathway in the energy metabolism of brain neurons. Therefore, whether TLR7 specific ligand Loxoribine or TLR7/8 ligand CL075 (3M-002) affects the expression of GLUTs and MCTs in the cultured neurons was studied in the second part of the experiment.Loxoribine or CL075 (3M-002) and neuronal cells were co-incubated for a certain time. Then the cells were collected, and the RNA and proteins were extracted to detect the expression of MCTs and GLUTs using real-time quantitative polymerase chain reaction (Real-time qPCR) and western blotting (WB). The results showed that Loxoribine could down-regulate the expression levels of MCT2, GLUT3 and GLUT8 mRNAs, but CL075 (3M-002) could up-regulate the expression levels of MCT2 and GLUT3 mRNAs. The expression levels of MCT2, GLUT3 and GLUT8 proteins in the cortical neurons decreased after stimulating with Loxoribine, but CL075 (3M-002) only increased MCT2 protein expression in the cortical neurons.The cortical neurons were treated with TLR7 siRNA in advance, and then stimulated with Loxoribine or CL075 (3M-002). The expression levels of MCT2, GLUT3 and GLUT8 mRNAs and proteins in Loxoribine stimulated group had no significant difference compared with the control group. The expression level of MCT2 in CL075 (3M-002) stimulated group had no obvious change compared with the control group. These results indicate that the stimulation with Loxoribine reduces the expression of MCT2, GLUT3 and GLUT8 mRNAs and proteins in neurons, while CL075 (3M-002) only affects MCT2 expression level. The activation of TLR7 and TLR8 is involved in glucose and lactic acid transport and the energy metabolism process of the brain neurons.Ⅲ. Mechanisms affecting the expression of GLUTs and MCTs in neurons by TLR7/8In order to study the mechanisms affecting the expression of GLUTs and MCTs in neurons after TLR7/8 activation, the mRNA expression of interferon-beta (IFN-β), interferon-alpha (IFN-α), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were detected with Real-time qPCR method after the co-incubation of Loxoribine or CL075 (3M-002) with neurons. The levels of cytokines secreted by the neurons were detected using enzyme linked immunosorbent assay (ELISA). The phosphorylation levels of TLR7/8 signaling pathways related molecules including interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7) and p65 were tested by western blot. The mRNA and protein expression levels of GLUTs and MCTs in neurons were tested with Real-time qPCR and western blot method after Loxoribine or CL075 (3M-002) stimulation following the pretreatment with NF-kappa B signaling pathway inhibitor ammonium pyrrolidinedithiocarbamate (PDTC).The results showed that Loxoribine or CL075 (3M-002) up-regulated the expression of TNF-α, IFN-α, IFN-β and IL-6 mRNAs in the neurons with different degree and enhanced the secretion levels of IFN-β and IL-6. The phosphorylation levels of IRF3, IRF7 and p65 in the cultured neurons stimulated by Loxoribine increased. After pretreating the neurons with NF-κB signaling pathway inhibitor PDTC, the down-regulatory effects of Loxoribine on the expression of MCT2, GLUT3 and GLUT8 mRNAs and proteins in the neurons could be partly inhibited, and only the up-regulatory effect of CL075 (3M-002) on the expression of MCT2 mRNA and protein could be partly suppressed while GLUT3 expression was not impacted.This part of the experimental results suggests that TLR7 may through MyD88 signaling pathways activated the NF-κB to suppress the expression of MCT2 and GLUT3 and GLUT8, causing the energy metabolism disorder in neurons and, in turn, affecing the survival of the neurons. IFN-β and IL-6 may be involved in this process. But the effect mechanism of TLR8 on the MCT2 expression of the neurons remains to be further studied.