The Role Of NLRC5 In Dendritic Development And Synapse Formation In Hippocampal Neurons And It’s Effects On Mouse Behavior

NOD-like receptors(NLRs)are one of the pattern recognition receptors(PRRs)that accurately recognize invading pathogens through pathogen-associated molecular patterns(PAMPs),thereby triggering the body’s inflammatory response and antiviral signaling pathways.NLRC5 is the largest member of the NLR family,expressed and highly conserved in a variety of species,and plays an important role in the immune system.NLRC5 mediate innate and antiviral immune responses.It is involved in tumor immune surveillance as MHC class I transactivator(CITA).In recent years,it has been found that NLRC5 exerts non-immunological functions in the central nervous system and participate in the regulation of a series of neurological and psychiatric diseases.NLRC5 may play a neuroprotective role in cerebral ischemia-reperfusion(I/R)injury,perform antiviral functions in nervous system infections such as Neuro HIV and neurotropic virus,and is involved in malignant progression of glioblastoma(GBM).However,people know nothing about how NLRC5 is expressed and located in the brain,whether it can affect the development of the nervous system.The main findings are as follows:1.The expression pattern of NLRC5 in the hippocampus Objective: To explore the expression pattern of NLRC5 in mouse brain.Methods: The spatiotemporal expression pattern of NLRC5 in mouse hippocampus and its localization in different cell types were tested by Western Blot and Immunofluorescence;The expression level of NLRC5 was detected in cultured microglia and neurons.Results: In the hippocampus,NLRC5 protein could be detected from P0.It’s expression gradually stabilized with the development of mice.NLRC5 colocalized extensively with the neuron-specific marker Neu N in the cortex and hippocampus.NLRC5 was hardly expressed in astrocytes,however,it was also found in primary cultured microglia.The above findings suggested that NLRC5 might play an important role in the development of the brain,especially the hippocampus.2.Effect of NLRC5 on dendrite development in mouse hippocampal neurons Objective: NLRC5 is highly expressed in mouse hippocampus and neurons.Therefore,it is necessary to study the effect of NLRC5 on neuronal development and dendritic growth.Methods: Golgi staining and Immunofluorescence were used to explore the effect of NLRC5 on the dendritic morphology of neurons in vitro and in vivo.The possible regulatory mechanisms were explored in primary neurons by virus infection.Results: Studies in vivo suggested that NLRC5 regulate dendritic development of pyramidal neurons in the hippocampal CA1 region.Nlrc5-/-mice exhibited increased dendritic length and complexity during development,however,there is no significant difference in adult neurons.The number of dendritic spines in NLRC5-knockout hippocampal neurons was relatively normal.In cultured Nlrc5-/-hippocampal neurons,dendritic length and complexity were also Increased at DIV 3 and 8.These results suggested that NLRC5,as an important regulator,is involved in the development of neuronal dendrites.Overexpression of Classical MHC class I molecules in NLRC5 knockout neurons ameliorated the abnormal dendritic morphology,suggesting that NLRC5 regulated dendritic development through classical MHC class I molecules.3.Role of NLRC5 in synapse formation in the developing mouse hippocampus Objective: The dendrites of the hippocampal neurons of Nlrc5-/-mice are abnormal during development,therefore it is necessary to further study the function of NLRC5 on synapse formation.Methods: The expression levels of proteins related to excitatory synapses and inhibitory synapses in the mouse hippocampus were detected by Western Blot and tissue immunofluorescence;Synapse structures in the CA region of the mouse hippocampus were observed by electron microscopy;The frequency and amplitude of m EPSC and m IPSC in physiological state of mouse hippocampal CA area were detected by electrophysiology.Results: PSD95 expression of Nlrc5-/-mice hippocampus was abnormal during development.Staining in vivo showed that NLRC5 colocalized with excitatory synapses and inhibitory synapses.Statistically,it was found that the formation of excitatory and inhibitory synapses was impaired in Nlrc5-/-mice at P15.Electron microscopy results showed that the number of excitatory synapses,the number of synaptic vesicles and the width of synaptic clefts increased significantly in Nlrc5-/-mice during development.Thus,synapse formation in developing hippocampal neurons was regulated by NLRC5.The frequency and amplitude of m EPSC and m IPSC in hippocampal CA1 neurons of Nlrc5-/-mice at P40 were consisitent with those of wild mice.4.The effect of NLRC5 on the physiological functions of miceObjective: Since NLRC5 affects dendritic branching and synapse formation in developing hippocampal neurons,whether physiological functions of Nlrc5-/-mice are impaired remains to be confirmed.Methods: Behavioral experiments,such as open field test,elevated zero maze test,tail suspension test and sugar preference test,three-chambered social test,Y maze and water maze test,were used to explore whether Nlrc5-/-mice had physiological dysfunction.Results: We came to the following conclusions: Nlrc5-/-mice have normal motor ability,but their anxiety and depression levels were elevated;Their ability of social interaction and social novelty were impaired;However,there was no obvious abnormality in cognitive ability such as spatial learning and memory in Nlrc5-/-mice.