The Role Of NLRC5 On The Expression Regulation Of Classical MHC Class?molecules In Neurons And The Development Of Neurons

Major histocompatibility complex?(MHC?)molecules are cell surface receptors,which are widely expressed on the surface of nucleated cells and play a key role in the regulation of adaptive cytotoxic immune response.Recent studies have shown that MHC class?molecules are expressed in the cerebral cortex,hippocampus,cerebellum of developing and adult mammals.They participate in important events such as synapse formation,synaptic plasticity,and motor learning and memory,and play a role completely different from the immune system.The expression pattern of classical MHC class?molecules in the central nervous system(CNS)is a specific spatial-temporal constitutive expression during the developmental period,which plays a physiological role in the process of synaptic plasticity and its dependent motor learning.In addition,MHC class?molecules expression can be induced in neurons during several pathological conditions such as stroke.Although many important functions of MHC class?molecules in developing neurons have been investigated,the mechanism that govern neuronal MHC class?molecules regulation remains unknown.NLRC5 is the largest member of the NLR protein family.Both in vitro and in vivo studies have shown that NLRC5 is a key regulator of the expression of MHC class?molecules in the immune system and its regulation is cellspecific.Whether NLRC5 is expressed in neurons and regulates MHC class?molecules,and its role in neuron development has not been reported.In this study,we examined the expression and localization of NLRC5 in hippocampal neurons,and explored the regulatory mechanism of NLRC5 on the expression of classical MHC class?molecules in neurons in vitro and in vivo.In addition,Nlrc5-/-mice exhibited abnormal neuronal dendritic branch development.Our results include the following several parts:1.The expression trend of m RNA and protein of classical MHC class?molecules in the hippocampus of mice from postnatal to adult(P0 to P60)was inconsistent.The expression levels of classical MHC?(H2-K and H2-D)m RNA gradually increased from P0 to P60,and MHC?proteins were increased from P0 to P15 and peaked at P8 to P15,then significantly declined at later ages while H2-K and H2-D m RNA remained steady at high level.The m RNA and protein expression trends of classical MHC class?molecules from P0 to P15 were consistent,suggesting that this period was mainly regulated by the transcription level.In addition,we detected that NLRC5 was widely expressed in hippocampal tissues,and the expression trend of NLRC5 in the period from P0 to P15 was similar to that of classical MHC class?molecules.Further immunofluorescence results showed that NLRC5 was expressed in both cytoplasm and nucleus of hippocampal neurons,and co-localize with classical MHC class?molecules.These results suggest that NLRC5 may be involved in the regulation of the expression of classical MHC class?molecules during hippocampal development.2.The expression of H2-D m RNA and MHC class?protein were significantly increased upon NLRC5 expression in vitro.NLRC5 also induced the expression of MHC class?complex light chain gene(?2m)and also genes involved in MHC class?antigen presenting: Tap1,Tap2,Lmp2,Lmp7,Lmp10,and Tapasin.Nlrc5-/-mice showed a significant reduction of both H2-K,H2-D and ?2m expression from P0 to P60,while the expression of other antigen-presentingrelated molecules was not significantly affected.These results suggest that NLRC5 plays an important role in the constitutive expression of classical MHC class?molecules in neurons.3.The expression of NLRC5 significantly increased H2-D and H2-K luciferase reporter expression in Neuro-2a cells.In addition,deletion of any of the three motifs(W/S,X,or Y)individually,markedly attenuated NLRC5-induced H2-D promoter activity.Expression of the RFX complex(RFX5,RFXAP,RFXANK)or CREB1 on X1 box markedly enhanced NLRC5-induced transactivation of H2-D.Furthermore,knockdown of RFX complex or CREB1 in Neuro-2a cells significantly decreased the transcriptional activity of H2-D.These findings suggest that the X motif is crucial for NLRC5-mediated transactivation in neurons.The result of Immunoprecipitation experiments showed that His-RFX5 and His-RFXANK each coprecipitated with FLAG-NLRC5,suggesting that RFXANK and RFX5 were essential for recruiting NLRC5 to the protein complex in neuronal cells.4.NLRC5 was expressed in the soma,dendrites and growth cones of hippocampal neurons in early primary culture.The dendrites of wild type and Nlrc5-/-mice in primary culture were analyzed from 3 DIV,8 DIV and 15 DIV.The results of Sholl analysis showed that the complexity of dendritic branches in Nlrc5-/-mice increased significantly.In addition,the expression of classical MHC class I molecules H2-K and H2-D were impaired in primary hippocampal neurons of Nlrc5-/-mice.The rescue of H2-D expression in Nlrc5-/-mice with FUGW-H2-D lentivirus can significantly improve the phenotype of abnormal dendritic branch development.These results suggest that NLRC5 may regulate dendritic branch development through classical MHC class?molecules.These results indicate that NLRC5 is widely expressed in mouse hippocampal neurons and regulates the up-regulated expression of classical MHC class?molecules in neurons.NLRC5 induces the transcriptional activation of MHC class?genes required the W/S-X-Y motifs.The transcription factor RFX complex and CREB1 on the X motif are essential for NLRC5 mediated transcription activation of MHC class?genes.RFX5 and RFXANK in the RFX complex are responsible for recruiting NLRC5 to the MHC class?gene promoter region.In addition,we found that NLRC5 is expressed in the dendrites and dendrite growth cones of neurons,and regulates the development of dendritic branches of hippocampal neurons in vitro by classical MHC class?molecules.This study analyzed the key regulatory factors and regulatory mechanisms of up-regulation of classical MHC class?molecules in hippocampal neurons,and preliminarily explored the new functions of the immune molecule NLRC5 in the central nervous system,providing an important experimental basis for further understanding of neuron development mechanism and neuro immune mechanism.