The Role And The Molecular Mechanism Of Classical MHC ? In Determining Neurite Growth Of Primary Cortical Neurons

It has been reported that many kinds of proteins,which play an important role in immune system,can be expressed in the developing and adult brain.Among them,studies on the expression and function of classical MHC class ? molecules in the central nervous system(CNS)have become hot spots in the field of brain development and neuroimmunology.The temporal and spatial patterns of classical MHC class ? molecules expression suggest that MHC class ? plays an important role in manipulation of CNS synase plasticity.A recent paper has reported that basal dendrites of L2/3 cortical neurons are more highly branched in the MHC class ? knockout mice.In our previous study,we found that classical MHC class ?molecules are expressed by neurons in mouse cerebral cortex during the early postnatal period before development of synaptic connections.We have also found that neurons cultured from MHC class ? knockout mouse displayed faster neurite outgrowth and developed more primary neuritis than neurons from wild type mouse.The results suggest that classical MHC class ? molecules can modulate neuritogenesis before development of synaptic connections.In the present study,we aim to elucidate the roles and molecular mechanisms of classical MHC class ? in neurite outgrowth by viral infection,immunostaining,western-blot and RT-PCR in primary cultured cortical neurons.The results were shown as follows:1.In the critical period of neurite growth in primary cultured neuron,double-staining experiments with MHC class ? antibody(0X18)and TRITC-phalloidin that visualized the F-actin was performed on 2 DIV,4 DIV and 7 DIV neurons.At 1-2 DIV,a stage corresponds to the emergence of few minor neuritis.At 4-5 DIV,a stage was characterized by continuing axonal growth and differentiation of minor neurites into dendrites.By 7-10 DIV,a stage was marked by continued maturation of axonal and dendritic arbors.The results showed that MHC class ? were expressed in cell bodies,dendrites and growth cones of neurons during the three periods of neurite outgrowth.Furthermore,MHC class ? immunoreactivity largely overlapped with the F-actin in the neurite formation site and growth cone.2.Overexpression of MHC class ? by viral vectors in primary cultured cortical neurons of C57BL/6 MHC class ? knockout mouse(H2-DbKb-/? mouse)inhibited the formation of neurite.The specific antibodies(0X18)of MHC class ?,the antibody and ectodomain of PirB were added to the primary cultured neurons of C57BL/6 wild type mouse.Quantitative analysis showed that the number of dendritic arborization increased significantly after blocking the signal pathway,suggesting that classical MHC class I molecules can inhibit the neurite outgrowth in primary cultured cortical neurons.3.The classical MHC class ?-related signal pathway was blocked by the specific antibody(0X18)in the primary cortical neurons of C57BL/6 wild-type mouse.The expression of cytoskeletal related molecules was detected by western blot.The results showed that the phosphorylation level of Cofilin and LIMK1 was down-regulated.4.The expression of GAP43,GPRIN1 and STMN2 were detected by RT-qPCR in the primary cortical neurons of C57BL/6 wild type and MHC class ? knockout mouse.Results showed that the mRNA expression levels of the three genes were significantly increased in MHC class ? knockout neurons cultured in vitro for 6 days.5.A cultured developing cortical neuronal epilepsy model was established by exposing the neurons to Mg-free media for 3 h and then returning to regular media.After 16h with regular media,the expression of MHC class I was up-regulated.The results of immunofluorescence showed that the number of dendritic branches increased after induction with Mg-free media and the increase of dendritic branch was most obvious after 16 h with regular media.However,the number of dendritic branches did not change after induction with Mg-free media in the primary cultured cortical neurons of MHC class ? knockout neurons.These results suggest that classical MHC class ? molecules can inhibit the formation of neurite processes during the development of cortical neurons and may interact with some receptors to influence the activity of cytoskeletal proteins.In the cultured developing cortical neuronal epilepsy model,the abnormal formation of dendritic arborization is related to the up-regulation of classical MHC ? molecules.Our study provides an experimental basis for elucidating deeply the molecular mechanism of MHC class ? molecules in inhibiting neurite outgrowth and the function of MHC class ? molecules in the disease state.It will provide new insights into the physiological and pathological functions of classical MHC class ?molecules in CNS.