Preliminary Study Of The Expression And Fuction Of CD226 In The Murine Brain

CD226, a member of immunoglobulin superfamily, is a ~65kDa transmembrane glycoprotein. As a cell adhesion molecule (CAM), CD226 has been widely studied about its gene, structure, expression and localization in the immune system and has been identified to be involved in several important biological functions including T cell proliferation and differentiation, platelet activation and aggregation, NK cell cytotoxicity and so on. However, the presence of CD226 in the other systems beside immune system has not been reported yet. But recently, more and more members of CAM, which were initially present in the immune system, have been identified to be present in the central nervous system (CNS) and play important roles in synaptogenesis and synaptic plasticity.CAMs are mainly membrane glycoproteins that mediates cell-cell adhesion and play essential roles in various functions, including embryonic development, cell signal transduction, inflammation, and tumor metastasis. During the last two decades, an increasing number of the CAM family members, such as neurexin, neuroligin, cadherin, Nectin, NCAM(neural cell adhesion molecule), and SynCAM, have been demonstrated to be located in the CNS and play important roles not only in initial target recognition as well as in synapse specification and maturation during synaptogenesis, but also in synaptic plasticity during learning and memory processes.Although the expression of CD226 in the CNS has not been reported yet, several lines of evidence have indicated that CD226 might be present and have important roles in the CNS. As a member of the immunoglobulin superfamily, CD226 possesses the typical structure of the other identified members of CAM reported in the CNS: single-spanning membrane proteins with several Ig(immunoglobulin)-like extracellular domains and an intracellular tail containing a PDZ-binding motif characterized by a conserved sequence. Furthermore, CD226 has recently been identified as the receptor for Nectin-2/CD112 and Necl (Nectin-like molecule) family member Necl-5/CD155 while many Nectin and Necl family members have been identified to be localized on the pre- and post-synaptic membrane.Molecular biological, immunological and morphological methods have been employed in our present research and the major achievements are: CD226 has been identified to be expressed and localized in the murine brain during adulthood and postnatal development. Exogenous CD226 and CD155 would not only evidently promote the growth and development of hippocampal neurons but also assist target recognition during neurite growth in vitro. The level of CD226 in the hippocampal neurons gradually decreased after hypoxia while the level of soluble CD226 in the serum of cerebral ischemic and tumor patients increased. The details are as following:1. CD226 mRNA and protein in the murine hippocampus and cerebellum was detected by RT-PCR and Western blotting, respectively. Furthermore, immunohistochemical staining revealed that CD226 was mainly distributed in the hilus of the dentate gyrus and stratum lucidum aligned along the pyramidal cells in the hippocampal CA3 area, the interspaces of granular cells and the somata of the Purkinje cells in the cerebellar cortex during adulthood. Double-staining results revealed that CD226 co-localized well with synaptic marker proteins including Synaptophysin, Syntaxin and PSD-95 while it didn't co-localize with NeuN or GFAP (glial fibrillary acidic protein). Ultrastructural analysis revealed that CD226 was located both on the pre-synaptic membrane and post-synaptic membrane. During postnatal development, CD226 could not be detected at its adult locations until postnatal day 12; however, it was temporally expressed in the somata of neighboring or distant nuclei associated with its adult location.2. Freshly dissected hippocampal neurons of embryonic mouse were successfully cultured in vitro. Immunofluonrescent staining was employed to observe the CD226 expression in the hippocampal neurons at different time point during culture in vitro. Two different plasmids, mCD226(murine CD226)-GFP or mCD155(murine CD155)-RFP, was successfully transfected into Hela cells by liposome transfection method. Then the transfected Hela cells were co-cultured with the freshly dissected hippocampal neurons of embryonic mouse. The co-culture of transfected Hela cells and neurons indicated that exogenous CD226 and CD155 would not only evidently promote the growth and development of hippocampal neurons by increasing the quantity and length of the neurite but also assist target recognition for the neurite by guiding them to head for the transfected Hela cells and establish fiber connections. 3. Hypoxia model of freshly dissected hippocampal neurons was successfully established and immunofluonrescent staining was used to observe the morphological changes of the hippocampal neurons and CD226 expression in them at different time point after hypoxia. Western blotting analysis revealed that CD226 expression in the hippocampal neurons was gradually down-regulated after hypoxia and vanished at 24h after hypoxia. Sandwich enzyme linked immunosorbent assay (ELISA) results indicated that the soluble CD226 in the serum of cerebral ischemic and tumor patients was evidently up-regulated when compared with that of the healthy control.In conclusion, our present study identified the expression and distribution of CD226 in the CNS for the first time, investigated the possible role of CD226 during the growth and development of hippocampal neurons and demonstrated the changes of CD226 expression in different diseases, which established the foundation for further exploration of the physiological functions and molecule mechanism of CD226 in the CNS.