Studies On The Distribution Of NgR In Rat Astrocytes And Oligodendrocytes

One big difference existing between central nervous system (CNS) and peripheral nervous system (PNS) is that injuried axons of CNS could not regenerate any more while nerves in PNS could. The specific and unfriendly surroundings of CNS,especially CNS myelin,which is quite different from that of PNS,contribute a lot to the regeneration failure.Several inhibitory proteins have been extracted from CNS myelin. These proteins,which mainly refer to Nogo-A,MAG ( myelin associated glycoprotein) and OMgp (oligodendrocyte myelin glycoprotein),are recognized as MAIFs (myelin associated inhibitory factors).MAIFs cause growth cone collapse and block axonal sprouting,and they have been found to transmit signals intracellularly through binding the same receptor-NgR (Nogo-66 receptor) . Therefore, as a potent target of treating CNS injury,NgR is getting more and more attention.NgR is a GPI ( Glycosylphosphatidyl nositol) -linked cell surface protein ,boasting eight leucine repeat regions (LRR) . NgR and other two molecules-NgRH1 and NgRH2,which have high homologywith NgR,constitute the NgR family.NgR is expressed prodominantly in brain and only moderately in several other organs such as heart and kidney.In CNS of normal adult rat, NgR has been found widely distributed in many classes of neurons and presented through axons surrounded by myelin.And it is NgR on the neural surface that binds MAIFs and take inhibitory effect.Recently, evidence have been offered to suggest that NgR is also expressed in glial cells.Our previous study has showed through immunohistochemical staining and immuno-electron microscopy that in spinal cord and cerebellum of adult normal rat ,NgR is found in glial cells of neuropils.Xiao ZC and his colleagues in Singapore reported having detected NgR protein in human U87MG glioma cells.However,more detailed studies of NgR in glial cells are needed.Based on these findings,we attempted to investigate the expression and distribution of NgR in glial cells systemicly.Our main results are as following:1. Identification of reactivity and specificity of several NgR antibodies which recognize different epitopes. In Blots, AB5615, sc-25659 and NgRPAb were all shown to react with NgR specifically, but not with its family members, NgRH1 or NgRH2. Double immunofluorescence staining with Flag and several NgR antibodies in Flag-NgR tansfected COS cells showed that, besides Ngr11-A, NgR antibodies can stain predominantly the plasma membrane and perinuclear organelles labeled by Flag antibody. By RT-PCR and Western blot, PC12 and C17.2 cells were confirmed to express mRNA and protein of NgR, then indirect immunofluorescence technique revealed intracellular localization of NgR protein in PC12, C17.2 and CGN cells.The results showed that all the NgR antibodies we used could react with NgR specifically. Different NgR antibodies behaved differently in Western blot and immunofluorescence staining.2. NgR is expressed in rat astrocytes.Specific NgR cDNA product could...