Studies On The Distribution Of NgR Protein In The White Matter Of Adult Rat Spinal Cord

Myelin has been proved to play a key role in inhibition of axonal sprouting and regeneration after injury in the central nervous system (CNS) of adult mammals. Since Nogo gene was cloned in 2000, extensive attention has been paid to Nogo molecule, the most important myelin-associated inhibitor. In 2001, the receptor of Nogo extracellular 66-amino-acid fragment (Nogo-66), named as Nogo receptor (NgR), was identified. The interaction between Nogo-A in the oligodendrocytes and NgR in the neurons is thought to be the major factor responsible for the failure of axonal regeneration in the CNS. Interestingly, it has been reported that at least three myelin-associated proteins, including Nogo-A, myelin associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) function through NgR, resulting in growth cone collapse and neurite outgrowth inhibition. Therefore, NgR may act as a coreceptor to trigger intracellular signal transduction by combiningwith different myelin-associated proteins. The convergence of these three disparate myelin-derived inhibitors onto one receptor suggests that NgR could be a crucial regulator of neurite outgrowth.NgR is a GPI-linked cell surface protein of 473 amino acids. It has been found widely distributed in many classes of CNS neurons and is present through axons surrounded by myelin. So far there has been no evidence revealing the expression of NgR in glial cells in vivo. Our immunohistochemical staining of adult rat spinal cord, however, clearly showed that NgR-immunoreactivity appeared in glial cells as well, with bright spots at the periphery of the cell bodies and along their processes. A detailed study was then carried out to identify the characteristics of the NgR-irty in the glia. Double immuno-staining showed that some of the punctate NgR-ir fluorescences were colocalized with the GFAP-ir astrocytic or RIP-ir oligodendritic cell bodies or processes. Therefore, we made a further detailed study by using immuno-electron microscopy. Ultrastructurally the NgR-irty was localized at the gap junctions between astrocytes, oligodendrocytes, or between astrocyte and oligodendrocyte. In addition, NgR-ir product was found at the cytoplasmic side of the junction. Immunocytochemistry analysis suggested cultured astrocytes and oligodendrocytes also expressed NgR protein in vitro. NgR-irty was localized in the cytoplasm and on the membrane of glia. Especially in the astrocyte, punctate NgR-irty was discontinuous in the processes and prominent at the convergent points of processes, which was similar to the distribution of Cx43 in astrocytes.In summary, immunochemical studies with light microscopy, confocal microscopy, electron microscopy andimmunocytochemistry analysis were used to examine thedistribution of NgR protein in the white matter of adult rat spinal cord. Our observation that NgR protein is distributed along the membrane of glia-glia gap junction in the white matter of spinal cord suggests its possible modulatory function on intercellular communication between glial cells.