Localisation and expression pattern of the Nogo receptor and its ligand, Nogo-A in cells of the mammalian central nervous system

Includes bibliographical references (leaves 62-84).Axon regeneration failure in the adult mammalian central nervous system is partly due to inhibitory molecules associated with myelin. The Nogo receptor plays a role in this process through an extraordinary degree of cross reactivity with three structurally unrelated myelin-associated inhibitory ligands namely; Nogo-A, myelin associated glycoprotein and oligodendrocyte myelin glycoprotein. The major aim of the study was to investigate the expression pattern of Nogo receptor and one of its ligands, Nogo-A in the mammalian nervous system, and also investigate whether Nogo receptor is located in neuronal lipid rafts by linking it to flotillins, known lipid raft markers. We therefore generated a rabbit polyclonal Nogo receptor antibody from the leucine rich repeat number 9 domain of Nogo receptor polypeptide chain. Together with a commercially available polyclonal antibody specific for Nogo receptor, and in conjunction with double labelling immunofluorescence methods on crysections and cell cultures, Nogo receptor immunoreactivity was also observed in brain, spinal cord, and dorsal ganglia. In cellular populations, it was confined to neuronal cell bodies and their processes. Nogo receptor was localised on the surface of extending dorsal root ganglion intact axons and growth cones in live staining experiments. Nogo-A, an important axon growth inhibitory molecule and member of the reticulon family protein, was widely distributed in the mammalian brain, spinal cord, and dorsal root ganglia. Intense Nogo-A immunoreactivity was dete cted in oligodendrocyte cell bodies and their myelin sheaths in nerve fibre tracts of the central nervous system. Furthermore, numerous populations of neurons in the brain and spinal cord expressed Nogo-A to a variable extent in their cell bodies and neurites, suggesting additional, as-yet-unknown, functions of this protein. In cell culture, cytoplasmic staining with anti-Nogo-A antibody was observed after fixation oligodendrocytes and neurones, but intracellular structures that presumably represent endoplasmic reticulum were also strongly labelled in fibroblasts. These results confirm results obtained by other researchers with different set of antibodies. However, they also raise the question of the mechanism and circumstances under which the Nogo receptor interacts with Nogo-A, as this protein appears to be confined to the cytoplasm and can therefore not be expected to bind Nogo receptor on the axon surface. To investigate whether Nogo receptor is localised in neuronal lipid rafts, commercial and local antibodies specific for Nogo receptor, in conjunction with flotillin (a known lipid raft-associated protein) were used in double-immunofluorescence, co-immunoprecipitation and western blotting experiments. Results revealed substantial immunofluorescent colocalisation of Nogo receptor and flotillin in membranes of axons and PC-12 cells. Further more, extraction Nogo receptor antigen from rat brain using receptor bound protein-A sepharose beads, followed by probing with anti-flotillin antibody, established the link between lipid rafts and Nogo receptor