The Expression Of Nogo-A In Rat CNS

During the past year, a major advance in the study of axon regeneration in CNS was the molecular cloning of Nogo. Nogo protein expressed by CNS myelin may be a major factor in the failure of CNS axon regeneration. The interest in this protein stems principally from the effects of a monoclonal antibody (IN-1) that was rasied against NI-250 (Nogo-A) and recognizes both NI-35 and NI-250 (Nogo-A). In vivo, this function-blocking antibody was shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes, and to result in long-distance fiber regeneration in the lesioned adult mammalian CNS and in a recovery of specific reflex and locomotor functions after spinal cord injury.Three major transcipts (Nogo-A, -B, -C) originate from the Nogo gene by alternative splicing. The three isoforms have a common carboxy-terminai domain of 188 amino acids, and this region is highly homologous to the reticulon protein family. The carboxyl portion of the Nogo protein contains two transmembrane domains, which are seperated by an extracellular 66-amino-acid fragment (Nogo-66). Analysis of Nogo-A suggests that both the long acidic amino-terminal domain and the Nogo-66 fragment have strong inhibitory activity. And a brain-specific, leucine-rich-repeat glycophosphatidylinositol-linked protein (NgR), recently cloned, may be a receptor that mediates Nogo-66 inhibition of axonaloutgrowth.Northern blot analysis and in situ hybridization showed subtypes of neurones expressed the Nogo-A transcript in brain, spinal cord and peripheral ganglia besides oligodendrocytes. Neuronal labelling was particularly conspicuous in the developing nervous system. The presence of Nogo-A in certain neuron populations within the brain had not been revealed by the results of McAb IN-1, while the identity of this protein remained elusive until recently. These differences will affect directly the correlation of the functions of Nogo-A molecule and the function-blocking effects of IN-1. To this, the celluar and subcellular expression pattern of Nogo-A in the developing and adult CNS of rat was explored. The process and main results are as followed:1. The full-length sequence of Nogo-A (KIAA0886) was generously provided by the Kazusa cDNA Project. The full-length coding sequence was amlified by polymerase chain reaction (PCR) and ligated into the pGEX-KG GST fusion and the pEGFP Nl EGFP-tagged vector (Clontech) to generate a plasmid encoding Nogo-A fusion protein. Same approach was applied to Nogo-B, -C and Nogo-66 receptor (NgR). Occasionally, a short form of Nogo-A deleted proline-rich region was cloned from the Clontech Fetal Brain Quick-Clone cDNA Library (Genbank accession No.AF320999). And we got the rat homolog to human NgR from rat brain cDNA library (Genbank accession No.AY028438). All ampliers prescribed previously were confirmed by sequencing.2. Rabbit polyclbnal antibody was rasied againt purified GST-Nogo and its specificity was characterized with western Blot analysis. In blots, anti-Nogo-A could specific recognized huamn and rat Nogo-A, not Nogo-B or Nogo-C. The primary cultured oligodendrocytes were labelled with anti-Nogo antibody.3. Immuofluorescence staining on ault rat CNS tissue secetion showed that there are complex celluar distribution and subcellular localization of Nogo-A.The oligodendrocytes were found to be Nogo-A-immunoreactive (ir). Also immunoreactive were many neurons. The Purkinje cells of the cerebellum and the spinal motor neurons were strongly Nogo-A-ir. The Nogo-A immune-reactivitycould be identified in the nucleus, cytoplasm, as well as the cell membrane. Often the nucleus stood out prominently, as was also demonstrated with Western blotting of the nucleus fraction.4. At various developing stage of rat spinal cord, mRNA and protein expression of Nogo-A were revealed a steady level by RT-PCR and Western Blot. Compared to MAG, the expression pattern of Nogo-A shown was not to be correlated well with the time course of myelin maturing as previously prescribed by IN-1.