Is the interaction of CNP with tubulin and microtubules required for process extension in oligodendrocytes?

Oligodendrocytes (ODC) differentiate and undergo vast membrane synthesis and process extension to form myelin. What are the molecular mechanisms underlying these events? The myelin protein, CNP, appears to play an important role. Recent work from our lab and others provide strong evidence that CNP is associated with the cytoskeleton and may be involved in process extension. Intriguingly, CNP has an enzymatic activity, whose in vivo relevance is unknown, since substrates with 2′,3′‐cyclic termini have not yet been determined for CNP. To elucidate the function of CNP, a 55‐kDa polypeptide, which copurifies with CNP by immunoprecipitation from rat brain, was identified as tubulin by mass spectrometry. Cultured ODC and CNP‐transfected COS‐7 cells showed extensive microtubule colocalization along the main processes and the cell body. COS‐7 cells expressing high levels of CNP exhibited major morphological changes similar to cultured ODC: rounding of the cell body, process extension, and extensive branching of the processes. Deletion mutant studies reveal that tubulin binding and cell transformation are mediated by the C‐terminal catalytic domain. However, the catalytic activity of CNP is not responsible for these morphological changes, since expression of enzymatic inactive mutants in non‐neuronal cells resembled cells expressing wild‐type CNP. Furthermore, RICH (Regeneration Induced CNPase Homologue), a protein that is expressed in fish retinal ganglion cells and shares extensive homology with CNP in the C‐terminal domain, can bind to tubulin and cause similar cytoskeletal transformations in non‐neuronal cells. Our results suggest that binding of CNP to microtubules facilitates cytoskeletal rearrangements necessary for process extension and myelination in ODC. Acknowledgements:  Supported by the CIHR and FCAR.