Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons

Abstract

The possibility that proteins are modified during axoplasmic transport in CNS axons was examined by analyzing neurofilament proteins (200,000, 140,000 and 70,000 MW) along the mouse primary optic pathway (optic nerve and optic tract). The major neurofilament proteins (NFP) exhibited considerable microheterogeneity. At least 3 forms of the 140,000 neurofilament protein differing in MW by SDS PAGE [sodium dodecyl sulfate/polyacrylamide gel electrophoresis] (140,000-145,000 MW) were identified. The 140,000 proteins, and their counterparts in purified neurofilament preparations, displayed similar isoelectric points and the same peptide maps. The 140,000 NFP exhibited regional heterogeneity when consecutive segments of the optic pathway were separately examined on polyacrylamide gels. Two major species (145,000 and 140,000 MW) were present along the entire length of the optic pathway. The 3rd protein (143,000 MW) was absent proximally but became increasingly prominent in distal segments. After intravitreal injection of [3H]proline, newly synthesized radiolabled proteins in the 140,000 MW region entered proximal mouse retinal ganglion cell (RGC) axons as 2 major species corresponding to the 145,000 and 140,000 MW NFP observed on stained gels. When transported NFP reached more distal axonal regions (30 d [days] postinjection or longer), a 143,000 MW protein appeared that was similar in isoelectric point and peptide map to the 145,000 and 140,000 MW species. The composition of CNS neurofilaments, particularly the 140,000 component, is probably more complex than previously recognized. Retinal ganglion cell axons display regional differentiation with respect to these cytoskeletal proteins. Structural heterogeneity of 140,000 NFP arise, at least in part, from posttranslational modification during axoplasmic transport. When excised but intact optic pathways were incubated in vitro at pH 7.4, a 143,000 NFP was rapidly formed by a Ca-dependent enzymatic process active at endogenous Ca levels. Changes in major proteins other than those in the 145,000-140,000 MW region were minimal. In optic pathways from mice injected intravitreally with L-[3H]proline, tritiated 143,000 MW NFP formed rapidly in vitro if radioactively labeled NFP were present in distal RGC axonal regions (31 d postinjection). By contrast, no 143,000 MW NFP was generated if radioactively labeled NFP were only present proximally in RGC axons (6 d post injection). The enzymatic process that generates 143,000 MW NFP in vitro, therefore, appears to have a nonuniform distribution along the RGC axons. CNS axons may be regionally specialized with respect to structure and function.