Axotomy-induced changes in the expression of a type III neuronal intermediate filament gene

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Abstract
The effect of axotomy on the expression of the 57 kDa neuronal intermediate filament (IF) protein in adult rat dorsal root ganglion (DRG) neurons was examined. This IF protein is known to have an exclusively neuronal localization but is considerably more limited in its distribution in the nervous system than the neurofilament (NF) triplet proteins. The 57 kDa neuronal IF protein is similar (and perhaps identical) to the protein “peripherin” and is known to be the product of a Type III IF gene. Since the down-regulated expression of NF proteins (products of type IV IF genes) has been well established, it was of interest to determine whether the novel 57 kDa IF protein was regulated in a similar or different manner from that of the NFs in axotomized neurons. In vitro pulse-labeling of DRGs with 35S- methionine: cysteine followed by 2-dimensional gel electrophoresis/fluorography revealed that the synthesis of the 57 kDa neuronal IF protein was increased 2 weeks after sciatic nerve crush. Immunocytochemical studies using a polyclonal antibody to the 57 kDa neuronal IF protein showed that the immunodetectable levels of this protein increased in DRG neurons after peripheral axotomy. In the normal DRG, staining was localized almost exclusively to small-sized neurons. At 2 weeks after axotomy, however, large- and medium-sized neurons also became immunoreactive; in addition, the overall level of staining in the DRG was greater than normal. Quantitative analysis of in situ hybridizations of DRG neurons with a 35S-labeled cDNA probe specific for the 57 kDa neuronal IF protein revealed a significant increase in the level of 57 kDa IF mRNA in the large-sized (greater than 1000 microns2) neurons 2 weeks after axotomy; the level of 57 kDa IF mRNA in the small neurons was not different from normal at that time. Finally, using a newly developed paradigm for examining the composition of regenerating axons by axonal transport, we determined that significant amounts of the 57 kDa neuronal IF protein were conveyed into the regrowing axonal sprouts of DRG neurons. When DRG neurons were conditioned by a previous axotomy (a crush axotomy of the distal sciatic nerve 2 weeks earlier) and then stimulated to regenerate axons by a second crush axotomy located very close to the DRG, the regenerating sprouts incorporated and conveyed significantly more 57 kDa IF protein by slow axonal transport than did those elaborated by unprimed DRG neurons.(ABSTRACT TRUNCATED AT 400 WORDS)