Sensory neurons selectively upregulate synthesis and transport of the βIII-tubulin protein during axonal regeneration

Abstract

The effects of peripheral nerve injury on the content, synthesis, and axonal transport of the class III β-tubulin protein in adult rat dorsal root ganglion (DRG) neurons were examined. Recent reports of selective increases in the steady-state levels of the βIII-tubulin mRNA during axonal regeneration (Moskowitz et al., 1993) led to the hypothesis that upregulated levels of expression of the βIII-tubulin isotype that alter the composition of neuronal microtubules is important for effective axonal regrowth. If this is the case, the increases in mRNA levels must be translated into increased βIII-tubulin protein levels and subsequently modify the axonal cytoskeleton via axonal transport mechanisms. The present study assessed whether or not this occurs by examining βIII-tubulin protein content in adult rat lumbar DRG neurons at different times (1-14 d) after a distal sciatic nerve crush (∼55 mm from the DRG) by Western blotting and immunocytochemistry with a βIII-tubulin specific monoclonal antibody. These studies showed substantial increases in βIII-tubulin content in DRG neurons, as well as in proximal regions of peripheral sensory axons (0-6 mm from the DRG), from 1-2 weeks after a distal nerve injury. Pulse labeling of DRG neurons with 35S-methionine and 35S-cysteine and immunoprecipitation of labeled βIII-tubulin indicated that the synthesis of βIII-tubulin was increased in the DRG after axotomy. Studies of axonal transport, wherein L5 DRG proteins were labeled with 35S-methionine and 35S-cysteine by microinjection, revealed that slow component b (SCb) of axonal transport conveyed more labeled tubulin moving at apparently faster rates through the intact regions of sciatic nerve axons in response to crush injury of the distal sciatic nerve. Immunoprecipitation experiments using proximal peripheral nerve segments showed that SCb in distally injured DRG neurons was enriched in the βIII-tubulin isotype. These findings demonstrate that the augmented synthesis of βIII-tubulin after axotomy alters the composition of the axonally transported cytoskeleton that moves with SCb. The increased amounts and rate of delivery of βIII-tubulin in axons of regenerating DRG neurons suggest that the altered pattern of tubulin gene expression that is initiated by axotomy impacts on the composition and organization of the axonal cytoskeleton in a manner that can facilitate axonal regrowth.