Sensory neurons selectively upregulate synthesis and transport of the beta sub(III)-tubulin protein during axonal regeneration

The effects of peripheral nerve injury on the content, synthesis, and axonal transport of the class III beta -tubulin protein in adult rat dorsal root ganglion (DRG) neurons were examined. Recent reports of selective increases in the steady-state levels of the beta sub(III)-tubulin mRNA during axonal regeneration led to the hypothesis that upregulated levels of expression of the beta sub(III)-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 beta sub(III)-tubulin protein levels and subsequently modify the axonal cytoskeleton via axonal transport mechanisms. The present study assessed whether or not this occurs by examining beta sub(III)-tubulin protein content in adult rat lumbar DRG neurons at different times (1-14 d) after a distal sciatic nerve crush ( similar to 55 mm from the DRG) by Western blotting and immunocytochemistry with a beta sub(III)-tubulin specific monoclonal antibody. These studies showed substantial increases in beta sub(III)-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 super(35)S-methionine and super(35)S-cysteine and immunoprecipitation of labeled beta sub(III)-tubulin indicated that the synthesis of beta sub(III)-tubulin was increased in the DRG after axotomy. Studies of axonal transport, wherein L5 DRG proteins were labeled with super(35)S-methionine and super(35)S-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 is distally injured DRG neurons was enriched in the beta sub(III)-tubulin isotype. These findings demonstrate that the augmented synthesis of beta sub(III)-tubulin after axotomy alters the composition of the axonally transported cytoskeleton that moves with SCb. The increased amounts and rate of delivery of beta sub(III)-tubulin in axons in 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 fa