Moskowitz P F, Oblinger M M
Department of Cell Biology and Anatomy, Chicago Medical School, Illinois 60064.
J Neurosci. 1995 Feb;15(2):1545-55. doi: 10.1523/JNEUROSCI.15-02-01545.1995.
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 III-tubulin mRNA during axonal regeneration (Moskowitz et al., 1993) led to the hypothesis that upregulated levels of expression of the beta 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 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 III-tubulin protein content in adult rat lumbar DRG neurons at different times (1-14 d) after a distal sciatic nerve crush (approximately 55 mm from the DRG) by Western blotting and immunocytochemistry with a beta III-tubulin specific monoclonal antibody. These studies showed substantial increases in beta 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 35S-methionine and 35S-cysteine and immunoprecipitation of labeled beta III-tubulin indicated that the synthesis of beta III-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 beta III-tubulin isotype. These findings demonstrate that the augmented synthesis of beta 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 III-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.
研究了周围神经损伤对成年大鼠背根神经节（DRG）神经元中III类β-微管蛋白的含量、合成及轴突运输的影响。最近有报道称，轴突再生过程中βIII-微管蛋白mRNA的稳态水平有选择性增加（Moskowitz等人，1993年），由此提出假说：βIII-微管蛋白亚型表达上调，改变神经元微管的组成，对有效的轴突再生很重要。如果是这样，mRNA水平的增加必须转化为βIII-微管蛋白水平的增加，并随后通过轴突运输机制改变轴突细胞骨架。本研究通过蛋白质免疫印迹法和使用βIII-微管蛋白特异性单克隆抗体的免疫细胞化学方法，检测成年大鼠腰段DRG神经元在坐骨神经远端挤压伤（距DRG约55毫米）后不同时间（1-14天）的βIII-微管蛋白含量，以评估是否发生这种情况。这些研究表明，在远端神经损伤后1-2周，DRG神经元以及外周感觉轴突的近端区域（距DRG 0-6毫米）中βIII-微管蛋白含量大幅增加。用35S-甲硫氨酸和35S-半胱氨酸对DRG神经元进行脉冲标记，并对标记的βIII-微管蛋白进行免疫沉淀，结果表明轴突切断后DRG中βIII-微管蛋白的合成增加。通过微注射用35S-甲硫氨酸和35S-半胱氨酸标记L5 DRG蛋白进行轴突运输研究，结果显示，坐骨神经远端挤压伤后，轴突运输的慢成分b（SCb）输送更多标记的微管蛋白，其在坐骨神经轴突的完整区域中移动速度明显加快。使用外周神经近端节段进行的免疫沉淀实验表明，远端损伤的DRG神经元中的SCb富含βIII-微管蛋白亚型。这些发现表明，轴突切断后βIII-微管蛋白合成增加，改变了与SCb一起移动的轴突运输细胞骨架的组成。再生DRG神经元轴突中βIII-微管蛋白数量和输送速率的增加表明，轴突切断引发的微管蛋白基因表达改变模式，以一种促进轴突再生的方式影响轴突细胞骨架的组成和组织。
