Verkade P, Verkleij A J, Gispen W H, Oestreicher A B
Rudolf Magnus Institute for Neurosciences, University of Utrecht, The Netherlands.
J Neurocytol. 1996 Oct;25(10):583-95. doi: 10.1007/BF02284826.
Following peripheral nerve injury, neurons respond with synthesis of proteins required for axonal regeneration. Newly synthesized membrane proteins, like B-50/GAP-43, are transported with the fast component of anterograde axonal transport. Structural proteins and calmodulin are transported by the slow component. Since B-50/GAP-43 can bind calmodulin, it has been hypothesised that B-50/GAP-43 may act as a carrier for fast anterograde transport of calmodulin, so that both proteins are delivered rapidly to the distally outgrowing axons ('the fast carrier hypothesis'). We have investigated whether this hypothesis is valid in myelinated axons of the regenerating rat sciatic nerve. Seven days after crush, the nerve was ligated to accumulate fast transported proteins. Nerve pieces were dissected proximal to the ligation and processed for immunofluorescence and quantitative electron microscopy by postembedding single and double immunogold labelling. By light microscopy, we observed a qualitative increase in B-50/GAP-43 immunofluorescence in the axonal element immediately proximal to the nerve ligation (termed 'accumulated') compared to an upstream site (termed 'regenerating') closer to the cell body. The immunofluorescence for calmodulin appeared to be the same at both sites. Using electron microscopy, we observed that organelles had collected at the 'accumulated' site, moreover the density of B-50/GAP-43 immunolabelling was significantly increased compared to the 'regenerating' site, where the axoplasmic structure was undisturbed. The increase in B-50/GAP-43 immunolabelling was largely associated with vesicles. The density of calmodulin immunolabelling was similar at both sites. Approximately 25% of the total B-50/GAP-43 was associated with vesicles of which only 15% also contained labelling for calmodulin. Thus, ligation of the nerve resulted in accumulation of vesicles, including those carrying B-50/GAP-43, largely without calmodulin. Therefore, contrary to 'the fast carrier hypothesis', the bulk of calmodulin is not co-transported with B-50/GAP-43 in myelinated axons of the sciatic nerve.
周围神经损伤后，神经元会合成轴突再生所需的蛋白质。新合成的膜蛋白，如B-50/GAP-43，通过轴突顺向快速运输成分进行运输。结构蛋白和钙调蛋白则由慢速运输成分运输。由于B-50/GAP-43能结合钙调蛋白，因此有人提出假说，认为B-50/GAP-43可能作为钙调蛋白快速顺向运输的载体，从而使这两种蛋白质都能迅速输送到向远端生长的轴突（“快速载体假说”）。我们研究了该假说在再生大鼠坐骨神经的有髓轴突中是否成立。挤压损伤7天后，将神经结扎以积累快速运输的蛋白质。在结扎近端解剖神经片段，并通过包埋后单重和双重免疫金标记进行免疫荧光和定量电子显微镜检查。通过光学显微镜，我们观察到与更靠近细胞体的上游部位（称为“再生部位”）相比，紧邻神经结扎处的轴突成分中B-50/GAP-43免疫荧光在质量上有所增加（称为“积累部位”）。钙调蛋白的免疫荧光在这两个部位似乎相同。利用电子显微镜，我们观察到细胞器在“积累部位”聚集，此外，与轴浆结构未受干扰的“再生部位”相比，B-50/GAP-43免疫标记的密度显著增加。B-50/GAP-43免疫标记的增加主要与囊泡有关。钙调蛋白免疫标记的密度在这两个部位相似。总的B-50/GAP-43中约25%与囊泡相关，其中只有15%也含有钙调蛋白标记。因此，神经结扎导致囊泡积累，包括携带B-50/GAP-43的囊泡，其中大部分不含钙调蛋白。所以，与“快速载体假说”相反，在坐骨神经的有髓轴突中，大部分钙调蛋白并非与B-50/GAP-43共同运输。
