Ultrastructural evidence for the lack of co-transport of B-50/GAP-43 and calmodulin in myelinated axons of the regenerating rat sciatic nerve

Abstract

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.

Urokinase-type plasminogen activator (uPA) regulates the expression and function of growth-associated protein 43 (GAP-43) in the synapse

“…Our data indicate that pGAP-43 colocalizes with synaptic vesicles in uPA-treated neurons and that this is followed by synapsin I phosphorylation at Ser-9 and mobilization of synaptic vesicles to the AZ. These observations agree with reports from other groups indicating that, following its association with synaptic vesicles (59,60), pGAP-43 activates the synaptic vesicle cycle and induces release of neurotransmitters (1). Importantly, our results indicate that these events require entry of calcium through open presynaptic NMDA receptors.…”

Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43

“…Previous electron microscopic studies revealed that the majority of GAP43 is associated with the plasma membrane and that only ∼25% of GAP43 is associated with vesicles (Verkade et al ., 1996b). This poses the question of how GAP43 gets sorted to the distal process under conditions in which only a minority is localized to vesicles at steady state.…”

“…About 30% of GAP43 was found to travel at fast axonal transport rate in regenerating sciatic nerves in vivo (Tetzlaff et al ., 1989), whereas GAP43 was found mostly to be localized at the cytosolic face of the plasma membrane in the adult pyramidal tract (Gorgels et al ., 1989). Ultrastructural analysis revealed that a minor portion of GAP43 is also associated with vesicles (Verkade et al ., 1996b), where it colocalizes with the synaptic vesicle marker synaptophysin (Verkade et al ., 1996a). This poses the question how GAP43 gets sorted to the distal process under conditions in which only a minority is localized to vesicles.…”

A swift expanding trend of extracellular vesicles in spinal cord injury research: a bibliometric analysis

“…The magnitude of the keyword circle within each cluster is commensurate with the frequency of its appearance (Additional file 3: Table S2). The main terms comprising cluster 1 are "axon" [52,53], "day" [54,55], "immunoreactivity" [56,57] and "glial cell" [58,59]. Meanwhile, cluster 2 is characterized by the core keywords "vesicle" [60][61][62], "source" [63,64], "miRNAs" [65,66] and "biomarker" [67][68][69].…”