Redistribution of GAP-43 during growth cone developmentin vitro; immunocytochemical studies

Abstract

The growth-associated protein GAP-43 (B-50, F1, pp46), has been found in elongating axons during development and regeneration, and has also been associated with synaptic plasticity in mature neurons. We have examined the loss of GAP-43 labelling from cerebellar granule cells with immunocytochemical localization of a polyclonal antibody to GAP-43. One day after plating, the plasma membrane of cell bodies, neurites and growth cones were all labelled with anti-GAP-43. By 10 days, most of the cell body labelling was lost, and by 20 days the neuritic and growth cone labelling was greatly reduced. Beginning at six days, anti-GAP-43 labelling of growth cones, which was initially uniform, became clustered. When growth cones were double-labelled with antibodies to GAP-43 and the synaptic vesicle protein, p65, inverse changes in the distribution of label was observed. While growth cone labelling with anti-p65 increased from three to 20 days in culture, GAP-43 label began to be lost from some growth cones by six days and showed continuing decline through 20 days. For individual growth cones, the loss of GAP-43 appeared to parallel the accumulation of p65, and first growth cones to lose GAP-43 appeared to be the first to accumulate p65 label. When cultures were grown on a substrate of basement membrane material, the time frames of neuritic outgrowth, loss of GAP-43 labelling, and increase in p65 labelling were all accelerated. At five days, labelling for GAP-43 was weak and labelling for p65 was strong, in a pattern comparable to that seen in older cultures on a polylysine substrate. These results suggest several conclusions concerning the expression and loss of GAP-43 in cultured cerebellar granule neurons. First, GAP-43 label is initially distributed in all parts of these cells. With increasing time in culture the label is first lost from cell bodies and later from neurites and growth cones. Second, the loss of GAP-43 label from growth cones is correlated with the appearance of the synaptic vesicle protein p65. Finally, in vitro developmental changes in the loss of GAP-43 can be altered by changing the growth substrate.

GAP‐43 mRNA in growth cones is associated with HuD and ribosomes

“…Selective distribution of GAP-43 in elongating axons is the result of developing neuronal polarity. During neuronal differentiation, GAP-43 protein is lost from the cell body, distributed to distal axons, and concentrated in growth cones (Burry et al, 1991(Burry et al, , 1992Goslin et al, 1988Przyborski and Cambray-Deakin, 1994). Transport of GAP-43 protein associated with vesicles from the cell body to growth cones has been well documented (Benowitz et al, 1981;Skene and Willard, 1981a,b,c).…”

“…Expression of GAP-43 has been correlated with axon elongation in developing and regenerating neurons (Skene and Willard, 1981a). When axon growth cones reach their targets, the levels of GAP-43 protein fall as they stop elongation and form presynaptic terminals (Burry et al, 1991;Caroni and Becker, 1992;Meiri et al, 1986;Perrone-Bizzozero et al, 1986). Thus, GAP-43 expression is considered an excellent marker of active axonal growth during development and nerve regeneration.…”

Extracellular adenosine triphosphate affects neural cell adhesion molecule (NCAM)-mediated cell adhesion and neurite outgrowth

“…Twenty-four hours after plating the medium was removed, and a suspension of dissociated hippocampal neurons in defined medium (see ''Microwell culture'' section) was seeded on monolayers of L-cells with or without NCAM-140 expression at a density of 5,000 cells/well. After 24 hr incubation at 37°C, 5% CO 2 , neurons were visualized using immunohistochemical staining for GAP-43 (Burry et al, 1991). Briefly, the cells were fixed for 30 min in 4% w/v paraformaldehyde in PBS, and then incubated in rabbit anti-GAP-43 antibodies (Mosevitsky et al, 1994) diluted 1:100 in PBS, 1% FCS, 0.1% BSA, 50 mM glycine, 0.02% NaN 3 , 0.2% saponin at 4°C overnight.…”

Transitional elements with characteristics of both growth cones and presynaptic terminals observed in cell cultures of cerebellar neurons

“…In cerebellar cultures, GAP-43 has been found in axons and growth cones (Burry et al, 1991). Thisstudy also showed transitional elements in cultures during the first week labelled for GAP-43.…”