Jeanette J. Norden

Light-microscopic immunolocalization of the growth- and plasticity-associated protein GAP-43 in the developing rat brain

Growth-associated protein-43 (GAP-43) is a developmentally regulated, fast-axonally transported phosphoprotein whose synthesis and transport are enhanced during periods of growth and synaptic terminal formation. GAP-43 is a substrate of protein kinase C and is identical to protein F1, a phosphoprotein which is regulated during long-term potentiation in the hippocampus. In order to characterize the cellular localization of GAP-43, we have raised a specific antiserum against it, and used this as a probe to show that GAP-43 is neuron-specific, and is localized to growing neuronal processes in developing rat brain, and to presynaptic terminals in both the peripheral and central nervous system. In the mature CNS, GAP-43 immunoreactivity is present in most neuropil areas, but is especially dense in the molecular layers of the cerebellum, neocortex, and the hippocampus, structures known to exhibit synaptic plasticity. Its localization, together with biochemical data concerning the dynamics of its synthesis and its identity as protein F1, suggest that GAP-43 may be involved in axon growth in the developing nervous system, and in some aspect of synaptic plasticity in the mature CNS. These data also suggest that axon growth and synaptic plasticity in the brain may be regulated by a common mechanism, both involving the protein kinase C-mediated phosphorylation of GAP-43.

S100B, a neurotropic protein that modulates neuronal protein phosphorylation, is upregulated during lesion-induced collateral sprouting and reactive synaptogenesis

Using light and electron microscopic immunocytochemistry, we examined the expression of the Ca2+-binding protein S100B in the dentate gyrus of adult rats during lesion-induced sprouting and reactive synaptogenesis. Nine days following unilateral lesioning of the entorhinal cortex, S100B was upregulated in cells primarily in the outer part of the molecular layer of the ipsilateral dentate gyrus. When examined with electron microscopy, numerous astrocytes and synapses containing S100B were identified. These data show that during lesion-induced sprouting and reactive synaptogenesis, S100B is upregulated in astrocytes and can be found in pre- and post-synaptic compartments where it might influence neuronal protein phosphorylation.

Localization of α-bungarotoxin binding sites to the goldfish retinotectal projection

Abstract The optic tectum of the goldfishCarassius auratus is a rich source of α-bungarotoxin (α-Btx) binding protein. In order to determine whether some fraction of these receptors is present at retinotectal synapses, we have compared the histological distribution of receptors revealed by the use of [125Iα-Btx radioautography to the distribution of optic nerve terminals revealed by the use of cobalt and horseradish peroxidase (HRP) techniques. The majority of α-Btx binding is concentrated in those tectal layers containing primary retinotectal synapses. The same layers contain high concentrations of acetylcholinesterase (AChE), revealed histochemically. Following enucleation of one eye, there is a loss of α-Btx binding in the contralateral tectum, observed both by radioautography and by a quantitative binding assay of α-Btx binding. Approximately 40% of the α-Btx binding sites are lost within two weeks following enucleation. By contrast, no significant change in AChE activity could be demonstrated up to 6 months enucleation. These results are discussed in light of recent studies which show that the α-Btx binding protein and the nicotinic acetylcholine receptor are probably identical in goldfish tectum. We conclude that the 3 main classes of retinal ganglion cells projecting to the goldfish tectum are nicotinic cholinergic and that little or no postdenervation hypersensitivity due to receptor proliferation occurs in tectal neurons following denervation of the retinal input.

Ultrastructural study of degeneration and regeneration in the amphibian tectum.

The processes of degeneration and reinnervation in the optic tectum of Xenopus laevis have been detailed by quantitative ultrastructural analysis. The tecta were denervated either permanently, by removing the contralateral eye, or temporarily, by cutting the optic tract. After denervation the total number of synapses decreases rapidly within 4 days of a stable level of about 40% of the original number. Vacated postsynaptic sites are subsequently removed by phagocytosis. When regenerating axons arrive in the tectum they make synaptic contact by inducing new postsynaptic membrane specializations. The ultrastructural sequence of reinnervation by optic axons resembles initial synaptogenesis.

Inhibition of protein kinase C- and casein kinase II-mediated phosphorylation of GAP-43 by S100β

Abstract The effect of the glial-derived protein, S100β, on the in vitro phosphorylation of the growth-associated protein GAP-43 was investigated. S100β inhibited in a dose dependent manner the phosphorylation of GAP-43 by protein kinase C (PKC) or by casein kinase II (CKII). S100β appeared to slow down the rate and the degree to which GAP-43 can be phosphorylated by either kinase. The specificity of the inhibition was demonstrated by the observation that the phosphorylation of two other CKII substrates, casein and a selective peptide substrate, was not inhibited by S100β. The marked inhibitory effect of S100β required the presence of calcium in the phosphorylation reactions. In addition, S100β inhibition of GAP-43 phosphorylation was seen with GAP-43 purified under a variety of conditions that alter acylation, suggesting that the acylation state of GAP-43 does not affect the ability of S100β to modulate CKII- or PKC-mediated phosphoryation of GAP-43.

Regulation of specific neuronal and nonneuronal proteins during development and following injury in the rat central nervous system.

Publisher Summary This chapter presents studies on growth-associated protein, GAP-43, to further characterize this protein using both anatomical and biochemical methods. It discusses a specific antibody to the rat GAP-43 and this antibody is used as a probe to examine the developmentally regulated pattern of the expression of GAP-43 in the rat hippocampus. The most prominent neuronal protein whose synthesis and transport is selectively enhanced during nerve growth is a protein designated as GAP-43 for its apparent molecular weight by two-dimensional polyacrylamide gel electrophoresis. It was initially reported that the synthesis of this protein was enhanced 15- to 20-fold during the regeneration of the optic nerve of the toad as compared to the levels of this protein found in normal adult nerves. The increased transport of a newly synthesized protein of the same approximate molecular weight and isoelectric point was subsequently identified during the regeneration of the hypoglossal nerve in the rabbit and corticospinal tract in neonatal hamsters and during the development of the optic nerve in rabbits. The synthesis of a similar protein is also increased during the regeneration of the optic nerve in goldfish and during the development of the optic nerve in rats. Taken together, these results suggest that GAP-43 may play a special role in neuronal growth.

RAPID ARREST OF AXON ELONGATION BY BREFELDIN A : A ROLE FOR THE SMALL GTP-BINDING PROTEIN ARF IN NEURONAL GROWTH CONES

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.

Chapter 5: Expression of the growth- and plasticity-associated

Publisher Summary This chapter summarizes observations on nerve growth factor (NGF) induction of GAP-43 in PC12 cells. It characterizes this NGF response in terms of its time course, dose–response relationship, sensitivity to variations in culture conditions, which affect neurite outgrowth, dependence on protein synthesis, and sensitivity to methyltransferase inhibitors and glucocorticoids. Many of these parameters have also been examined for a number of other agents that were found to increase GAP-43 expression. To determine whether GAP-43 in PC12 cells is also primarily localized to neurites, NGF-treated cells were stained immunohistochemically for GAP-43. Specific immunoreactivity is present throughout the neurites, but is not evident in cell bodies. Recent immunogold labeling experiments have shown that the very low levels of GAP-43 in unstimulated PC12 cells is mostly associated with lysosomal structures and Golgi apparatus. In stimulated cells, the much higher GAP-43 content is localized mostly to the plasma membrane, especially around processes, and to membranous structures in neurites.

Up-regulation of fast-axonally transported proteins in retinal ganglion cells of adult rats with optic–peroneal nerve grafts

Metabolic labeling and quantitative 2D gel fluorography were used to assess changes in the synthesis and transport of five fast-axonally transported and developmentally regulated proteins (GAP-43, SNAP-25, and proteins of 18, 22, and 23/24 kDa) after grafting of a peroneal nerve segment onto a transected optic nerve in adult rats. After optic nerve transection alone, only GAP-43 was up-regulated significantly compared to normal adult controls. The other proteins showed little change or were down-regulated following axotomy. By 4 weeks following optic nerve transection and peroneal nerve grafting, however, GAP-43, proteins 22 and 23/24 kDa showed a sustained up-regulation in synthesis and transport compared to normal controls; SNAP-25 and protein 18 kDa showed levels of expression similar to or slightly greater than normal controls. Increased expression of GAP-43 in retinal ganglion cells was also examined with immunocytochemistry. While a transient up-regulation of GAP-43 in retinal ganglion cells was observed following optic nerve transection, a sustained increase in GAP-43 immunoreactivity was present only in animals with nerve grafts. Backfilling of retinal ganglion cells from the grafts with horseradish peroxidase combined with GAP-43 immunocytochemistry revealed that all retinal ganglion cells with axons growing into the grafts were positive for GAP-43, but not all retinal ganglion cells showing GAP-43 immunoreactivity were extending axons into the grafts. We conclude that the presence of a nerve graft sustains the up-regulation of a number of proteins including GAP-43, and that this up-regulation is correlated with an increased potential for nerve growth, but other as yet unknown factors or conditions appear to play a role in determining if this growth potential will be realized.

Retinotectal synapses formed by ipsilaterally projecting fibers in the doubly innervated goldfish tectum

When one tectum of an adult goldfish is removed, the severed retinal fibers regenerate ipsilaterally into the remaining tectal lobe. Initially fibers from the two eyes overlap in the tectum but EM-HRP data suggest that few mature retinal synapses are formed between the ipsilateral eye and tectum at this time. At longer time periods, when some fibers appear to segregate into eye-specific termination bands, our data suggest that a significant number of synapses from the ipsilateral eye are present. These findings have important implications for how eye-specific termination bands are formed in doubly innervated tecta.