Developmental study of the expression of B50/GAP-43 in rat retina

B50/GAP-43 has been implicated in neural plasticity, development, and regeneration. Several studies of axonally transported proteins in the optic nerve have shown that this protein is synthesized by developing and regenerating retinal ganglion cells in mammals, amphibians, and fish. However, previous studies using immunohistochemistry to localize B50/GAP-43 in retina have shown that this protein is found in the inner plexiform layer in adults. Since the inner plexiform layer contains the processes of amacrine cells, ganglion cells, and bipolar cells to determine which cells in the retina express B50/GAP-43, we have now used in situ hybridization to localize the mRNA that codes for this protein in the developing rat retina. We have found that B50/GAP-43 is expressed primarily by cells in the retinal ganglion cell layer as early as embryonic day 15, and until 3 weeks postnatal. Some cells in the inner nuclear layer, possibly a subclass of amacrine cells, also express B50/GAP-43 protein and mRNA; however, the other retinal neurons–bipolar cells, photoreceptors, and horizontal cells express little, if any, B50/GAP-43 at any stage in their development. Early in development, the protein appears in the somata and axons of ganglion cells, while later in development, B50/GAP-43 becomes concentrated in the inner plexiform layer, where it continues to be expressed in adult animals. These results are discussed in terms of previous proposals as to the functions of this molecule. © 1993 John Wiley & Sons, Inc.     

GAP-43 phosphorylation is dynamically regulated in individual growth cones

In vivo, kinase C phosphorylation of the growth-associated protein GAP-43 is spatially and temproally associated with the proximity of growing axons to their targets. Here we have used dissociated dorsal root ganglia (DRG)s and an antibody specific for the phosphorylated form of GAP-43 to demonstrate that neurite regeneration in culture also begins in the absence of detectable levels of phosphorylated GAP-43. Since the β isoform of kinase C was found to be enriched in growth cones before stably phosphorylated GAP-43 was detected, it may normally be inactive during initial neurite outgrowth; however, premature phosphorylation of GAP-43 could be stimulated in newly dissociated DRGs by plating them on cultures in which phosphorylation had already been initiated; media conditioned by such cultures caused no response suggesting an effect of either cell-cell or cell-substrate contact. Increased GAP-43 phosphorylation correlated with a reduced extent of neurite outgrowth but not with the rate at which individual growth cones translocated so that motile growth cones contained very low levels of phosphorylated GAP-43, whereas stationary growth cones showed much more immunoreactivity. Downregulation of kinase C by phorbol ester prevented increased GAP-43 phosphorylation and led to growth cone collapse. Finally, phosphorylated GAP-43 was found to be differently distributed within growth cones. Increased immunoreactivity was frequently observed in the neck of the growth cone and was heterogeneously distributed in lamellae and filopodia. These results, which demonstrate the dynamic regulation of GAP-43 phosphorylation in individual growth cones, are discussed with reference to the association between changes in growth cone shape and the ability to translocate and change direction. © 1992 John Wiley & Sons, Inc.     

Overexpression of HuD, but not of its truncated form HuD I+II, promotes GAP-43 gene expression and neurite outgrowth in PC12 cells in the absence of nerve growth factor

We have previously shown that the RNA-binding protein HuD binds to a regulatory element in the growth-associated protein (GAP)-43 mRNA and that this interaction involves its first two RNA recognition motifs (RRMs). In this study, we investigated the functional significance of this interaction by overexpression of human HuD protein (pcHuD) or its truncated form lacking the third RRM (pcHuD I+II) in PC12 cells. Morphological analysis revealed that pcHuD cells extended short neurites containing GAP-43-positive growth cones in the absence of nerve growth factor (NGF). These processes also contained tubulin and F-actin filaments but were not stained with antibodies against neurofilament M protein. In correlation with this phenotype, pcHuD cells contained higher levels of GAP-43 without changes in levels of other NGF-induced proteins, such as SNAP-25 and tau. In mRNA decay studies, HuD stabilized the GAP-43 mRNA, whereas HuD I+II did not have any effect either on GAP-43 mRNA stability or on the levels of GAP-43 protein. Likewise, pcHuD I+II cells showed no spontaneous neurite outgrowth and deficient outgrowth in response to NGF. Our results indicate that HuD is sufficient to increase GAP-43 gene expression and neurite outgrowth in the absence of NGF and that the third RRM in the protein is critical for this function.     

Chicken Growth-Associated Protein GAP-43 Is Tightly Bound to the Actin-Rich Neuronal Membrane Skeleton

We have identified the chicken equivalent of growth-associated protein GAP-43 in a detergent-resistant membrane skeleton from cultures of chick neurones and embryonic chick brain. Antisera to the membrane skeleton protein, the 3D5 antigen, precipitate the translation product of chick GAP-43 cDNA, and the 3D5 antigen is also detected by antisera against synthetic peptides from the known amino acid sequence of rat GAP-43. The chick protein and the rat GAP-43 are biochemically similar proteins that both serve as major targets of phosphorylation by endogenous protein kinase C. The detergent-resistant complex in which GAP-43 is found also contains actin (approximately 5% of the total protein) and a neurone-specific cell surface glycoprotein. We suggest that the membrane skeleton of neurones may be a primary site of action of GAP-43.     

The 21-Aminosteroid U-74389F Attenuates Hyperexpression of GAP-43 and NADPH-Diaphorase in the Spinal Cord of Wobbler Mouse,a Model for Amyotrophic Lateral Sclerosis

The wobbler mouse suffers an autosomal recessive mutation producing severe neurodegeneration and astrogliosis in spinal cord. It has been considered a model for amyotrophic lateral sclerosis. We have studied in these animals the expression of two proteins, the growth-associated protein (GAP-43) and the NADPH-diaphorase, the nitric oxide synthesizing enzyme, employing immunocytochemistry and histochemistry. We found higher expression of GAP-43 immunoreactivity in dorsal horn, Lamina X, corticospinal tract and ventral horn motoneurons in wobbler mice compared to controls. Weak NADPH-diaphorase activity was present in control motoneurons, in contrast to intense labeling of the wobbler group. No differences in diaphorase activity was measured in the rest of the spinal cord between control and mutant mice. A group of animals received subcutaneously for 4 days a 50 mg pellet of U-74389F, a glucocorticoid-derived 21-aminosteroid with antioxidant properties but without glucocorticoid activity. U-74389F slightly attenuated GAP-43 immunostaining in dorsal regions of the spinal cord from wobblers but not in controls. However, in motoneurons of wobbler mice number of GAP-43 immunopositive neurons, cell processes and reaction intensity were reduced by U-74389F. The aminosteroid reduced by 50% motoneuron NADPH-diaphorase activity. Hyperexpression of GAP-43 immunoreactivity in wobbler mice may represent an exaggerated neuronal response to advancing degeneration or muscle denervation. It may also be linked to increased nitric oxide levels. U-74389F may stop neurodegeneration and/or increase muscle trophism and stop oxidative stress, consequently GAP-43 hyperexpression was attenuated. Wobbler mice may be important models to evaluate the use of antioxidant steroid therapy with a view to its use in human motoneuron disease.     

Differential Regulation of Fast Axonally Transported Proteins During the Early Response of Rat Retinal Ganglion Cells to Axotomy

Abstract: We have found that the early response of axotomized rat retinal ganglion cells is characterized by the differential regulation of a number of fast axonally transported proteins. The abundance of 23 radiolabeled fast transported proteins was analyzed at 2 and 5 days after axotomy using two-dimensional gel electrophoresis. Corresponding changes in retinal GAP-43 mRNA were measured using northern analysis. Within 2 days of injury, >40% of the transported proteins analyzed, including GAP-43, showed increased labeling above control levels. Approximately 13% of transported proteins decreased below control levels, whereas the remainder did not change. Five days after axotomy, only GAP-43 and another fast transported protein, C3, continued to sustain measurable increased labeling above control levels; all previously elevated proteins appeared to have been down-regulated by this time, which corresponds to the onset of cell death. These differential changes were accompanied by parallel increases in GAP-43 mRNA. These results suggest that the molecular changes within rat retinal ganglion cells are differentially regulated within two stages subsequent to damage, initial regenerative growth followed by cell death.     

Mutagenesis of ser41 to ala inhibits the association of GAP-43 with the membrane skeleton of GAP-43-deficient PC12B cells: Effects on cell adhesion and the composition of neurite cytoskeleton and membrane

To investigate the molecular basis for GAP-43 function in axon outgrowth, we produced a mutant, GAP-43 (Ala₄₁), whose interaction with calmodulin in vitro was unaffected by increasing Ca²⁺ concentrations, and stably transfected it into GAP-43-deficient PC12B cells. Several lines that expressed wild-type or mutant protein at levels that resembled endogenous GAP-43 expression in PC12 controls were subcloned and characterized. GAP-43 (Ala₄₁) was significantly more extractable with Nonidet P-40 and less tightly associated with the membrane skeleton than the wild-type protein. Furthermore, GAP-43 (Ala₄₁) expression by PC12B cells profoundly affected their phenotype: First, observation of living cells using video-enhanced microscopy revealed irregular plasma membranes with numerous blebs and protrusions and neurites that appeared thin and varicose. Second, both the cells' ability to remain attached to laminin substrates and the amount of α1β1 integrin expressed on the cell surface was significantly decreased. Finally, peripherin transport, which is abnormal in PC12B cells, could be rescued by transfection of wild-type GAP-43 but not the GAP-43 (Ala₄₁) mutant. The phenotypic abnormalities resemble other cell types in which membrane skeleton/plasma membrane interactions have been functionally decoupled, and our results are consistent with the notion that these interactions may be abnormal in GAP-43 (Ala₄₁)-expressing PC12B cells, either as a direct consequence of the mutation or arising secondarily to the altered availability of calmodulin in the growing neurite. © 1996 John Wiley & Sons, Inc.     

M-calpain-mediated cleavage of GAP-43 near Ser41 is negatively regulated by protein kinase C, calmodulin and calpain-inhibiting fragment GAP-43-3

Neuronal protein GAP-43 performs multiple functions in axon guidance, synaptic plasticity and regulation of neuronal death and survival. However, the molecular mechanisms of its action in these processes are poorly understood. We have shown that in axon terminals GAP-43 is a substrate for calcium-activated cysteine protease m-calpain, which participates in repulsion of axonal growth cones and induction of neuronal death. In pre-synaptic terminals in vivo, in synaptosomes, and in vitro, m-calpain cleaved GAP-43 in a small region near Ser41, on either side of this residue. In contrast, micro-calpain cleaved GAP-43 in vitro at several other sites, besides Ser41. Phosphorylation of Ser41 by protein kinase C or GAP-43 binding to calmodulin strongly suppressed GAP-43 proteolysis by m-calpain. A GAP-43 fragment, lacking about forty N-terminal residues (named GAP-43-3), was produced by m-calpain-mediated cleavage of GAP-43 and inhibited m-calpain, but not micro-calpain. This fragment prevented complete cleavage of intact GAP-43 by m-calpain as a negative feedback. GAP-43-3 also blocked m-calpain activity against casein, a model calpain substrate. This implies that GAP-43-3, which is present in axon terminals in high amount, can play important role in regulation of m-calpain activity in neurons. We suggest that GAP-43-3 and another (N-terminal) GAP-43 fragment produced by m-calpain participate in modulation of neuronal response to repulsive and apoptotic signals.