Absence of persistent spreading, branching, and adhesion in GAP-43- depleted growth cones

The growth-associated protein GAP-43 is a major protein kinase C substrate of growth cones and developing nerve terminals. In the growth cone, it accumulates near the plasma membrane, where it associates with the cortical cytoskeleton and membranes. The role of GAP-43 in neurite outgrowth is not yet clear, but recent findings suggest that it may be a crucial competence factor in this process. To define the role of GAP- 43 in growth cone activity, we have analyzed neurite outgrowth and growth cone activity in primary sensory neurons depleted of GAP-43 by a specific antisense oligonucleotide procedure. Under optimal culture conditions, but in the absence of GAP-43, growth cones adhered poorly, displayed highly dynamic but unstable lamellar extensions, and were strikingly devoid of local f-actin concentrations. Upon stimulation, they failed to produce NGF-induced spreading or insulin-like growth factor-1-induced branching, whereas growth factor-induced phosphotyrosine immunoreactivity and acceleration of neurite elongation were not impaired. Unlike their GAP-43-expressing counterparts, they readily retracted when exposed to inhibitory central nervous system myelin-derived liposomes. Frequency and extent of induced retraction were attenuated by NGF. Our results indicate that GAP-43 can promote f- actin accumulation, evoked morphogenic activity, and resistance to retraction of the growth cone, suggesting that it may promote regulated neurite outgrowth during development and regeneration.     

Model system for live imaging of neuronal responses to injury and repair

Although it has been well established that induction of growth-associated protein-43 (GAP-43) during development coincides with axonal outgrowth and early synapse formation, the existence of neuronal plasticity and neurite outgrowth in the adult central nervous system after injuries is more controversial. To visualize the processes of neuronal injury and repair in living animals, we generated reporter mice for bioluminescence and fluorescence imaging bearing the luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine GAP-43 promoter. Reporter functionality was first observed during the development of transgenic embryos. Using in vivo bioluminescence and fluorescence imaging, we visualized induction of the GAP-43 signals from live embryos starting at E10.5, as well as neuronal responses to brain and peripheral nerve injuries (the signals peaked at 14 days postinjury). Moreover, three-dimensional analysis of the GAP-43 bioluminescent signal confirmed that it originated from brain structures affected by ischemic injury. The analysis of fluorescence signal at cellular level revealed colocalization between endogenous protein and the GAP-43-driven gfp transgene. Taken together, our results suggest that the GAP-43-luc/gfp reporter mouse represents a valid model system for real-time analysis of neurite outgrowth and the capacity of the adult nervous system to regenerate after injuries.     

Inhibition of nerve growth factor-induced B-50/GAP-43 expression by antisense oligomers interferes with neurite outgrowth of PC12 cells.

Substantial evidence has now been gathered for the involvement of B-50/GAP-43 in neuronal development and regeneration. The precise role of this protein, however, is still debated. In an earlier study, a linear correlation between NGF-induced neurite outgrowth and B-50/GAP-43 levels was observed in PC12 cells. To establish the involvement of B-50/GAP-43 expression in neurite outgrowth in these cells, we interfered with the expression by antisense oligomers and measured the outgrowth. In the present study, a B-50/GAP-43 antisense 5'-oligomer interfered both with the NGF-induced increase in B-50/GAP-43 and with neurite outgrowth, whereas an antisense 3'-oligomer was ineffective. We conclude, that in PC12 cells under normal conditions B-50/GAP-43 expression and neurite outgrowth are or become coupled upon NGF-induction, in contrast to the situation in PC12 clones with no or very low B-50/GAP-43 expression.     

GAP-43 regulates NCAM-180-mediated neurite outgrowth

The neural cell adhesion molecule (NCAM), and the growth-associated protein (GAP-43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP-43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP-43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP-43. We demonstrate that phosphorylation of GAP-43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM-induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP-43, NCAM-induced neurite outgrowth requires functional association of NCAM-180/spectrin/GAP-43, whereas in the absence of GAP-43, the NCAM-140/non-receptor tyrosine kinase (Fyn)-associated signaling pathway is pivotal. Thus, expression of GAP-43 presumably acts as a functional switch for NCAM-180-induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP-43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM-180/spectrin-mediated modulation of the actin cytoskeleton, NCAM-140-mediated activation of Fyn, or both.     

The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells

The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.     

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.     

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.     

Increase in Neurite Formation and Acetylene line Release by Transfection of Growth-Associated Protein-43 cDNA into NG108–15 Cells

Abstract: We previously reported that growth-associated protein-43 (GAP-43) could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in accumulation of GAP-43. In the present study, to clarify the functional significance of GAP-43 for neurite maintenance and acetylcholine (ACh) release, we prepared NG-G11 cells by transfection of GAP-43 cDNA into NG108-15 cells. NG-G11 cells expressed GAP-43 mRNA at levels approximately twice that in nontransfected or vector-transfected cells under control conditions and after treatment with dibutyryl cyclic AMP (diBu-cAMP) or 12-O-tetradecanoylphorbol 13-acetate (TPA) plus diBu-cAMP. Neurite outgrowth after addition of diBu-cAMP was greater in NG-G11 than in control cells. In NG-G11 cells, neurites elongated by treatment with diBu-cAMP for 72 h were maintained after removal of the drug. Treatment with TPA plus diBu-cAMP for 24 h induced neurite outgrowth in NG-G11 cells, although control cells required 72 h. Depolarization by 50 m M KCI induced ACh release in both NG-G11 and control cells treated with diBu-cAMP or TPA/diBu-cAMP. Although removal of the drugs following diBu-cAMP treatment reversed ACh release to nontreated levels in control cells, a high-K⁺-induced level of ACh release remained in NG-G11 cells after removal of diBu-cAMP. ACh release induced by TPA plus diBu-cAMP for 24 h was further enhanced after removal of the drugs in NG-G11 cells, but it was not seen in control cells. These results suggest that levels of GAP-43 mRNA are correlated with neurite maintenance and the level of ACh release. Thus, GAP-43 may be involved in neuronal differentiation in NG108-15 cells.