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.     

Early vertebrate evolution

Debate over the origin and evolution of vertebrates has occupied biologists and palaeontologists alike for centuries. This debate has been refined by molecular phylogenetics, which has resolved the place of vertebrates among their invertebrate chordate relatives, and that of chordates among their deuterostome relatives. The origin of vertebrates is characterized by wide‐ranging genomic, embryologic and phenotypic evolutionary change. Analyses based on living lineages suggest dramatic shifts in the tempo of evolutionary change at the origin of vertebrates and gnathostomes, coincident with whole‐genome duplication events. However, the enriched perspective provided by the fossil record demonstrates that these apparent bursts of anatomical evolution and taxic richness are an artefact of the extinction of phylogenetic intermediates whose fossil remains evidence the gradual assembly of crown gnathostome characters in particular. A more refined understanding of the timing, tempo and mode of early vertebrate evolution rests with: (1) better genome assemblies for living cyclostomes; (2) a better understanding of the anatomical characteristics of key fossil groups, especially the anaspids, thelodonts, galeaspids and pituriaspids; (3) tests of the monophyly of traditional groups; and (4) the application of divergence time methods that integrate not just molecular data from living species, but also morphological data and extinct species. The resulting framework will provide for rigorous tests of rates of character evolution and diversification, and of hypotheses of long‐term trends in ecological evolution that themselves suffer for lack of quantitative functional tests. The fossil record has been silent on the nature of the transition from jawless vertebrates to the jawed vertebrates that have dominated communities since the middle Palaeozoic. Elucidation of this most formative of episodes likely rests with the overhaul of early vertebrate systematics that we propose, but perhaps more fundamentally with fossil grades that await discovery.     

Transient increase in CSF GAP-43 concentration after ischemic stroke

Background

Cerebrospinal fluid (CSF) biomarkers reflect ongoing processes in the brain. Growth-associated protein 43 (GAP-43) is highly upregulated in brain tissue shortly after experimental ischemia suggesting the CSF GAP-43 concentration may be altered in ischemic brain disorders. CSF GAP-43 concentration is elevated in Alzheimer’s disease patients; however, patients suffering from stroke have not been studied previously.

Methods

The concentration of GAP-43 was measured in longitudinal CSF samples from 28 stroke patients prospectively collected on days 0–1, 2–4, 7–9, 3?weeks, and 3–5?months after ischemia and cross-sectionally in 19 controls. The stroke patients were clinically evaluated using a stroke severity score system. The extent of the brain lesion, including injury size and degrees of white matter lesions and atrophy were evaluated by CT and magnetic resonance imaging.

Results

Increased GAP-43 concentration was detected from day 7–9 to 3?weeks after stroke, compared to day 1–4 and to levels in the control group (P?=?0.02 and P?=?0.007). At 3–5?months after stroke GAP-43 returned to admission levels. The initial increase in GAP-43 during the nine first days was associated to stroke severity, the degree of white matter lesions and atrophy and correlated positively with infarct size (r_s?=?0.65, P?=?0.001).

Conclusions

The transient increase of CSF GAP-43 is important to take into account when used as a biomarker for other neurodegenerative diseases such as Alzheimer’s disease. Furthermore, GAP-43 may be a marker of neuronal responses after stroke and additional studies confirming the potential of CSF GAP-43 to reflect severity and outcome of stroke in larger cohorts are warranted.

     

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.     

Neurite outgrowth in PC12 cells deficient in GAP-43.

The neuronal cell line PC12 undergoes a well-documented morphological and biochemical differentiation when treated with NGF and other growth factors. A hallmark of this growth factor-mediated differentiation is the induction of the growth-associated protein, GAP-43. Here we show that a PC12 cell line which is capable of NGF-, bFGF-, and cAMP-mediated neurite outgrowth is deficient in GAP-43 protein and full-length mRNA, as measured by immunocytochemistry, Western blot, Northern blot, and PCR analyses, respectively. We propose that the GAP-43 protein may not be essential for the initial extension and maintenance of neurites induced by these neuritogenic factors; rather, its role may lie predominantly in growth cone function and in the operation of the presynaptic terminal.     

Stimulation and conformational change of Goa induced by GAP-43

GAP-43, a major component of the neuronal growth cone is closely correlated with neural development and regeneration[1—5]. Go is the predominant noncytoskeletal protein in the growth cone membrane[6]. It is a kind of heterotrimeric GTP-binding proteins, which transduce signals across the plasma membrane by coupling between receptors and effectors[7]. Stimulation or inhibi-tion of G proteins altered neurite outgrowth[3]. Mastoparan, which activates heterotrimeric G pro-teins of the Go and …     

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.     

ADP-Ribosylation of the Neuronal Phosphoprotein B-50/GAP-43

Abstract: The neuronal phosphoprotein B-50/GAP-43 is associated with growth and regeneration within the nervous system and its posttranslational status can be correlated with its cellular localization during growth and regeneration. Recently, B-50 has been shown to interact with certain G protein subunits. Regulation of G protein-mediated signal transduction may involve ADP-ribosylation in vivo. In the present study we have demonstrated that B-50 is a substrate for endogenous ADP-ribosyltransferases. The results are discussed with respect to the possible interaction of B-50 with G proteins, but also with regard to the posttranslational modification of B-50 by all major regulatory mechanisms that act at, or through, the neuronal membrane.     

Understanding vertebrate brain evolution

Four major questions can be asked about vertebrate brain evolution:1) What major changes have occurred in neural organization andfunction? 2) When did these changes occur? 3) By what mechanismsdid these changes occur? 4) Why did these changes occur? Comparativeneurobiologists have been very successful in recognizing majorchanges in brain structure. They have also made progress inunderstanding the functional significance of these changes,although this understanding is primarily limited to sensorycenters, rather than integrative or motor centers, because ofthe relative ease of manipulating the relevant stimuli. Althoughneuropaleontology continues to provide important insights intowhen changes occurred, this approach is generally limited torecognizing variation in overall brain size, and sometimes brainregions, as interpreted from the surface of an endocranial cast.In recent years, most new information regarding when neuralchanges occurred has been based on cladistical analysis of neuralfeatures in extant taxa. Historically, neurobiologists havemade little progress in understanding how and why brains evolve.The emerging field of evolutionary developmental biology appearsto be the most promising approach for revealing how changesin development and its processes produce neural changes, includingthe emergence of novel features. Why neural changes have occurredis the most difficult question and one that has been the mostignored, in large part because its investigation requires abroad interdisciplinary approach involving both behavior andecology.     

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.     

GAP-43 as a plasticity protein in neuronal form and repair

Neurons exhibit a remarkable plasticity of form, both during neural development and during the subsequent remodelling of synaptic connectivity. Here we review work on GAP-43 and G₀, and focus upon the thesis that their interaction may endow neurons with such plasticity. We also present new data on the role of G proteins in neurite growth, and on the interaction of GAP-43 and actin. GAP-43 is a protein induced during periods of axonal extension and highly enriched on the inner surface of the growth cone membrane. Its membrane localization is primarily due to a short amino terminal sequence which is subject to palmitoylation. Binding to actin filaments may also assist in restricting the protein to specific cellular domains. Consistent with its role as a ?plasticity protein,”? there is evidence that GAP-43 can directly alter cell shape and neurite extension, and several theses have been advanced for how it might do so. Two other prominent components of the growth cone membrane are the α and β subunits of G₀. GAP-43 regulates their guanine nucleotide exchange, which is an unusual role for an intracellular protein. We speculate that GAP-43 may adjust the ?set point”? of responsiveness for G₀ stimulation by receptors, thereby altering the neuronal propensity to growth, without actually causing growth. To begin to address how G protein activity affects axon growth, we have developed a means to introduce guanine nucleotide analogs into sympathetic neurons. Stimulation of G proteins with GTP-γ-S retards axon growth, whereas GDP-β-S enhances it. This is compatible with G protein registration of inhibitory signals. © 1992 John Wiley & Sons, Inc.     

Role of GAP-43 in sequestering phosphatidylinositol 4,5-bisphosphate to Raft bilayers

The lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) is critical for a number of physiological functions, and its presence in membrane microdomains (rafts) appears to be important for several of these spatially localized events. However, lipids like PIP₂ that contain polyunsaturated hydrocarbon chains are usually excluded from rafts, which are enriched in phospholipids (such as sphingomyelin) containing saturated or monounsaturated chains. Here we tested a mechanism by which multivalent PIP₂ molecules could be transferred into rafts through electrostatic interactions with polybasic cytoplasmic proteins, such as GAP-43, which bind to rafts via their acylated N-termini. We analyzed the interactions between lipid membranes containing raft microdomains and a peptide (GAP-43P) containing the linked N-terminus and the basic effector domain of GAP-43. In the absence or presence of nonacylated GAP-43P, PIP₂ was found primarily in detergent-soluble membranes thought to correspond to nonraft microdomains. However, when GAP-43P was acylated by palmitoyl coenzyme A, both the peptide and PIP₂ were greatly enriched in detergent-resistant membranes that correspond to rafts; acylation of GAP-43P changed the free energy of transfer of PIP₂ from detergent-soluble membranes to detergent-resistant membranes by −1.3 kcal/mol. Confocal microscopy of intact giant unilamellar vesicles verified that in the absence of GAP-43P PIP₂ was in nonraft microdomains, whereas acylated GAP-43P laterally sequestered PIP₂ into rafts. These data indicate that sequestration of PIP₂ to raft microdomains could involve interactions with acylated basic proteins such as GAP-43.     

Distribution of GAP-43-immunoreactive structures in the human fetal amygdala.

The growth-associated protein GAP-43 is a developmentally regulated protein which is involved in the formation of neuronal contacts. In immunohistochemical studies, GAP-43 is detected within axons during their elongation; thus a fibrous immunoreactivity is visible. After axonal growth is completed there is a shift from a fibrous to a punctate immunoreactivity. The latter has been shown to correlate with synaptogenesis. In the amygdala of the 5th gestational month, a fibrous GAP-43-immunoreactivity is seen in the basolateral nuclei, whereas the corticomedial nuclei exclusively show a punctate immunoreactivity. In the 7th month, all amygdaloid nuclei display immunoreactive puncta, but no fibers. In the 9th month GAP-43-immunoreactivity is no longer visible within the amygdala. The results demonstrate the differential distribution of GAP-43-immunoreactive structures in the amygdaloid nuclei. The nuclear specific immunostaining and its changes may indicate the sequential appearance of the monoaminergic innervation of the amygdala, as GAP-43 is known to occur in monoaminergic systems. Nuclei involved in high levels of the cortical processing hierarchy such as the lateral or basal nucleus display a late occurrence of GAP-43-immunoreactivity. In general, anti-GAP-43 has been shown to be an appropriate tool to investigate axonal growth and synaptogenesis in the developing human brain.     

The evolution of vertebrate flight

Flight–defined as the ability to produce useful aerodynamic forces by flapping the wings–is one of the most striking adaptations in vertebrates. Its origin has been surrounded by considerable controversy, due in part to terminological inconsistencies, in part to phylogenetic uncertainty over the sister groups and relationships of birds, bats and pterosaurs, and in part to disagreement over the interpretation of the available fossil evidence and over the relative importance of morphological, mechanical and ecological specializations. Study of the correlation between functional morphology and mechanics in contemporary birds and bats, and in particular of the aerodynamics of flapping wings, clarifies the mechanical changes needed in the course of the evolution of flight. This strongly favours a gliding origin of tetrapod flight, and on mechanical and ecological grounds the alternative cursorial and fluttering hypotheses (neither of which is at present well-defined) may be discounted. The argument is particularly strong in bats, but weaker in birds owing to apparent inconsistencies with the fossil evidence. However, study of the fossils of the Jurassic theropod dinosaur Archaeopteryx , the sister-group of the stem-group proto-birds, supports this view. Its morphology indicates adaptation for flapping flight at the moderately high speeds which would be associated with gliding, but not for the slow speeds which would be required for incipient flight in a running cursor, where the wingbeat is aerodynamically and kinematically considerably more complex. Slow flight in birds and bats is a more derived condition, and vertebrate flapping flight apparently evolved through a gliding stage.     

The neural crest in vertebrate evolution

Vertebrates belong to the group of chordates characterized by a dorsal neural tube and an anteroposterior axis, the notochord. They are the only chordates to possess an embryonic and pluripotent structure associated with their neural primordium, the neural crest (NC). The NC is at the origin of multiple cell types and plays a major role in the construction of the head, which has been an important asset in the evolutionary success of vertebrates. We discuss here the contribution of the rostral domain of the NC to craniofacial skeletogenesis. Moreover, recent data show that cephalic NC cells regulate the activity of secondary brain organizers, hence being critical for preotic brain development, a role that had not been suspected before.