GAP-43 dependency defines distinct effects of netrin-1 on cortical and spinal neurite outgrowth and directional guidance.

Growth-associated protein-43 (GAP-43) is a major nervous system protein whose phosphorylation by protein kinase C regulates growth cone responses to extracellular guidance cues via F-actin. GAP-43 is essential for axon pathfinding in both cortical afferents and efferents: when it is genetically deleted, somatosensory, auditory and visual somatotopic maps fail to form, and telencephalic commissural axons fail to cross the midline. Here we investigated whether the midline guidance cue netrin-1 depends on GAP-43 for its functions in neurite growth and guidance. We used 3-dimensional collagen gel co-cultures to show that both endogenous netrin-1, expressed by the spinal cord floor plate, and recombinant netrin-1, expressed by transfected COS7 cells, stimulate neurite outgrowth and chemotropic guidance of neocortical callosal axons. In contrast both were significantly inhibited in GAP-43 (-/-) neocortical callosal axons, mimicking the in vivo phenotype. Conversely, neither netrin-1-stimulated neurite outgrowth nor guidance of dorsal spinal cord commissure axons were affected when GAP-43 was absent, again consistent with in vivo phenotype but suggesting fundamental differences in how neocortical and spinal cord axons respond to netrin-1. In addition, differences in GAP-43 dependency also distinguished how ventrolateral cortical efferents respond to netrin-1: in contrast to callosal neurites, in which netrin-1 required GAP-43 in order to stimulate both outgrowth and guidance, in ventrolateral efferents, netrin-1 required GAP-43 only to stimulate outgrowth, but not guidance. Moreover, netrin-1 increased the numbers of both types of cortical, but not spinal neurites. The results demonstrate previously unappreciated diversity in how different classes of neurons respond to the same guidance cue.

Emergence of layer IV barrel cytoarchitecture is delayed in somatosensory cortex of GAP-43 deficient mice following delayed development of dendritic asymmetry.

The emergence of barrel cytoarchitecture in mouse somatosensory cortex is extremely well defined. However, mechanisms underlying the development of this cellular organization are not completely understood. While it is generally accepted that hollows emerge via passive displacement of cortical cells by dense thalamocortical afferent clusters in barrel centers, it is not known what causes cellular segregation of barrel sides and septa. Here, we hypothesized that the emergence of sides and septa is related to the progressive asymmetry of dendrites from the cells of the barrel side toward the barrel hollow during development. We tested this hypothesis in the barrel cortex of growth-associated protein-43 heterozygous mice (GAP43 (+/-) mice) that display a 2-day delay in retraction of septally oriented dendrites compared to (+/+) littermates. We predicted that this delayed retraction would result in a subsequent 2-day delay in the emergence of barrel sides and septa. Using cresyl violet staining of barrel cortex, we found that initial emergence of hollows was not different between GAP43 (+/-) mice and (+/+) littermate controls. However, the emergence of sides and septa was delayed by 2 days, supporting our hypothesis that the emergence of barrel sides and septa is related to, and perhaps reliant upon, the developmental step of dendritic orientation toward barrel hollows. This process, which is mechanistically distinct from the emergence of barrel hollows, is likely due to both active and passive events resulting from asymmetric cell orientation.

Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity.

Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of ß-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of ß-actin mRNA, and introducing GFP with the 3'UTR of ß-actin mRNA depletes axons of endogenous ß-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of ß-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.

Postsynaptic deregulation in GAP-43 heterozygous mouse barrel cortex.

Formation of whisker-related barrels in primary somatosensory cortex (S1) requires communication between presynaptic thalamocortical afferents (TCAs) and postsynaptic cortical neurons. GAP-43 is crucially involved in targeting TCAs to postsynaptic S1 neurons but its influence on the interactions between these 2 elements has not been explored. Here, we tested the hypothesis that reduced early expression of presynaptic GAP-43 (GAP-43 heterozygous [HZ] mice) alters postsynaptic differentiation of barrel cells. We found a transient increase in cytochrome oxidase staining between P6 and P14 in HZ animals, indicative of increased metabolic activity in barrel cortex during this time. Golgi impregnation and microtubule-associated protein 2 immunohistochemistry showed anomalous dendritic patterning in GAP-43 HZ cortex at P5, with altered dendritic length and branching and abnormal retention of dendrites that extend into developing septa. This deficiency was no longer apparent at P7, suggesting partial recovery of dendritic pruning processes. Finally, we showed early defects in synaptogenesis from P4 to P5 with increased colocalization of NR1 and GluR1 staining in HZ mice. By P7, this colocalization had normalized to wild type levels. Taken together, our findings suggest abnormal postsynaptic differentiation in GAP-43 HZ cortex during early barrel development, followed by adaptive compensation and partial phenotypic rescue.

Both cell-autonomous and cell non-autonomous functions of GAP-43 are required for normal patterning of the cerebellum in vivo.

Growth-associated protein 43 (GAP-43) is required for development of a functional cerebral cortex in vertebrates; however, its role in cerebellar development is not well understood. Recently, we showed that absence of GAP-43 caused defects in proliferation, differentiation, and polarization of cerebellar granule cells. In this paper, we show that absence of GAP-43 causes defects in cerebellar patterning that reflect both cell-autonomous and non-autonomous functions. Cell-autonomous effects of GAP-43 impact precursor proliferation and axon targeting: In its absence, (1) proliferation of granule cell precursors in response to sonic hedgehog and fibroblast growth factor is inhibited, (2) proliferation of neuroepithelial precursors is inhibited, and (3) targeting of climbing fibers to the central lobe is disrupted. Cell non-autonomous effects of GAP-43 impact differentiated Purkinje cells in which GAP-43 has been downregulated: In its absence, both maturation and mediolateral patterning of Purkinje cells are inhibited. Both cell-autonomous and non-autonomous functions of GAP-43 involve its phosphorylation by protein kinase C. GAP-43 is phosphorylated in granule cell precursors in response to sonic hedgehog in vitro, and phosphorylated GAP-43 is also found in proliferating neuroepithelium and climbing fibers. Phosphorylated GAP-43 is specifically enriched in the presynaptic terminals of parallel and climbing fibers that innervate Purkinje cell bodies and dendrites. The cell-autonomous and non-autonomous effects of GAP-43 converge on the central lobe. The multiple effects of GAP-43 on cerebellar development suggest that it is a critical downstream transducer of signaling mechanisms that integrate generation of cerebellar structure with functional parcellation at the central lobe.

GAP-43 is key to mitotic spindle control and centrosome-based polarization in neurons.

In neurons, the position of the centrosome during final mitosis marks the point of emergence of the future axon. However, the molecular underpinnings linking centrosome position to axon emergence are unknown. GAP-43 is a calmodulin-binding IQ motif protein that regulates neuronal cytoskeletal architecture by interacting with F-actin in a phosphorylation dependent manner. Here we show that GAP-43 is associated with the centrosome and plays a critical role in mitosis and acquisition of neuronal polarity in cerebellar granule neurons. In the absence of GAP-43, the centrosome position is delinked from process outgrowth and is only capable of mediating morphological polarization, however molecular specification of the axonal compartment does not take place. These results show that GAP-43 is required to link centrosome position to process outgrowth in order to generate neuronal polarity in cerebellar granule cells.

Increased thalamocortical synaptic response and decreased layer IV innervation in GAP-43 knockout mice.

The growth-associated protein, GAP-43, is an axonally localized neuronal protein with high expression in the developing brain and in regenerating neurites. Mice that lack GAP-43 (GAP-43 -/-) fail to form a whisker-related barrel map. In this study, we use GAP-43 -/- mice to examine GAP-43 synaptic function in the context of thalamocortical synapse development and cortical barrel map formation. Examination of thalamocortical synaptic currents in an acute brain slice preparation and in autaptic thalamic neurons reveals that GAP-43 -/- synapses have larger alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor (AMPAR)-mediated currents than controls despite similar AMPAR function and normal probability of vesicular release. Interestingly, GAP-43 -/- synapses are less sensitive to blockade by a competitive glutamate receptor antagonist, suggesting higher levels of neurotransmitter in the cleft during synaptic transmission. Field excitatory postsynaptic potentials (EPSPs) from GAP-43 -/- thalamocortical synapses reveal a reduced fiber response, and anatomical analysis shows reduced thalamic innervation of barrel cortex in GAP-43 -/- mice. Despite this fact synaptic responses in the field EPSPs are similar in GAP-43 -/- mice and wild-type littermate controls, and the ratio of AMPAR-mediated to N-methyl-d-aspartate receptor (NMDAR)-mediated currents (AMPAR:NMDAR ratio) is larger than normal. This suggests that GAP-43 -/- mice form fewer thalamocortical synapses in layer IV because of decreased anatomical innervation of the cortex, but the remaining contacts are individually stronger possibly due to increased neurotransmitter concentration in the synaptic cleft. Together, these results indicate that in addition to its well known role in axonal pathfinding GAP-43 plays a functional role in regulating neurotransmitter release.

Anomalous functional organization of barrel cortex in GAP-43 deficient mice.

Growth associated protein 43 (GAP-43), found only in the nervous system, regulates the response of neurons to axon guidance signals. It is also critical for establishing normal somatotopy. Mice lacking GAP-43 (KO) show aberrant pathfinding by thalamocortical afferents, and do not form cortical whisker/barrels. GAP-43 heterozygous (HZ) mice show more subtle deficits--delayed barrel segregation and enlarged barrels at postnatal day 7. Here, we used cortical intrinsic signal imaging to characterize adult somatotopy in wildtype (WT), GAP-43 KO, and HZ mice. We found clear foci of activation in GAP-43 KO cortex in response to single-whisker stimulation. However, the KO spatial activation patterns showed severe anomalies, indicating a loss of functional somatotopy. In some cases, multiple foci were activated by single whiskers, while in other cases, the same cortical zone was activated by several whiskers. The results are consistent with our previous findings of aberrant pathfinding and clustering by thalamocortical afferent axons, and absence of barrel patterning. Our findings indicate that cortex acts to cluster afferents from a given whisker, even in the absence of normal topography. By contrast, single-whisker stimulation revealed normal adult topographic organization in WT and HZ mice. However, we found that functional representations of adult HZ barrels are larger than those found in WT mice. Since histological HZ barrels recover normal dimensions by postnatal day 26, the altered circuit function in GAP-43 HZ cortex could be a secondary consequence of the rescue of barrel dimensions.

Behavioral characterization in a comprehensive mouse test battery reveals motor and sensory impairments in growth-associated protein-43 null mutant mice.

The growth-associated protein (GAP)-43 is a major neuronal protein associated with axonal growth, neuronal plasticity and learning. The observation that only 5-10% of mice with a full GAP-43 gene deletion survive weaning suggests that basic neural functions are disturbed. Here we used a comprehensive test battery to characterise and quantify the motor and sensory function of surviving adult homozygous GAP-43 (-/-) mice as compared with GAP-43 (+/-) and wild-type animals. The test battery was comprised of motor, sensory, and reflex tests producing 25 measures of locomotion, as well as epicritic, auditory, olfactory and visual function. The analysis revealed significant impairments in muscle strength, limb coordination and balance in GAP-43 (-/-) mice. Furthermore, GAP-43 (-/-) animals were hyperactive and showed reduced anxiety as measured by open field and light dark tests. In sensory tests, GAP-43 (-/-) mice were tested for impaired tactile and labyrinthine function. Abnormal reflexes were found in the contact and vibrissa placing responses, and in the crossed extensor reflex. GAP-43 (+/-) animals showed only moderate abnormalities as compared with wild-type animals. We conclude that GAP-43 is necessary for the development and function of a variety of neuronal systems. The results also show that the comprehensive test battery used in the present study represents a sensitive approach to assess the functional integrity of ascending and descending pathways in genetically manipulated mice.

Failure to express GAP-43 leads to disruption of a multipotent precursor and inhibits astrocyte differentiation.

The nervous system-specific protein GAP-43 is significantly upregulated in neurons and glia that are differentiating. In P19 EC cells that do not express GAP-43, neurogenesis is inhibited; many immature neurons apoptose and the survivors do not mature morphologically. Here we show that the initial defect is in an early precursor with characteristics of a neural stem cell, which failed to respond normally to retinoic acid (RA). As a consequence, its progeny had altered cell fates: In addition to the neuronal defects previously reported, RC1-labeled radial glia failed to exit the cell cycle, accumulated, and failed to acquire GFAP immunoreactivity. However, leukemia inhibitory factor (LIF) could stimulate GFAP expression suggesting that astrocytes not derived from radial glia are less affected by absence of GAP-43. Differentiation of radial glia-derived astrocytes was also inhibited in glial cultures from GAP-43 (-/-) cerebellum, and in GAP-43 (-/-) telencephalon in vivo, differentiation of astrocytes derived from both radial and nonradial glia lineages were both affected: In the glial wedge, GFAP-labeled radial glia-derived astrocytes were reduced consistent with the interpretation that they may be unable to deflect GAP-43 (-/-) commissural axons toward the midline. At the midline, both radial and nonradial glia-derived astrocytes were also decreased although it fused normally. The results demonstrate that GAP-43 expressed in multipotent precursors is required for appropriate cell fate commitment, and that its absence affects astrocyte as well as neuronal differentiation.

A point mutant of GAP-43 induces enhanced short-term and long-term hippocampal plasticity.

The growth-associated protein GAP-43 (or neuromodulin or B-50) plays a critical role during development in mechanisms of axonal growth and formation of synaptic networks. At later times, GAP-43 has also been implicated in the regulation of synaptic transmission and properties of plasticity such as long-term potentiation. In a molecular approach, we have analyzed transgenic mice overexpressing different mutated forms of GAP-43 or deficient in GAP-43 to investigate the role of the molecule in short-term and long-term plasticity. We report that overexpression of a mutated form of GAP-43 that mimics constitutively phosphorylated GAP-43 results in an enhancement of long-term potentiation in CA1 hippocampal slices. This effect is specific, because LTP was affected neither in transgenic mice overexpressing mutated forms of non-phosphorylatable GAP-43 nor in GAP-43 deficient mice. The increased LTP observed in transgenic mice expressing a constitutively phosphorylated GAP-43 was associated with an increased paired-pulse facilitation as well as an increased summation of responses during high frequency bursts. These results indicate that, while GAP-43 is not necessary for LTP induction, its phosphorylation may regulate presynaptic properties, thereby affecting synaptic plasticity and the induction of LTP.

GAP-43 is critical for normal development of the serotonergic innervation in forebrain.

Serotonergic (5-HT) axons from the raphe nuclei are among the earliest afferents to innervate the developing forebrain. The present study examined whether GAP-43, a growth-associated protein expressed on growing 5-HT axons, is necessary for normal 5-HT axonal outgrowth and terminal arborization during the perinatal period. We found a nearly complete failure of 5-HT immunoreactive axons to innervate the cortex and hippocampus in GAP-43-null (GAP43-/-) mice. Abnormal ingrowth of 5-HT axons was apparent on postnatal day 0 (P0); quantitative analysis of P7 brains revealed significant reductions in the density of 5-HT axons in the cortex and hippocampus of GAP43-/- mice relative to wild-type (WT) controls. In contrast, 5-HT axon density was normal in the striatum, septum, and amygdala and dramatically higher than normal in the thalamus of GAP43-/- mice. Concentrations of serotonin and its metabolite, 5-hydroxyindolacetic acid, and norepinephrine were decreased markedly in the anterior and posterior cerebrum but increased in the brainstem of GAP43-/- mice. Cell loss could not account for these abnormalities, because unbiased stereological analysis showed no significant difference in the number of 5-HT dorsal raphe neurons in P7 GAP43-/- versus WT mice. The aberrant 5-HT innervation pattern persisted at P21, indicating a long-term alteration of 5-HT projections to forebrain in the absence of GAP-43. In heterozygotes, the density and morphology of 5-HT axons was intermediate between WT and homozygous GAP43-/- mice. These results suggest that GAP-43 is a key regulator in normal pathfinding and arborization of 5-HT axons during early brain development.

Targeting the "species gene ensemble".

The members of a given species display a normal distribution of gene expression which is here termed the species gene ensemble. This more specific designation of the 'genetic background' leads to a consideration of why gene targeting outcomes are regulated by the species gene ensemble. One consequence is the utility of the heterozygous knockout which buffers the drastic compensatory reactions of the homozygous knockout thereby revealing subtle but yet consistent alterations in hippocampally-dependent behaviors.

Growth-associated protein-43 is required for commissural axon guidance in the developing vertebrate nervous system.

Growth-associated protein-43 (GAP-43) is a major growth cone protein whose phosphorylation by PKC in response to extracellular guidance cues can regulate F-actin behavior. Here we show that 100% of homozygote GAP-43 (-/-) mice failed to form the anterior commissure (AC), hippocampal commissure (HC), and corpus callosum (CC) in vivo. Instead, although midline fusion was normal, selective fasciculation between commissural axons was inhibited, and TAG-1-labeled axons tangled bilaterally into Probst's bundles. Moreover, their growth cones had significantly smaller lamellas and reduced levels of F-actin in vitro. Likewise, 100% of GAP-43 (+/-) mice with one disrupted allele also showed defects in HC and CC, whereas the AC was unaffected. Individual GAP-43 (+/-) mice could be assigned to two groups based on the amount that PKC phosphorylation of GAP-43 was reduced in neocortical neurons. In mice with approximately 1% phosphorylation, the HC and CC were absent, whereas in mice with approximately 10% phosphorylation, the HC and CC were smaller. Both results suggest that PKC-mediated signaling in commissural axons may be defective. However, although Probst's bundles formed consistently at the location of the glial wedge, both GAP-43 (-/-) and GAP-43 (+/+) cortical axons were still repulsed by Slit-2 in vitro, precluding failure of this deflective signal from the glial wedge as the source of the phenotype. Nonetheless, the data show that a functional threshold of GAP-43 is required for commissure formation and suggests that failure to regulate F-actin in commissural growth cones may be related to inhibited PKC phosphorylation of GAP-43.

Failure to express GAP-43 during neurogenesis affects cell cycle regulation and differentiation of neural precursors and stimulates apoptosis of neurons.

GAP-43 is first expressed in proliferating neuroblasts and is required for maturation of neurons. When GAP-43 is not expressed in differentiating embryonal carcinoma P19 cells, reduced numbers of neurons were generated. Here we show that neuronal differentiation is initially disrupted at the onset of cell-cycle arrest in aggregated, proliferating neuronal precursors. The ratio of nestin:beta-tubulin-labeled progeny generated at this stage suggests that the differentiation is asymmetric. Apoptosis of immature neurons subsequently produced was also significantly induced. In vivo, too, proliferation of neuroblasts was significantly reduced in cortex of GAP-43(-/-) mice at E14.5. These data demonstrate that when GAP-43 is not expressed in proliferating neuroblasts, neural differentiation is not initiated appropriately, inducing apoptosis. Moreover, the concurrent inhibition of Ca2+-dependent adhesion between differentiating P19 cells in aggregates implicates GAP-43 in CAM-mediated signaling during neurogenesis, as has been previously shown in growth cones.

Absence of GAP-43 can protect neurons from death.

The main function of GAP-43 is thought to be regulating growth cone motility and axon guidance signals. GAP-43 is highly expressed during development and in regenerating nerves and in particular regions of the adult brain. We here present the first evidence that GAP-43 can modulate guidance signals emanating from Semaphorin III (SemaIII) in cultured NGF-dependent sensory neurons. We further show that absence of GAP-43 dramatically increases resistance of specific sensory neurons to apoptotic stimuli in vitro. NGF-dependent sensory neurons from GAP-43 (+/-) and null mutant mice are strongly protected against SemaIII-induced death. Furthermore, NGF- and BDNF-dependent neurons, but not NT-3-dependent neurons, from GAP-43 null mutant mice are much more resistant to apoptosis induced by trophic factor deprivation. We also show that early postnatal Purkinje cells from GAP-43 (+/-) mice are more resistant to cell death in organotypic cultures. We conclude that GAP-43 can influence neuronal survival and modulate repulsive axon guidance signals.

GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation.

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.

Altered midline axon pathways and ectopic neurons in the developing hypothalamus of netrin-1- and DCC-deficient mice.

Optic nerve formation in mouse involves interactions between netrin-1 at the optic disk and the netrin-1 receptor DCC (deleted in colorectal cancer) expressed on retinal ganglion cell (RGC) axons. Deficiency in either protein causes RGC pathfinding defects at the disk leading to optic nerve hypoplasia (). Here we show that further along the visual pathway, RGC axons in netrin-1- or DCC-deficient mice grow in unusually angular trajectories within the ventral hypothalamus. In heterozygous Sey(neu) mice that also have a small optic nerve, RGC axon trajectories appear normal, indicating that the altered RGC axon trajectories in netrin-1 and DCC mutants are not secondarily caused by optic nerve hypoplasia. Intrinsic hypothalamic patterning is also affected in netrin-1 and DCC mutants, including a severe reduction in the posterior axon projections of gonadotropin-releasing hormone neurons. In addition to axon pathway defects, antidiuretic hormone and oxytocin neurons are found ectopically in the ventromedial hypothalamus, apparently no longer confined to the supraoptic nucleus in mutants. In summary, netrin-1 and DCC, presumably via direct interactions, govern both axon pathway formation and neuronal position during hypothalamic development, and loss of netrin-1 or DCC function affects both visual and neuroendocrine systems. Netrin protein localization also indicates that unlike in more caudal CNS, guidance about the hypothalamic ventral midline does not require midline expression of netrin.

Disrupted cortical map and absence of cortical barrels in growth-associated protein (GAP)-43 knockout mice.

There is strong evidence that growth-associated protein (GAP-43), a protein found only in the nervous system, regulates the response of neurons to axonal guidance signals. However, its role in complex spatial patterning in cerebral cortex has not been explored. We show that mice lacking GAP-43 expression (-/-) fail to establish the ordered whisker representation (barrel array) normally found in layer IV of rodent primary somatosensory cortex. Thalamocortical afferents to -/- cortex form irregular patches in layer IV within a poorly defined cortical field, which varies between hemispheres, rather than the stereotypic, whisker-specific, segregated map seen in normal animals. Furthermore, many thalamocortical afferents project abnormally to widely separated cortical targets. Taken together, our findings indicate a loss of identifiable whisker territories in the GAP-43 -/- mouse cortex. Here, we present a disrupted somatotopic map phenotype in cortex, in clear contrast to the blurring of boundaries within an ordered whisker map in other barrelless mutants. Our results indicate that GAP-43 expression is critical for the normal establishment of ordered topography in barrel cortex.