Induce Growth Cone Collapse Research Articles

Polysialylated NCAM and EphrinA/EphA Regulate Synaptic Development of GABAergic Interneurons in Prefrontal Cortex

A novel function for the neural cell adhesion molecule (NCAM) was identified in ephrinA/EphA-mediated repulsion as an important regulatory mechanism for development of GABAergic inhibitory synaptic connections in mouse prefrontal cortex. Deletion of NCAM, EphA3, or ephrinA2/3/5 in null mutant mice increased the numbers and size of perisomatic synapses between GABAergic basket interneurons and pyramidal cells in the developing cingulate cortex (layers II/III). A functional consequence of NCAM loss was increased amplitudes and faster kinetics of miniature inhibitory postsynaptic currents in NCAM null cingulate cortex. NCAM and EphA3 formed a molecular complex and colocalized with the inhibitory presynaptic marker vesicular GABA transporter (VGAT) in perisomatic puncta and neuropil in the cingulate cortex. EphrinA5 treatment promoted axon remodeling of enhanced green fluorescent protein-labeled basket interneurons in cortical slice cultures and induced growth cone collapse in wild-type but not NCAM null mutant neurons. NCAM modified with polysialic acid (PSA) was required to promote ephrinA5-induced axon remodeling of basket interneurons in cortical slices, likely by providing a permissive environment for ephrinA5/EphA3 signaling. These results reveal a new mechanism in which NCAM and ephrinAs/EphA3 coordinate to constrain GABAergic interneuronal arborization and perisomatic innervation, potentially contributing to excitatory/inhibitory balance in prefrontal cortical circuitry.

Matrix Metalloproteinase-8 Is Involved in Dermal Nerve Growth: Implications for Possible Application to Pruritus from In Vitro Models

Cutaneous nerve density is related to abnormal itch perception in dermatoses, such as atopic dermatitis and xerosis. However, the mechanisms underlying the elongation of dermal nerve fibers within the interstitial collagen (CoL) matrix are poorly understood. In this study, a culture system of rat dorsal root ganglion neurons consisting of type I CoL and a Boyden chamber containing a nerve growth factor (NGF) concentration gradient was used. Nerve fibers penetrating into type I CoL gel were observed in the presence of the NGF concentration gradient. Levels of matrix metalloproteinase-8 (MMP-8) mRNA and protein were increased in the cultured neurons and the conditioned medium, respectively. The nerve fiber penetration was dose dependently inhibited by MMP-8 blockers. Moreover, MMP-8 immunoreactivity was partially localized at growth cones in NGF-responsive nerve fibers. Semaphorin 3A stimulation also showed the opposite effects on these NGF-dependent events. Intriguingly, MMP-8 expression was upregulated by type I and III CoLs, which are substrates for this enzyme. These results suggested that MMP-8 is involved in sensory nerve growth within the interstitial CoL matrix through modulation by the axonal guidance molecules and/or extracellular matrix components. These findings provide insight into the development of pruritus involving skin nerve density.

Activated Microglia Inhibit Axonal Growth through RGMa

By causing damage to neural networks, spinal cord injuries (SCI) often result in severe motor and sensory dysfunction. Functional recovery requires axonal regrowth and regeneration of neural network, processes that are quite limited in the adult central nervous system (CNS). Previous work has shown that SCI lesions contain an accumulation of activated microglia, which can have multiple pathophysiological influences. Here, we show that activated microglia inhibit axonal growth via repulsive guidance molecule a (RGMa). We found that microglia activated by lipopolysaccharide (LPS) inhibited neurite outgrowth and induced growth cone collapse of cortical neurons in vitro—a pattern that was only observed when there was direct contact between microglia and neurons. After microglia were activated by LPS, they increased expression of RGMa; however, treatment with RGMa-neutralizing antibodies or transfection of RGMa siRNA attenuated the inhibitory effects of microglia on axonal outgrowth. Furthermore, minocycline, an inhibitor of microglial activation, attenuated the effects of microglia and RGMa expression. Finally, we examined whether these in vitro patterns could also be observed in vivo. Indeed, in a mouse SCI model, minocycline treatment reduced the accumulation of microglia and decreased RGMa expression after SCI, leading to reduced dieback in injured corticospinal tracts. These results suggest that activated microglia play a major role in inhibiting axon regeneration via RGMa in the injured CNS.

Proteolytic cleavage of Reelin within its C-terminal region regulates its ability to induce growth cone collapse

Proteolytic cleavage of Reelin within its C-terminal region regulates its ability to induce growth cone collapse

FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons

Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A-Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.

Versican V0 and V1 Direct the Growth of Peripheral Axons in the Developing Chick Hindlimb

Peanut agglutinin-binding disaccharides and chondroitin sulfate mark transient mesenchymal barriers to advancing motor and sensory axons innervating the hindlimbs during chick development. Here we show that the vast majority of these carbohydrates are at the critical stage and location attached to the versican splice variants V0 and V1. We reveal that the isolated isoforms of this extracellular matrix proteoglycan suppress axon extension at low concentrations and induce growth cone collapse and rapid retraction at higher levels. Moreover, we demonstrate that versican V0 and/or V1, recombinantly expressed in collagen-I gels or ectopically deposited in the hindlimbs of chicken embryos in ovo, cause untimely defasciculation and axon stalling. Consequently, severe disturbances of nerve patterning are observed in the versican-treated embryos. Our experiments emphasize the inhibitory capacity of versicans V0 and V1 in axonal growth and evidence for their function as basic guidance cues during development of the peripheral nervous system.

Different spatial and temporal protein expressions of repulsive guidance molecule a and neogenin in the rat optic nerve after optic nerve crush with and without lens injury

The failure of lesioned mammalian CNS neurons to regenerate their axons remains a challenge. Evidence is emerging that repulsive proteins contribute to this failure. The repulsive guidance molecule A (RGMA) induces growth cone collapse in vitro, accumulates in the scar after spinal cord injury, and is up-regulated in glaucoma. In this study, we evaluated the spatial and temporal localization pattern of RGMA and its receptor neogenin in the optic nerve after optic nerve crush (ONC) without or with lens injury (LI) at up to nine time points (6 hr to 20 days) postsurgery by performing immunohistochemistry and Western blots. We found RGMA at the crush site (CS) and in the developing scar of ONC rats at every time point investigated, whereas it was absent in the CS of ONC + LI rats. Independent of the model, many cells were RGMA(+) in the ON: nerve fibers, blood vessels, astrocytes, oligodendrocytes, some microglia, some macrophages, and the sheath of the ON. Western blots showed a significantly lowered amount of RGMA in ONC + LI animals at 2, 4, and 6 days after crush compared with ONC animals. Furthermore, LI in sham-operated animals showed an increase of RGMA in six of eight time points compared with the sham-operated animals. Moreover, the effects of LI on the morphology of the ON were characterized at a level of detail never reported before. Our results show that RGMA is present and might contribute to the inhibitory environment in the ON, especially in and around the CS after ONC.

Neurite formation by neurons derived from adult rat hippocampal progenitor cells is susceptible to myelin inhibition

Myelin-associated inhibitors expressed following injury to the adult central nervous system (CNS) induce growth cone collapse and retraction of the axonal cytoskeleton. Myelin-associated glycoprotein (MAG) is a bi-functional molecule that promotes neuritogenesis in some immature neurons during development then becomes inhibitory to neurite outgrowth as neurons mature. Progress is being made towards the elucidation of the downstream events that regulate myelin inhibition of regeneration in neuronal populations. However it is not known how adult-derived neural stem cells or progenitors respond to myelin during neuronal differentiation and neuritogenesis. Here we examine the effect of MAG on neurons derived from an adult rat hippocampal progenitor cell line (AHPCs). We show that, unlike their developmental counterparts, AHPC-derived neurons are susceptible to MAG inhibition of neuritogenesis during differentiation and display a 57% reduction in neurite outgrowth when compared with controls. We demonstrate that this effect can be overcome (by up to 69%) by activation of the neurotrophin, cyclic AMP and protein kinase A pathways or by Rho-kinase suppression. We also demonstrate that combination of these factors enhanced neurite outgrowth from differentiating neurons in the presence of MAG. This work provides important information for the successful generation of new neurons from adult neural stem cell populations within compromised adult circuitry and is thus directly relevant to endogenous repair and regeneration of the adult CNS.

Semaphorin 4D/Plexin-B1 Stimulates PTEN Activity through R-Ras GTPase-activating Protein Activity, Inducing Growth Cone Collapse in Hippocampal Neurons

Plexins are receptors for axonal guidance molecules semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). Here we examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. We also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 alpha activity. Thus, we propose that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse.

Cellular responses to extracellular guidance cues

Extracellular guidance cues have a key role in orchestrating cell behaviour. They can take many forms, including soluble and cell-bound ligands (proteins, lipids, peptides or small molecules) and insoluble matrix substrates, but to act as guidance cues, they must be presented to the cell in a spatially restricted manner. Cells that recognize such cues respond by activating intracellular signal transduction pathways in a spatially restricted manner and convert the extracellular information into intracellular polarity. Although extracellular cues influence a broad range of cell polarity decisions, such as mitotic spindle orientation during asymmetric cell division, or the establishment of apical-basal polarity in epithelia, this review will focus specifically on guidance cues that promote cell migration (chemotaxis), or localized cell shape changes (chemotropism).

EphA3 functions are regulated by collaborating phosphotyrosine residues

Ephrin ligands interact with Eph receptors to regulate a wide variety of biological and pathological processes. Recent studies have identified several downstream pathways that mediate the functions of these receptors. Activation of the receptors by ephrin binding results in the phosphorylation of the receptor tyrosine residues. These phosphorylated residues serve as docking sites for many of the downstream signaling pathways. However, the relative contributions of different phospho-tyrosine residues remain undefined. In the present study, we mutated each individual tyrosine residues in the cytoplasmic domain of EphA3 receptor and studied the effects using cell migration, process retraction, and growth cone collapse assays. Stimulation of the EphA3 receptor with ephrin-A5 inhibits 293 cell migration, reduces NG108-15 cell neurite outgrowth, and induces growth cone collapse in hippocampal neurons. Mutation of either Y602 or Y779 alone partially decreases EphA3-induced responses. Full abrogation can only be achieved with mutations of both Y602 and Y779. These observations suggest a collaborative model of different downstream pathways.

Antidepressant fluoxetine suppresses neuronal growth from both vertebrate and invertebrate neurons and perturbs synapse formation between Lymnaea neurons

Current treatment regimes for a variety of mental disorders involve various selective serotonin reuptake inhibitors such as Fluoxetine (Prozac). Although these drugs may 'manage' the patient better, there has not been a significant change in the treatment paradigm over the years and neither have the outcomes improved. There is also considerable debate as to the effectiveness of various selective serotonin reuptake inhibitors and their potential side-effects on neuronal architecture and function. In this study, using mammalian cortical neurons, a dorsal root ganglia cell line (F11 cells) and identified Lymnaea stagnalis neurons, we provide the first direct and unequivocal evidence that clinically relevant concentrations of Fluoxetine induce growth cone collapse and neurite retraction of both serotonergic and non-serotonergic neurons alike in a dose-dependent manner. Using intracellular recordings and calcium imaging techniques, we further demonstrate that the mechanism underlying Fluoxetine-induced effects on neurite retraction from Lymnaea neurons may involve lowering of intracellular calcium and a subsequent retardation of growth cone cytoskeleton. Using soma-soma synapses between identified presynaptic and postsynaptic Lymnaea neurons, we provide further direct evidence that clinically used concentrations of Fluoxetine also block synaptic transmission and synapse formation between cholinergic neurons. Our study raises alarms over potentially devastating side-effects of this antidepressant drug on neurite outgrowth and synapse formation in a developing/regenerating brain. Our data also demonstrate that drugs such as Fluoxetine may not just affect communication between serotonergic neurons but that the detrimental effects are widespread and involve neurons of various phenotypes from both vertebrate and invertebrate species.

Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling

Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system. The Nogo-A-specific fragment NogoDelta20 induces growth cone collapse, and inhibits neurite outgrowth and cell spreading by activating RhoA. Here, we show that NogoDelta20 is internalized into neuronal cells by a Pincher- and rac-dependent, but clathrin- and dynamin-independent, mechanism. Pincher-mediated macroendocytosis results in the formation of NogoDelta20-containing signalosomes that direct RhoA activation and growth cone collapse. In compartmentalized chamber cultures, NogoDelta20 is endocytosed into neurites and retrogradely transported to the cell bodies of dorsal root ganglion neurons, triggering RhoA activation en route and decreasing phosphorylated cAMP response element binding levels in cell bodies. Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.

Sema4A induces cell morphological changes through B-type plexin-mediated signaling

Semaphorins are a family of secreted and membrane-bound proteins known as axonal pathfinders. Sema4A, a member of class 4 semaphorins, induces growth cone collapse of hippocampal neurons. The binding of Sema4A to growth cones indicates the presence of receptors transmitting signals through the intracellular effectors to induce growth cone collapse in hippocampal neurons. Transfection experiments of the candidate receptor genes into COS-7 cells demonstrated that Sema4A binds to axonal guidance receptors Plexin-B1, -B2 and -B3. To identify the functional Sema4A receptor and the signal transduction machinery, COS-7 cell contraction assay was performed, in which intracellular signal transmission induced by Sema4A triggered cell contraction. Expression vectors encoding plexins and Rnd1, a Rho family GTPase, were transfected into COS-7 cells, and a proportion of contracted cells among the transfectants was determined after incubation with Sema4A. The results demonstrated that the combination of Rnd1 and Plexin-B1, -B2 or -B3 induced significant cell contraction, indicating that B-type plexins transmit an intracellular signal of Sema4A through Rnd1. To further study the mechanism of B-type plexin-mediated signaling in Sema4A-induced growth cone collapse, mouse hippocampal neurons transfected with a control or expression plasmid encoding a constitutively active mutant of R-Ras (R-RasQL) were stimulated with Sema4A, followed by the assessment of growth cone collapse. Expression of R-RasQL significantly blocked Sema4A-induced growth cone collapse in the hippocampal neurons compared with the control plasmid. Sema4A thus induces growth cone collapse through the down-regulation of R-Ras activity in mouse hippocampal neurons.

The Tumor Suppressor p53 Transcriptionally Regulates cGKI Expression during Neuronal Maturation and Is Required for cGMP-Dependent Growth Cone Collapse

The cGMP-dependent protein kinase type I (cGKI) has multiple functions including a role in axonal growth and pathfinding of sensory neurons, and counteracts Semaphorin 3A (Sema3A)-induced growth cone collapse. Within the nervous system, however, the transcriptional regulation of cGKI is still obscure. Recently, the transcription factor and tumor suppressor p53 has been reported to promote neurite outgrowth by regulating the gene expression of factors that promote growth cone extension, but specific p53 targets genes that may counteract growth cone collapse have not been identified so far. Here, we show that p53 promotes cGKI expression in neuronal-like PC-12 cells and primary neurons by occupying specific regulatory elements in a chromatin environment during neuronal maturation. Importantly, we demonstrate that p53-dependent expression of cGKI is required for the ability of cGMP to counteract growth cone collapse. Growth cone retraction mediated by Sema3A is overcome by cGMP only in wild-type, but not in p53-null dorsal root ganglia. Reconstitution of p53 levels is sufficient to recover both cGKI expression and the ability of cGMP to counteract growth cone collapse, while cGKI overexpression rescues growth cone collapse in p53-null primary neurons. In conclusion, this study identifies p53 as a transcription factor that regulates the expression of cGKI during neuronal maturation and cGMP-dependent inhibition of growth cone collapse.

Disruption of the cytoskeleton during Semaphorin 3A induced growth cone collapse correlates with differences in actin organization and associated binding proteins

Repulsive guidance cues induce growth cone collapse or collapse and retraction. Collapse results from disruption and loss of the actin cytoskeleton. Actin-rich regions of growth cones contain binding proteins that influence filament organization, such as Arp2/3, cortactin, and fascin, but little is known about the role that these proteins play in collapse. Here, we show that Semaphorin 3A (Sema 3A), which is repulsive to mouse dorsal root ganglion neurons, has unequal effects on actin binding proteins and their associated filaments. The immunofluorescence staining intensity of Arp-2 and cortactin decreases relative to total protein; whereas in unextracted growth cones fascin increases. Fascin and myosin IIB staining redistribute and show increased overlap. The degree of actin filament loss during collapse correlates with filament superstructures detected by rotary shadow electron microscopy. Collapse results in the loss of branched f-actin meshworks, while actin bundles are partially retained to varying degrees. Taken together with the known affects of Sema 3A on actin, this suggests a model for collapse that follows a sequence; depolymerization of actin meshworks followed by partial depolymerization of fascin associated actin bundles and their movement to the neurite to complete collapse. The relocated fascin associated actin bundles may provide the substrate for actomyosin contractions that produce retraction.

Protein microarray analysis identifies cyclic nucleotide phosphodiesterase as an interactor of Nogo-A

Nogo-A, a neurite outgrowth inhibitor, is expressed exclusively on oligodendrocytes and neurons in the CNS. The central domain of Amino-Nogo spanning amino acids 567-748 in the human Nogo-A designated NIG, mediates persistent inhibition of axonal outgrowth and induces growth cone collapse by signaling through an as yet unidentified NIG receptor. We identified 82 NIG-interacting proteins by screening a high-density human protein microarray composed of 5000 proteins with a recombinant NIG protein as a probe. Following an intensive database search, we selected 12 neuron/oligodendrocyte-associated NIG interactors. Among them, we verified the molecular interaction of NIG with 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a cell type-specific marker of oligodendrocytes, by immunoprecipitation and cell imaging analysis. Although CNP located chiefly in the cytoplasm of oligodendrocytes might not serve as a cell-surface NIG receptor, it could act as a conformational stabilizer for the intrinsically unstructured large segment of Amino-Nogo.

Inactivation of Ras by p120GAP via Focal Adhesion Kinase Dephosphorylation Mediates RGMa-Induced Growth Cone Collapse

The repulsive guidance molecule RGMa performs several functions in the developing and adult CNSs. RGMa, through its receptor neogenin, induces growth cone collapse and neurite outgrowth inhibition. Here, we demonstrate that RGMa binding to neogenin leads to the inactivation of Ras, which is required for the RGMa-mediated repulsive function in cortical neurons. This signal transduction is mediated by the Ras-specific GTPase-activating protein (GAP) p120GAP. The SH2 domain of p120GAP interacts with focal adhesion kinase (FAK), which is phosphorylated at Tyr-397. When the cells are stimulated with RGMa, FAK undergoes dephosphorylation at Tyr-397 and is dissociated from p120GAP, and this dissociation is followed by an increase in the interaction between p120GAP and GTP-Ras. In addition, the knockdown of p120GAP prevents RGMa-induced growth cone collapse and neurite outgrowth inhibition. Furthermore, RGMa stimulation induces Akt inactivation through p120GAP, and the expression of the constitutively active Akt prevents RGMa-induced growth cone collapse. Thus, RGMa binding to neogenin regulates p120GAP activity through FAK Tyr-397 dephosphorylation, leading to the inactivation of Ras and its downstream effector Akt, and this signal transduction plays a role in the RGMa-mediated repulsive function.

A Novel Function for p53: Regulation of Growth Cone Motility through Interaction with Rho Kinase

The transcription factor p53 suppresses tumorgenesis by regulating cell proliferation and migration. We investigated whether p53 could also control cell motility in postmitotic neurons. p53 isoforms recognized by phospho-p53-specific (at Ser-15) or "mutant" conformation-specific antibodies were highly and specifically expressed in axons and axonal growth cones in primary hippocampal neurons. Inhibition of p53 function by inhibitors, small interfering RNAs, or by dominant-negative forms, induced axonal growth cone collapse, whereas p53 overexpression led to larger growth cones. Furthermore, deletion of the p53 nuclear export signal blocked its axonal distribution and induced growth cone collapse. p53 inhibition-induced axonal growth cone collapse was significantly reduced by the Rho kinase (ROCK) inhibitor, Y27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide]. Our results reveal a new function for p53 as a critical regulator of axonal growth cone behavior by suppressing ROCK activity.

In vitro guidance of retinal axons by a tectal lamina-specific glycoprotein Nel

Nel is a glycoprotein containing five chordin-like and six epidermal growth factor-like domains and is strongly expressed in the nervous system. In this study, we have examined expression patterns and in vitro functions of Nel in the chicken retinotectal system. We have found that in the developing tectum, expression of Nel is localized in specific laminae that retinal axons normally do not enter, including the border between the retinorecipient and non-retinorecipient laminae. Nel-binding activity is detected on retinal axons both in vivo and in vitro, suggesting that retinal axons express a receptor for Nel. In vitro, Nel inhibits retinal axon outgrowth and induces growth cone collapse and axon retraction. These results indicate that Nel acts as an inhibitory guidance cue for retinal axons, and suggest its roles in the establishment of the lamina-specificity in the retinotectal projection.