Semaphorin 3A inhibits ERM protein phosphorylation in growth cone filopodia through inactivation of PI3K

Ezrin-radixin-moesin (ERM) proteins are involved in the linkage of membranes to theactin filament (F-actin) cytoskeleton. Phosphorylation of the C-terminus activates the F-actin binding domain of ERM proteins by preventing the action of an autoinhibitory domain. In this study, we investigated whether a growth cone collapsing signal, semaphorin 3A (Sema3A), alters the state of ERM C-terminus phosphorylation. In the growth cones of dorsal root ganglion axons, phosphorylated ERM proteins localize to filopodia. We report that Sema3A inhibits ERM protein phosphorylation in growth cone filopodia. Significantly, Sema3A decreased ERM phosphorylation prior to the onset of growth cone collapse. Over-expression of the F-actin binding fragment of ERM proteins, which competes with endogenous ERM proteins for binding to F-actin, inhibited filopodial initiation and dynamics. Sema3A has been previously shown to inhibit phosphoinositide 3-kinase (PI3K) activity. Inhibition of PI3K resulted in the loss of phosphorylated ERM proteins from growth cone filopodia, and treatment with a PI3K activating peptide blocked the effects of Sema3A on ERM phosphorylation. Collectively, these observations demonstrate that inactivation of PI3K in response to Sema3A results in decreased phosphorylation of ERM proteins in filopodia thereby contributing to growth cone collapse.     

An inactive pool of GSK-3 at the leading edge of growth cones is implicated in Semaphorin 3A signaling

Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase that has been implicated in several aspects in embryonic development and several growth factor signaling cascades. We now report that an inactive phosphorylated pool of the enzyme colocalizes with F-actin in both neuronal and nonneuronal cells. Semaphorin 3A (Sema 3A), a molecule that inhibits axonal growth, activates GSK-3 at the leading edge of neuronal growth cones and in Sema 3A-responsive human breast cancer cells, suggesting that GSK-3 activity might play a role in coupling Sema 3A signaling to changes in cell motility. We show that three different GSK-3 antagonists (LiCl, SB-216763, and SB-415286) can inhibit the growth cone collapse response induced by Sema 3A. These studies reveal a novel compartmentalization of inactive GSK-3 in cells and demonstrate for the first time a requirement for GSK-3 activity in the Sema 3A signal transduction pathway.     

Developmental Upregulation of Ephrin-B1 Silences Sema3C/Neuropilin-1 Signaling during Post-crossing Navigation of Corpus Callosum Axons

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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. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Quantitative analysis of microtubule dynamics during adhesion-mediated growth cone guidance

During adhesion-mediated neuronal growth cone guidance microtubules undergo major rearrangements. However, it is unknown whether microtubules extend to adhesion sites because of changes in plus-end polymerization and/or translocation dynamics, because of changes in actin-microtubule interactions, or because they follow the reorganization of the actin cytoskeleton. Here, we used fluorescent speckle microscopy to directly quantify microtubule and actin dynamics in Aplysia growth cones as they turn towards beads coated with the cell adhesion molecule apCAM. During the initial phase of adhesion formation, dynamic microtubules in the peripheral domain preferentially explore apCAM-beads prior to changes in growth cone morphology and retrograde actin flow. Interestingly, these early microtubules have unchanged polymerization rates but spend less time in retrograde translocation due to uncoupling from actin flow. Furthermore, microtubules exploring the adhesion site spend less time in depolymerization. During the later phase of traction force generation, the central domain advances and more microtubules in the peripheral domain extend because of attenuation of actin flow and clearance of F-actin structures. Microtubules in the transition zone and central domain, however, translocate towards the adhesion site in concert with actin arcs and bundles, respectively. We conclude that adhesion molecules guide neuronal growth cones and underlying microtubule rearrangements largely by differentially regulating microtubule-actin coupling and actin movements according to growth cone region and not by controlling plus-end polymerization rates.     

Growth cone collapse through coincident loss of actin bundles and leading edge actin without actin depolymerization

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.     

Focal loss of actin bundles causes microtubule redistribution and growth cone turning

It is commonly believed that growth cone turning during pathfinding is initiated by reorganization of actin filaments in response to guidance cues, which then affects microtubule structure to complete the turning process. However, a major unanswered question is how changes in actin cytoskeleton are induced by guidance cues and how these changes are then translated into microtubule rearrangement. Here, we report that local and specific disruption of actin bundles from the growth cone peripheral domain induced repulsive growth cone turning. Meanwhile, dynamic microtubules within the peripheral domain were oriented into areas where actin bundles remained and were lost from areas where actin bundles disappeared. This resulted in directional microtubule extension leading to axon bending and growth cone turning. In addition, this local actin bundle loss coincided with localized growth cone collapse, as well as asymmetrical lamellipodial protrusion. Our results provide direct evidence, for the first time, that regional actin bundle reorganization can steer the growth cone by coordinating actin reorganization with microtubule dynamics. This suggests that actin bundles can be potential targets of signaling pathways downstream of guidance cues, providing a mechanism for coupling changes in leading edge actin with microtubules at the central domain during turning.     

Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. For the first time, we show that the ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5-treated retinal ganglion cell cultures, Rho was activated and Rac was downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduced the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway.     

Semaphorin3A regulates axon growth independently of growth cone repulsion via modulation of TrkA signaling

Regulation of axon growth is a critical event in neuronal development. Nerve growth factor (NGF) is a strong inducer of axon growth and survival in the dorsal root ganglia (DRG). Paradoxically, high concentrations of NGF are present in the target region where axon growth must slow down for axons to accurately identify their correct targets. Semaphorin3A (Sema3A), a powerful axonal repellent molecule for DRG neurons, is also situated in their target regions. NGF is a modulator of Sema3A-induced repulsion and death. We show that Sema3A is a regulator of NGF-induced neurite outgrowth via the TrkA receptor, independent of its growth cone repulsion activity. First, neurite outgrowth of DRG neurons is more sensitive to Sema3A than repulsion. Second, at concentrations sufficient to significantly inhibit Sema3A-induced repulsion, NGF has no effect on Sema3A-induced axon growth inhibition. Third, Sema3A-induced outgrowth inhibition, but not repulsion activity, is dependent on NGF stimulation. Fourth, Sema3A attenuates TrkA-mediated growth signaling, but not survival signaling, and over-expression of constitutively active TrkA blocks Sema3A-induced axon growth inhibition, suggesting that Sema3A activity is mediated via regulation of NGF/TrkA-induced growth. Finally, quantitative analysis of axon growth in vivo supports the possibility that Sema3A affects axon growth, in addition to its well-documented role in axon guidance. We suggest a model whereby NGF at high concentrations in the target region is important for survival, attraction and inhibition of Sema3A-induced repulsion, while Sema3A inhibits its growth-promoting activity. The combined and cross-modulatory effects of these two signaling molecules ensure the accuracy of the final stages in axon targeting.     

A role for semaphorin 3A signaling in the degeneration of hippocampal neurons during Alzheimer's disease

Among the earliest invariant neuropathological changes in Alzheimer's disease (AD) is the degeneration of vulnerable hippocampal CA1 and subicular pyramidal neurons. Semaphorin 3A (Sema3A) is a secreted protein that functions in signaling growth cone collapse, chemorepulsion and neuronal apoptosis during early development of the central nervous system. In this report we show that accumulation of an internalized form of Sema3A is associated with degeneration of neurons in vulnerable fields of the hippocampus during AD. Accumulation of Sema3A overlaps the appearance of phosphorylated MAP1B and tau in many neurons, suggesting that Sema3A signaling at some level may be coupled to these previously identified cytoskeletal markers of neurodegeneration. Consistent with this, we isolated and partially characterized a multiprotein complex from the hippocampus of patients with AD that contains phosphorylated MAP1B, collapsin-response mediator protein 2 (CRMP-2), Plexins A1 and A2, and a processed form of Sema3A. A model is presented in which aberrant release of Sema3A from expressing neurons in the subiculum during AD results in the internalization and transport of Sema3A from this field to CA1. Within the context of the myriad of potential insults that contribute to Alzheimer's and other neurodegenerative diseases, the bioactivity of Sema3A may contribute either directly to neurodegeneration by inducing neuronal collapse, or indirectly by abrogating the recovery capabilities of adult neurons faced with these insults.     

FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse

Axonal receptors for class 3 semaphorins (Sema3s) are heterocomplexes of neuropilins (Nrps) and Plexin-As signalling coreceptors. In the developing cerebral cortex, the Ig superfamily cell adhesion molecule L1 associates with Nrp1. Intriguingly, the genetic removal of L1 blocks axon responses of cortical neurons to Sema3A in vitro despite the expression of Plexin-As in the cortex, suggesting either that L1 substitutes for Plexin-As or that L1 and Plexin-A are both required and mediate distinct roles. We report that association of Nrp1 with L1 but not Plexin-As mediates the recruitment and activation of a Sema3A-induced focal adhesion kinase-mitogen-activated protein kinase cascade. This signalling downstream of L1 is needed for the disassembly of adherent points formed in growth cones and subsequently their collapse response to Sema3A. Plexin-As and L1 are coexpressed and present in common complexes in cortical neurons and both dominant-negative forms of Plexin-A and L1 impair their response to Sema3A. Consistently, Nrp1-expressing cortical projections are defective in mice lacking Plexin-A3, Plexin-A4 or L1. This reveals that specific signalling activities downstream of L1 and Plexin-As cooperate for mediating the axon guidance effects of Sema3A.     

Counteraction of axonal growth inhibitory properties of Semaphorin 3A and myelin-associated proteins by a synthetic neurotrophic compound

One of the reasons for the lack of nerve regeneration in the CNS is the formation of a glial scar over-expressing multiple inhibitory factors including myelin-associated proteins and members of the Semaphorin family. Innovative therapeutic strategies must stimulate axon extension across the lesion site despite this inhibitory molecular barrier. We recently developed a synthetic neurotrophic compound combining an omega-alkanol with a retinol-like cycle (3-(15-hydroxy-pentadecyl)-2,4,4,-trimethyl-cyclohexen-2-one (tCFA15)). Here, we demonstrate that tCFA15 is able to promote cortical axon outgrowth in vitro even in the presence of the inhibitory Semaphorin 3A and myelin extracts. This growth-promoting effect is selectively observed in axons and requires multiple growth-associated intracellular pathways. Our results illustrate the potential use of synthetic neurotrophic compounds to promote nerve regeneration by counteracting the axonal growth inhibition triggered by glial scar-associated inhibitory factors.     

Induction of axon growth arrest without growth cone collapse through the N-terminal region of four-transmembrane glycoprotein M6a

During development, axons elongate vigorously, carefully controlling their speed, to connect with their targets. In general, rapid axon growth is correlated with active growth cones driven by dynamic actin filaments. For example, when the actin-driven tip is collapsed by repulsive guidance molecules, axon growth is severely impaired. In this study, we report that axon growth can be suppressed, without destroying the actin-based structure or motility of the growth cones, when antibodies bind to the four-transmembrane glycoprotein M6a concentrated on the growth cone edge. Surprisingly, M6a-deficient axons grow actively but are not growth suppressed by the antibodies, arguing for an inductive action of the antibody. The binding of antibodies clusters and displaces M6a protein from the growth cone edge membrane, suggesting that the spatial rearrangement of this protein might underlie the unique growth cone behavior triggered by the antibodies. Molecular dissection of M6a suggested involvement for the N-terminal intracellular domain in this antibody-induced growth cone arrest.     

The region-specific activities of lipid rafts during axon growth and guidance

The generation and control of cell polarity is a fundamental mechanism for directed migration of the cell. In developing neurons, the axonal growth cone recognizes environmental molecular cues and migrates toward its correct target, thereby forming neuronal networks. The spatial information provided by environmental cues directs axon growth and guidance through generating polarity of intracellular signals and cytoskeletal organization in the growth cone. This polarization process is dependent on lipid rafts, specialized microdomains in the cell membrane. Lipid rafts in specific regions of the growth cone are involved in axon growth and guidance. For example, forward migration of the growth cone requires raft membranes in its leading front. Recent experiments have suggested that lipid rafts function as a platform for localized signaling downstream of adhesion molecules and guidance receptors. The rafts assemble into an active membrane domain that captures and reorganizes the cytoskeletal machinery. In this way, the spatial control of signaling through raft membranes plays a critical role in translating extracellular information into polarized motility of the growth cone.     

A stochastic model of neuronal growth cone guidance regulated by multiple sensors

Neuronal growth cones migrate directionally under the control of axon guidance molecules, thereby forming synapses in the developing brain. The signal transduction system by which a growth cone detects surrounding guidance molecules, analyzes the detected signals, and then determines the overall behavior remains undetermined. In this study, we describe a novel stochastic model of this behavior that utilizes multiple sensors on filopodia to respond to guidance molecules. Overall growth cone behavior is determined by using only the concentration gradients of guidance molecules in the immediate vicinity of each sensor. The detected signal at each sensor, which is treated as a vector quantity, is sent to the growth cone center and then integrated to determine axonal growth in the next step by means of a simple vector operation. We compared the results of computer simulations of axonal growth with observations of actual axonal growth from co-culture experiments using olfactory bulb and septum. The probabilistic distributions of axonal growth generated by the computer simulation were consistent with those obtained from the culture experiments, indicating that our model accurately simulates growth cone behavior. We believe that this model will be useful for elucidating the as yet unknown mechanisms responsible for axonal growth in vivo.     

Hypertonic conditions trigger transient plasmolysis,growth arrest and blockage of transporter endocytosis in Aspergillus nidulans and Saccharomyces cerevisiae

Abstract

By using Aspergillus nidulans strains expressing functional GFP-tagged transporters under hypertonic conditions, we noticed the rapid appearance of cortical, relatively static, fluorescent patches (0.5–2.3 μm). These patches do not correspond to transporter microdomains as they co-localize with other plasma membrane-associated molecules, such as the pleckstrin homology (PH) domain and the SsoA t-Snare, or the lipophilic markers FM4-64 and filipin. In addition, they do not show characteristics of lipid rafts, MCCs or other membrane microdomains. Deconvoluted microscopic images showed that fluorescent patches correspond to plasma membrane invaginations. Transporters remain fully active during this phenomenon of localized plasmolysis. Plasmolysis was however associated with reduced growth rate and a dramatic blockage in transporter and FM4-64 endocytosis. These phenomena are transient and rapidly reversible upon wash-out of hypertonic media. Based on the observation that block in endocytosis by hypertonic treatment altered dramatically the cellular localization of tropomyosin (GFP-TpmA), although it did not affect the cortical appearance of upstream (SlaB-GFP) or downstream (AbpA-mRFP) endocytic components, we conclude that hypertonicity modifies actin dynamics and thus acts indirectly on endocytosis. This was further supported by the effect of latrunculin B, an actin depolymerization agent, on endocytosis. We show that the phenomena observed in A. nidulans also occur in Saccharomyces cerevisiae, suggesting that they constitute basic homeostatic responses of ascomycetes to hypertonic shock. Finally, our work shows that hypertonic treatments can be used as physiological tools to study the endocytic down-regulation of transporters in A. nidulans, as non-conditional genetic blocks affecting endocytic internalization are lethal or severely debilitating.     

Neuronal NADPH oxidase 2 regulates growth cone guidance downstream of slit2/robo2

NADPH oxidases (Nox) are membrane‐bound multi‐subunit protein complexes producing reactive oxygen species (ROS) that regulate many cellular processes. Emerging evidence suggests that Nox‐derived ROS also control neuronal development and axonal outgrowth. However, whether Nox act downstream of receptors for axonal growth and guidance cues is presently unknown. To answer this question, we cultured retinal ganglion cells (RGCs) derived from zebrafish embryos and exposed these neurons to netrin‐1, slit2, and brain‐derived neurotrophic factor (BDNF). To test the role of Nox in cue‐mediated growth and guidance, we either pharmacologically inhibited Nox or investigated neurons from mutant fish that are deficient in Nox2. We found that slit2‐mediated growth cone collapse, and axonal retraction were eliminated by Nox inhibition. Though we did not see an effect of either BDNF or netrin‐1 on growth rates, growth in the presence of netrin‐1 was reduced by Nox inhibition. Furthermore, attractive and repulsive growth cone turning in response to gradients of BDNF, netrin‐1, and slit2, respectively, were eliminated when Nox was inhibited in vitro. ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms involving Nox2 activation. We also investigated the possible relationship between Nox2 and slit2/Robo2 signaling in vivo. astray/nox2 double heterozygote larvae exhibited decreased area of tectal innervation as compared to individual heterozygotes, suggesting both Nox2 and Robo2 are required for establishment of retinotectal connections. Our results provide evidence that Nox2 acts downstream of slit2/Robo2 by mediating growth and guidance of developing zebrafish RGC neurons.     

Computational studies of gramicidin permeation: An entryway sulfonate enhances cation occupancy at entry sites

The impact on the cation-transport free-energy profile of replacing the C-terminal ethanolamine in the gramicidin A channel with a taurine residue is studied using molecular dynamics simulations of gramicidin A (1JNO) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl saline solution. The potential of mean force for ion transport is obtained by umbrella sampling. The presence of a negatively charged sulfonate group at the entrance of the gramicidin channel affects the depth and the location of the binding sites, producing a strong attraction for the cations in the bulk. The potential of mean force by the sulfonate acting directly through electrostatics and van der Waals interactions on the test ion is highly modulated by indirect effects (i.e., sulfonate effects on other components of the system that, in turn, affect the ion free-energy profile in the channel). Because the “entry” sites are located symmetrically at both entry and exit of the channel, the deeper free-energy wells should inhibit exit. Given that the channel has increased conductance experimentally, the simulation results suggest that the channel conductance is normally entry limited.     

Semaphorin-3F attracts the growth cone of cerebellar granule cells through cGMP signaling pathway

Growth cone extension is guided by extracellular factors during the brain development but the underlying cellular mechanisms remain largely unclear. Here, we examined the potential function of class-3 semaphorins in cultured cerebellar granule cells. We found neuropilin-2 (NP2), the high-affinity receptor for semaphorin-3F (Sema3F), is highly expressed in cerebellar granule cells. An extracellular gradient of Sema3F triggered an NP2-dependent attractive turning of the growth cone of cultured cerebellar granule cells. This Sema3F-triggered growth cone attraction was abolished by inhibition of the cGMP signaling pathway and reduced by elevating the intracellular cGMP level. Furthermore, Sema3F partially rescued the collapse induced by inhibition of basal cGMP in granule cells. Thus, Sema3F may act as a chemoattractant for the growth cone of cerebellar granule cells through cGMP signaling pathway.     

A subset of signal transduction pathways is required for hippocampal growth cone collapse induced by ephrin-A5

The Eph family tyrosine kinase receptors and their ligands, ephrins, play key roles in a wide variety of physiological and pathological processes including tissue patterning, angiogenesis, bone development, carcinogenesis, axon guidance, and neural plasticity. However, the signaling mechanisms underlying these diverse functions of Eph receptors have not been well understood. In this study, effects of Eph receptor activation on several important signal transduction pathways are examined. In addition, the roles of these pathways in ephrin-A5-induced growth cone collapse were assessed with a combination of biochemical analyses, pharmacological inhibition, and overexpression of dominant-negative and constitutively active mutants. These analyses showed that ephrin-A5 inhibits Erk activity but activates c-Jun N-terminal kinase. However, regulation of these two pathways is not required for ephrin-A5-induced growth cone collapse in hippocampal neurons. Artificial Erk activation by expression of constitutively active Mek1 and B-Raf failed to block ephrin-A5 effects on growth cones, and inhibitors of the Erk pathway also failed to inhibit collapse by ephrin-A5. Inhibition of JNK had no effects on ephrin-A5-induced growth cone collapse either. In addition, inhibitors to PKA and PI3-K showed no effects on ephrin-A5-induced growth cone collapse. However, pharmacological blockade of phosphotyrosine phosphatase activity, the Src family kinases, cGMP-dependent protein kinase, and myosin light chain kinase significantly inhibited ephrin-A5-induced growth cone collapse. These observations indicate that only a subset of signal transduction pathways is required for ephrin-A5-induced growth cone collapse.