Osteoprotection by semaphorin 3A

The bony skeleton is maintained by local factors that regulate bone-forming osteoblasts and bone-resorbing osteoclasts, in addition to hormonal activity. Osteoprotegerin protects bone by inhibiting osteoclastic bone resorption, but no factor has yet been identified as a local determinant of bone mass that regulates both osteoclasts and osteoblasts. Here we show that semaphorin 3A (Sema3A) exerts an osteoprotective effect by both suppressing osteoclastic bone resorption and increasing osteoblastic bone formation. The binding of Sema3A to neuropilin-1 (Nrp1) inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by inhibiting the immunoreceptor tyrosine-based activation motif (ITAM) and RhoA signalling pathways. In addition, Sema3A and Nrp1 binding stimulated osteoblast and inhibited adipocyte differentiation through the canonical Wnt/β-catenin signalling pathway. The osteopenic phenotype in Sema3a?/? mice was recapitulated by mice in which the Sema3A-binding site of Nrp1 had been genetically disrupted. Intravenous Sema3A administration in mice increased bone volume and expedited bone regeneration. Thus, Sema3A is a promising new therapeutic agent in bone and joint diseases.     

Semaphorin 3A is a chemoattractant for cortical apical dendrites

The apical dendrites of pyramidal neurons integrate inputs from various cortical layers and are central to information processing. Here we show that the growth of apical dendrites towards the pial surface is regulated by a diffusible chemoattractant present at high levels near the marginal zone. A major component of this signal is semaphorin 3A (Sema3A), which was previously characterized as a chemorepellant for cortical axons. Soluble guanylate cyclase is asymmetrically localized to the developing apical dendrite, and is required for the chemoattractive effect of Sema3A. Thus the asymmetric localization of soluble guanylate cyclase confers distinct Sema3A responses to axons and dendrites. These observations reveal a mechanism by which a single chemotropic signal can pattern both axons and dendrites during development.     

Disoriented pathfinding by pioneer neurone growth cones deprived of filopodia by cytochalasin treatment

A major question in developmental neurobiology is how developing nerve cells accurately extend processes to establish connections with their target cells. This problem involves both the nature of cues for growth cone guidance and also the question of how growth cones survey their environment for cues and respond by altering their direction of migration. The filopodia which normally extend from neuronal growth cones have been shown to affect growth cone steering in vitro and it has been proposed that they function in vivo in the detection of and response to guidance cues. This hypothesis could be tested in vivo if growth cones which normally have filopodia could be induced to migrate in their absence. The pair of Ti1 neurones are the first neurones to extend axons through the limb buds of embryonic grasshoppers. We report here an examination of the migration of Ti1 pioneer growth cones deprived of filopodia by culture in agents which disrupt actin microfilaments. Under these conditions, axons continue to extend but a large percentage of growth cones are highly disoriented. Our results indicate that Ti1 filopodia are not necessary for axonal elongation in vivo but that they are important for correctly oriented growth cone steering.     

Adaptation in the chemotactic guidance of nerve growth cones

Pathfinding by growing axons in the developing nervous system may be guided by gradients of extracellular guidance factors. Analogous to the process of chemotaxis in microorganisms, we found that axonal growth cones of cultured Xenopus spinal neurons exhibit adaptation during chemotactic migration, undergoing consecutive phases of desensitization and resensitization in the presence of increasing basal concentrations of the guidance factor netrin-1 or brain-derived neurotrophic factor. The desensitization is specific to the guidance factor and is accompanied by a reduction of Ca2+ signalling, whereas resensitization requires activation of mitogen-associated protein kinase and local protein synthesis. Such adaptive behaviour allows the growth cone to re-adjust its sensitivity over a wide range of concentrations of the guidance factor, an essential feature for long-range chemotaxis.     

Assembly of microtubules at the tip of growing axons

The growth of axons in the developing nervous system depends on the elongation of the microtubules that form their principal longitudinal structural element. It is not known whether individual microtubules in the axon elongate at their proximal ends, close to the cell body, and then move forward into the lengthening axon, or whether tubulin subunits are transported to the tip of the axon and assembled there onto the free ends of microtubules. The former possibility is supported by studies of slow axonal transport in mature nerves from which it has been deduced that microtubule assembly occurs principally at the neuronal cell body. By contrast, the polarity of microtubules in axons, which have their 'plus' or 'fast-growing' ends distal to the cell body, suggests that assembly occurs at the growing tip, or growth cone, of the axon. We have addressed this question by topically applying Colcemid (N-desacetyl-N-methylcolchicine), and other drugs which alter microtubule stability, to different regions of isolated nerve cells growing in tissue culture. We find that the sensitivity to these drugs is greatest at the growth cone by at least two orders of magnitude, suggesting that this is a major site of microtubule assembly during axonal growth.     

Direct evidence that growth cones pull

There is controversy over whether axonal elongation is the result of a pulling growth cone and the role of tension in axonal elongation. Earlier in this decade, the consensus was that axons or neurites elongated from tension generated by forward motility of the growth cone. It was presumed that contractile filopodia were the source of the tension moving the growth cone. But this view was challenged by experiments showing that neurites elongate, albeit abnormally, in the presence of cytochalasin, which inhibits growth-cone and filopodial movements. Additionally, high resolution, video-enhanced observations of growth-cone activity argued against filopodial shortening as a source of tension, suggesting instead that an extrusion of cytoplasm rather than a pulling process, is the key event in neurite elongation. Studies of slow axonal transport, however, indicate that much slower cytoskeletal pushing underlies axonal elongation. We report here direct measurements of neurite force as a function of growth-cone advance which show that they are linearly related and accompanied by apparent neurite growth. No increase in force occurs in neurites whose growth cone fails to advance.     

Semaphorin 7A initiates T-cell-mediated inflammatory responses through alpha1beta1 integrin

Semaphorins are axon guidance factors that assist growing axons in finding appropriate targets and forming synapses. Emerging evidence suggests that semaphorins are involved not only in embryonic development but also in immune responses. Semaphorin 7A (Sema7A; also known as CD108), which is a glycosylphosphatidylinositol-anchored semaphorin, promotes axon outgrowth through beta1-integrin receptors and contributes to the formation of the lateral olfactory tract. Although Sema7A has been shown to stimulate human monocytes, its function as a negative regulator of T-cell responses has also been reported. Thus, the precise function of Sema7A in the immune system remains unclear. Here we show that Sema7A, which is expressed on activated T cells, stimulates cytokine production in monocytes and macrophages through alpha1beta1 integrin (also known as very late antigen-1) as a component of the immunological synapse, and is critical for the effector phase of the inflammatory immune response. Sema7A-deficient (Sema7a-/-) mice are defective in cell-mediated immune responses such as contact hypersensitivity and experimental autoimmune encephalomyelitis. Although antigen-specific and cytokine-producing effector T cells can develop and migrate into antigen-challenged sites in Sema7a-/- mice, Sema7a-/- T cells fail to induce contact hypersensitivity even when directly injected into the antigen-challenged sites. Thus, the interaction between Sema7A and alpha1beta1 integrin is crucial at the site of inflammation. These findings not only identify a function of Sema7A as an effector molecule in T-cell-mediated inflammation, but also reveal a mechanism of integrin-mediated immune regulation.     

Turning of nerve growth cones induced by localized increases in intracellular calcium ions

Guidance of developing axons involves turning of the motile tip, the growth cone, in response to a variety of extracellular cues. Little is known about the intracellular mechanism by which the directional signal is transduced. Ca2+ is a key second messenger in growth cone extension and has been implicated in growth-cone turning. Here I report that a direct, spatially restricted elevation of intracellular Ca2+ concentration ([Ca2+]i) on one side of the growth cone by focal laser-induced photolysis (FLIP) of caged Ca2+ consistently induced turning of the growth cone to the side with elevated [Ca2+]i (attraction). Furthermore, when the resting [Ca2+]i at the growth cone was decreased by the removal of extracellular Ca2+, the same focal elevation of [Ca2+]i by FLIP induced repulsion. These results provide direct evidence that a localized Ca2+ signal in the growth cone can provide the intracellular directional cue for extension and is sufficient to initiate both attraction and repulsion. By integrating local and global Ca2+ signals, a growth cone could thus generate different turning responses under different environmental conditions during guidance.     

A common mechanism of action for three mood-stabilizing drugs

Lithium, carbamazepine and valproic acid are effective mood-stabilizing treatments for bipolar affective disorder. The molecular mechanisms underlying the actions of these drugs and the illness itself are unknown. Berridge and colleagues suggested that inositol depletion may be the way that lithium works in bipolar affective disorder, but others have suggested that glycogen synthase kinase (GSK3) may be the relevant target. The action of valproic acid has been linked to both inositol depletion and to inhibition of histone deacetylase (HDAC). We show here that all three drugs inhibit the collapse of sensory neuron growth cones and increase growth cone area. These effects do not depend on GSK3 or HDAC inhibition. Inositol, however, reverses the effects of the drugs on growth cones, thus implicating inositol depletion in their action. Moreover, the development of Dictyostelium is sensitive to lithium and to valproic acid, but resistance to both is conferred by deletion of the gene that codes for prolyl oligopeptidase, which also regulates inositol metabolism. Inhibitors of prolyl oligopeptidase reverse the effects of all three drugs on sensory neuron growth cone area and collapse. These results suggest a molecular basis for both bipolar affective disorder and its treatment.     

Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones

Outgrowth of distinct axonal and dendritic processes is essential for the development of the functional polarity of nerve cells. In cultures of neurons from the hippocampus, where the differential outgrowth of axons and dendrites is readily discernible, we have sought molecules that might underlie the distinct modes of elongation of these two types of processes. One particularly interesting protein is GAP-43 (also termed B-50, F1 or P-57), a neuron-specific, membrane-associated phosphoprotein whose expression is dramatically elevated during neuronal development and regeneration. GAP-43 is among the most abundant proteins in neuronal growth cones, the motile structures that form the tips of advancing neurites, but its function in neuronal growth remains unknown. Using immunofluorescence staining, we show that GAP-43 is present in axons and concentrated in axonal growth cones of hippocampal neurons in culture. Surprisingly, we could not detect GAP-43 in growing dendrites and dendritic growth cones. These results show that GAP-43 is compartmentalized in developing nerve cells and provide the first direct evidence of important molecular differences between axonal and dendritic growth cones. The sorting and selective transport of GAP-43 may give axons and axonal growth cones certain of their distinctive properties, such as the ability to grow rapidly over long distances or the manner in which they recognize and respond to cues in their environment.     

Endothelins are vascular-derived axonal guidance cues for developing sympathetic neurons

During development, sympathetic neurons extend axons along a myriad of distinct trajectories, often consisting of arteries, to innervate one of a large variety of distinct final target tissues. Whether or not subsets of neurons within complex sympathetic ganglia are predetermined to innervate select end-organs is unknown. Here we demonstrate in mouse embryos that the endothelin family member Edn3 (ref. 1), acting through the endothelin receptor EdnrA (refs 2, 3), directs extension of axons of a subset of sympathetic neurons from the superior cervical ganglion to a preferred intermediate target, the external carotid artery, which serves as the gateway to select targets, including the salivary glands. These findings establish a previously unknown mechanism of axonal pathfinding involving vascular-derived endothelins, and have broad implications for endothelins as general mediators of axonal growth and guidance in the developing nervous system. Moreover, they suggest a model in which newborn sympathetic neurons distinguish and choose between distinct vascular trajectories to innervate their appropriate end organs.     

Calcium signalling in the guidance of nerve growth by netrin-1

Pathfinding by growing axons in the developing nervous system is guided by diffusible or bound factors that attract or repel the axonal growth cone. The cytoplasmic signalling mechanisms that trigger the responses of the growth cone to guidance factors are mostly unknown. Previous studies have shown that the level and temporal patterns of cytoplasmic Ca2+ can regulate the rate of growth-cone extension in vitro and in vivo. Here we report that Ca2+ also mediates the turning behaviour of the growth cones of cultured Xenopus neurons that are induced by an extracellular gradient of netrin-1, an established diffusible guidance factor in vivo. The netrin-1-induced turning response depends on Ca2+ influx through plasma membrane Ca2+ channels, as well as Ca2+-induced Ca2+ release from cytoplasmic stores. Reduction of Ca2+ signals by blocking either of these two Ca2+ sources converted the netrin-1-induced response from attraction to repulsion. Activation of Ca2+-induced Ca2+ release from internal stores with a gradient of ryanodine in the absence of netrin-1 was sufficient to trigger either attractive or repulsive responses, depending on the ryanodine concentration used. These results support the model that cytoplasmic Ca2+ signals mediate growth-cone guidance by netrin-1, and different patterns of Ca2+ elevation trigger attractive and repulsive turning responses.     

A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth

Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3sigma. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3sigma from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.     

Novel form of growth cone motility involving site-directed actin filament assembly.

Regulation of cytoskeletal structure and motility by extracellular signals is essential for all directed forms of cell movement and underlies the developmental process of axonal guidance in neuronal growth cones. Interaction with polycationic microbeads can trigger morphogenic changes in neurons and muscle cells normally associated with formation of pre- and postsynaptic specializations. Furthermore, when various types of microscopic particles are applied to the lamellar surface of a neuronal growth cone or motile cell they often exhibit retrograde movement at rates of 1-6 microns min-1 (refs 3-6). There is strong evidence that this form of particle movement results from translocation of membrane proteins associated with cortical F-actin networks, not from bulk retrograde lipid flow and may be a mechanism behind processes such as cell locomotion, growth cone migration and capping of cell-surface antigens. Here we report a new form of motility stimulated by polycationic bead interactions with the growth-cone membrane surface. Bead binding rapidly induces intracellular actin filament assembly, coincident with a production of force sufficient to drive bead movements. These extracellular bead movements resemble intracellular movements of bacterial parasites known to redirect host cell F-actin assembly for propulsion. Our results suggest that site-directed actin filament assembly may be a widespread cellular mechanism for generating force at membrane-cytoskeletal interfaces.     

Regulation of axon growth in vivo by activity-based competition

The formation of functional neural networks requires precise regulation of the growth and branching of the terminal arbors of axons, processes known to be influenced by early network electrical activity. Here we show that a rule of activity-based competition between neighbouring axons appears to govern the growth and branching of retinal ganglion cell (RGC) axon arbors in the developing optic tectum of zebrafish. Mosaic expression of an exogenous potassium channel or a dominant-negative SNARE protein was used to suppress electrical or neurosecretory activity in subsets of RGC axons. Imaging in vivo showed that these forms of activity suppression strongly inhibit both net growth and the formation of new branches by individually transfected RGC axon arbors. The inhibition is relieved when the activity of nearby 'competing' RGC axons is also suppressed. These results therefore identify a new form of activity-based competition rule that might be a key regulator of axon growth and branch initiation.     

Spatiotemporal dynamics of RhoA activity in migrating cells

Rho family GTPases regulate the actin and adhesion dynamics that control cell migration. Current models postulate that Rac promotes membrane protrusion at the leading edge and that RhoA regulates contractility in the cell body. However, there is evidence that RhoA also regulates membrane protrusion. Here we use a fluorescent biosensor, based on a novel design preserving reversible membrane interactions, to visualize the spatiotemporal dynamics of RhoA activity during cell migration. In randomly migrating cells, RhoA activity is concentrated in a sharp band directly at the edge of protrusions. It is observed sporadically in retracting tails, and is low in the cell body. RhoA activity is also associated with peripheral ruffles and pinocytic vesicles, but not with dorsal ruffles induced by platelet-derived growth factor (PDGF). In contrast to randomly migrating cells, PDGF-induced membrane protrusions have low RhoA activity, potentially because PDGF strongly activates Rac, which has previously been shown to antagonize RhoA activity. Our data therefore show that different extracellular cues induce distinct patterns of RhoA signalling during membrane protrusion.     

Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein

Adult mammalian axon regeneration is generally successful in the peripheral nervous system (PNS) but is dismally poor in the central nervous system (CNS). However, many classes of CNS axons can extend for long distances in peripheral nerve grafts. A comparison of myelin from the CNS and the PNS has revealed that CNS white matter is selectively inhibitory for axonal outgrowth. Several components of CNS white matter, NI35, NI250(Nogo) and MAG, that have inhibitory activity for axon extension have been described. The IN-1 antibody, which recognizes NI35 and NI250(Nogo), allows moderate degrees of axonal regeneration and functional recovery after spinal cord injury. Here we identify Nogo as a member of the Reticulon family, Reticulon 4-A. Nogo is expressed by oligodendrocytes but not by Schwann cells, and associates primarily with the endoplasmic reticulum. A 66-residue lumenal/extracellular domain inhibits axonal extension and collapses dorsal root ganglion growth cones. In contrast to Nogo, Reticulon 1 and 3 are not expressed by oligodendrocytes, and the 66-residue lumenal/extracellular domains from Reticulon 1, 2 and 3 do not inhibit axonal regeneration. These data provide a molecular basis to assess the contribution of Nogo to the failure of axonal regeneration in the adult CNS.     

Class IV semaphorin Sema4A enhances T-cell activation and interacts with Tim-2

Semaphorins are a family of phylogenetically conserved soluble and transmembrane proteins. Although many soluble semaphorins deliver guidance cues to migrating axons during neuronal development, some members are involved in immune responses. For example, CD100 (also known as Sema4D), a class IV transmembrane semaphorin, signals through CD72 to effect nonredundant roles in immune responses in a ligand-receptor system that is distinct from any seen previously in the nervous system. Here we report that the class IV semaphorin Sema4A, which is expressed in dendritic cells and B cells, enhances the in vitro activation and differentiation of T cells and the in vivo generation of antigen-specific T cells. Treating mice with monoclonal antibodies against Sema4A blocks the development of an experimental autoimmune encephalomyelitis that is induced by an antigenic peptide derived from myelin oligodendrocyte glycoprotein. In addition, expression cloning shows that the Sema4A receptor is Tim-2, a member of the family of T-cell immunoglobulin domain and mucin domain (Tim) proteins that is expressed on activated T cells.     

Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.