Anti-semaphorin 3A antibodies rescue retinal ganglion cells from cell death following optic nerve axotomy

Damage to the optic nerve in mammals induces retrograde degeneration and apoptosis of the retinal ganglion cell (RGC) bodies. The mechanisms that mediate the response of the neuronal cells to the axonal injury are still unknown. We have previously shown that semaphorins, axon guidance molecules with repulsive cues, are capable of mediating apoptosis in cultured neuronal cells (Shirvan, A., Ziv, I., Fleminger, G., Shina, R., He, Z., Brudo, I., Melamed, E., and Brazilai, A. (1999) J. Neurochem. 73, 961-971). In this study, we examined the involvement of semaphorins in an in vivo experimental animal model of complete axotomy of the rat optic nerve. We demonstrate that a marked induction of type III semaphorin proteins takes place in ipsilateral retinas at early stages following axotomy, well before any morphological signs of RGC apoptosis can be detected. Time course analysis revealed that a peak of expression occurred after 2-3 days and then declined. A small conserved peptide derived from semaphorin 3A that was previously shown to induce neuronal death in culture was capable of inducing RGC loss upon its intravitreous injection into the rat eye. Moreover, we demonstrate a marked inhibition of RGC loss when axotomized eyes were co-treated by intravitreous injection of function-blocking antibodies against the semaphorin 3A-derived peptide. Marked neuronal protection from degeneration was also observed when the antibodies were applied 24 h post-injury. We therefore suggest that semaphorins are key proteins that modulate the cell fate of axotomized RGC. Neutralization of the semaphorin repulsive function may serve as a promising new approach for treatment of traumatic injury in the adult mammalian central nervous system or of ophthalmologic diseases such as glaucoma and ischemic optic neuropathy that induce apoptotic RGC death.     

Collapsin-1/semaphorin D is a repellent for chick ganglion of Remak axons.

Chick collapsin-1/human semaphorin III/mouse semaphorin D is believed to guide the extension of specific axons by a repellent mechanism. Here we examine its role in the guidance of axons of the ganglion of Remak (Remak) in the developing chick intestine. Early in embryogenesis Remak axons extend parallel to, but do not enter, the intestine when collapsin-1 is expressed in the adjacent rectal wall. Remak axons later penetrate the peripheral portions of the rectal wall when collapsin-1 expression retreats from the outer muscle layer to the more internal submucosal and mucosal layers of the rectum. Extension of Remak neurites is repelled in vitro by rectum explants and also by 293T cells expressing collapsin-1. The rectal chemorepellent activity is blocked by anti-collapsin-1 antibodies. Our results suggest that collapsin-1 may help prevent Remak axons from projecting into the intestinal wall at early developmental times and later restricts Remak axon trajectories to the outer part of the intestinal muscle layer.     

Collapsin response mediator proteins of neonatal rat brain interact with chondroitin sulfate

Chondroitin sulfate proteoglycans are structurally and functionally important components of the extracellular matrix of the central nervous system. Their expression in the developing mammalian brain is precisely regulated, and cell culture experiments implicate these proteoglycans in the control of cell adhesion, neuron migration, neurite formation, neuronal polarization, and neuron survival. Here, we report that a monoclonal antibody against chondroitin sulfate-binding proteins from neonatal rat brain recognizes collapsin response mediator protein-4 (CRMP-4), which belongs to a family of proteins involved in collapsin/semaphorin 3A signaling. Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate affinity columns, and CRMP-specific antisera co-precipitated chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were found to be localized immuno-histochemically in overlapping distributions in the marginal zone and the subplate of the cerebral cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is strongly expressed in the upper cortical plate of neonatal rat where cell death is abundant. Therefore, naturally occurring cell death is a plausible mechanism that targets CRMPs to the extracellular matrix at certain stages of development. In summary, our data indicate that CRMPs, in addition to their role as cytosolic signal transduction molecules, may subserve as yet unknown functions in the developing brain as ligands of the extracellular matrix.     

Evidence for collapsin-1 functioning in the control of neural crest migration in both trunk and hindbrain regions

Collapsin-1 belongs to the Semaphorin family of molecules, several members of which have been implicated in the co-ordination of axon growth and guidance. Collapsin-1 can function as a selective chemorepellent for sensory neurons, however, its early expression within the somites and the cranial neural tube (Shepherd, I., Luo, Y. , Raper, J. A. and Chang, S. (1996) Dev. Biol. 173, 185-199) suggest that it might contribute to the control of additional developmental processes in the chick. We now report a detailed study on the expression of collapsin-1 as well as on the distribution of collapsin-1-binding sites in regions where neural crest cell migration occurs. collapsin-1 expression is detected in regions bordering neural crest migration pathways in both the trunk and hindbrain regions and a receptor for collapsin-1, neuropilin-1, is expressed by migrating crest cells derived from both regions. When added to crest cells in vitro, a collapsin-1-Fc chimeric protein induces morphological changes similar to those seen in neuronal growth cones. In order to test the function of collapsin-1 on the migration of neural crest cells, an in vitro assay was used in which collapsin-1-Fc was immobilised in alternating stripes consisting of collapsin-Fc/fibronectin versus fibronectin alone. Explanted neural crest cells derived from both trunk and hindbrain regions avoided the collapsin-Fc-containing substratum. These results suggest that collapsin-1 signalling can contribute to the patterning of neural crest cell migration in the developing chick.     

A novel action of collapsin: Collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse

Chick collapsin-1, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, we now demonstrate that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems. There are at least two distinct pathways through which collapsin exerts its actions in developing neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 316–328, 1997     

Role of CRMP-2 in neuronal polarity

Of the several types of polarized cells, the neuron is one of the most dramatic examples. It extends two distinctive processes, axon and dendrite. Polarization in neurons enables the two processes to play their functionally different roles, sending and receiving electrical signals in a vectorial fashion. While a catalog of structural, molecular, and functional differences between axon and dendrite is accumulating, the mechanisms involved in establishment of neuronal polarity are not well understood. Neuronal polarity formation begins with the elongation of one process as an axon in a symmetric cell phase. In this review, we describe recent advances in the understanding of several cellular events in the early development of axon and dendrite. We also discuss the involvement of the Rho family small GTPases, their upstream and downstream molecules, and collapsin response mediator protein-2 (CRMP-2) in the regulation of neuronal polarity.     

Semaphorin-1a acts in concert with the cell adhesion molecules fasciclin II and connectin to regulate axon fasciculation in Drosophila

Semaphorins comprise a large family of phylogenetically conserved secreted and transmembrane glycoproteins, many of which have been implicated in repulsive axon guidance events. The transmembrane semaphorin Sema-1a in Drosophila is expressed on motor axons and is required for the generation of neuromuscular connectivity. Sema-1a can function as an axonal repellent and mediates motor axon defasciculation. Here, by manipulating the levels of Sema-1a and the cell adhesion molecules fasciclin II (Fas II) and connectin (Conn) on motor axons, we provide further evidence that Sema-1a mediates axonal defasciculation events by acting as an axonally localized repellent and that correct motor axon guidance results from a balance between attractive and repulsive guidance cues expressed on motor neurons.     

Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165.

Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65-75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.     

Evidence that collapsin response mediator protein-2 is involved in the dynamics of microtubules

Collapsin response mediator protein-2 (CRMP-2) is a member of the CRMP/TOAD/Ulip/DRP family of cytosolic phosphoproteins involved in neuronal differentiation and axonal guidance. CRMP-2 mediates the intracellular response to collapsin 1/semaphorin 3A, a repulsive extracellular guidance cue for axonal outgrowth. The mutation of UNC-33, a Caenorhabditis elegans homolog of CRMP-2, results in abnormality of microtubules in neurites, but the mechanism of CRMP-2 action remains to be clarified. Here, we report that overexpression of human CRMP-2 in Neuro2a cells, a mouse neuroblastoma cell line, results in blebbing of the cytoplasm. Furthermore, some cells exhibited intranuclear inclusions, which were labeled with antibodies to CRMP-2 and tubulin. CRMP-2 was found to be associated with microtubule bundles in the spindles at the metaphase and in the midbodies at the late telophase in mitotic cells. Thus, it is most likely that failure of complete disassembly of the spindle microtubules during mitosis is responsible for the formation of these intranuclear inclusions. We suggest that CRMP-2 functions by regulating the dynamics of microtubules.     

Molecular basis of semaphorin-mediated axon guidance

The semaphorin family of proteins constitute one of the major cues for axonal guidance. The prototypic member of this family is Sema3A, previously designated semD/III or collapsin-1. Sema3A acts as a diffusible, repulsive guidance cue in vivo for the peripheral projections of embryonic dorsal root ganglion neurons. Sema3A binds with high affinity to neuropilin-1 on growth cone filopodial tips. Although neuropilin-1 is required for Sema3A action, it is incapable of transmitting a Sema3A signal to the growth cone interior. Instead, the Sema3A/neuropilin-1 complex interacts with another transmembrane protein, plexin, on the surface of growth cones. Certain semaphorins, other than Sema3A, can bind directly to plexins. The intracellular domain of plexin is responsible for initiating the signal transduction cascade leading to growth cone collapse, axon repulsion, or growth cone turning. This intracellular cascade involves the monomeric G-protein, Rac1, and a family of neuronal proteins, the CRMPs. Rac1 is likely to be involved in semaphorin-induced rearrangements of the actin cytoskeleton, but how plexin controls Rac1 activity is not known. Vertebrate CRMPs are homologous to the Caenorhabditis elegans unc-33 protein, which is required for proper axon morphology in worms. CRMPs are essential for Sema3A-induced, neuropilin-plexin-mediated growth cone collapse, but the molecular interactions of growth cone CRMPs are not well defined. Mechanistic aspects of plexin-based signaling for semaphorin guidance cues may have implications for other axon guidance events and for the basis of growth cone motility.     

Neuroprotection against traumatic brain injury by a peptide derived from the collapsin response mediator protein 2 (CRMP2)

Neurological disabilities following traumatic brain injury (TBI) may be due to excitotoxic neuronal loss. The excitotoxic loss of neurons following TBI occurs largely due to hyperactivation of N-methyl-d-aspartate receptors (NMDARs), leading to toxic levels of intracellular Ca(2+). The axon guidance and outgrowth protein collapsin response mediator protein 2 (CRMP2) has been linked to NMDAR trafficking and may be involved in neuronal survival following excitotoxicity. Lentivirus-mediated CRMP2 knockdown or treatment with a CRMP2 peptide fused to HIV TAT protein (TAT-CBD3) blocked neuronal death following glutamate exposure probably via blunting toxicity from delayed calcium deregulation. Application of TAT-CBD3 attenuated postsynaptic NMDAR-mediated currents in cortical slices. In exploring modulation of NMDARs by TAT-CBD3, we found that TAT-CBD3 induced NR2B internalization in dendritic spines without altering somal NR2B surface expression. Furthermore, TAT-CBD3 reduced NMDA-mediated Ca(2+) influx and currents in cultured neurons. Systemic administration of TAT-CBD3 following a controlled cortical impact model of TBI decreased hippocampal neuronal death. These findings support TAT-CBD3 as a novel neuroprotective agent that may increase neuronal survival following injury by reducing surface expression of dendritic NR2B receptors.     

Identification of a member of mouse semaphorin family

Grasshopper semaphorin I (Sema I) and its related proteins, chick collapsin and mouse Sema III contribute to the axon guidance by their repellent actions [5,9,12]. We have identified a member of semaphorin gene family from the mouse brain and named it M-Sema F. The N-terminal encodes a semaphorin domain that is similar between Sema I–III [6] followed by a single putative immunoglobulin-like domain, a transmembrane domain, and a proline-rich intracellular domain. M-Sema F mRNA is expressed widely in the nervous tissues during development. These suggest that M-Sema F is a transmembrane member of the semaphorin family of the vertebrate which may function in the developing neuronal network.     

Hydrogen peroxide induces neurite degeneration: Prevention by tocotrienols

Reactive oxygen species (ROS) may attack several types of tissues and chronic exposure to ROS may attenuate various biological functions and increase the risk of several types of serious disorders. It is known that treatments with ROS attack neurons and induce cell death. However, the mechanisms of neuronal change by ROS prior to induction of cell death are not yet understood. Here, it was found that treatment of neurons with low concentrations of hydrogen peroxide induced neurite injury, but not cell death. Unusual bands located above the original collapsin response mediator protein (CRMP)-2 protein were detected by western blotting. Treatment with tocopherol or tocotrienols significantly inhibited these changes in neuro2a cells and cerebellar granule neurons (CGCs). Furthermore, prevention by tocotrienols of hydrogen peroxide-induced neurite degeneration was stronger than that by tocopherol. These findings indicate that neurite beading is one of the early events of neuronal degeneration prior to induction of death of hydrogen peroxide-treated neurons. Treatment with tocotrienols may protect neurite function through its neuroprotective function.     

Collapsin response mediator protein-2 inhibits neuronal phospholipase D(2) activity by direct interaction.

Although the functional significance of neuronal phospholipase D (PLD) is being recognized, little is known about its regulatory role in neuronal cells. To elucidate the regulatory mechanism of neuronal PLD, we investigated PLD(2)-binding neuronal protein from rat brain cytosol. During the fractionation of rat brain cytosol by four-column chromatography, a 62-kDa PLD(2)-interacting protein was detected by PLD(2) overlay assay and identified as collapsin response mediator protein-2 (CRMP-2), which controls neuronal axon guidance and outgrowth. Using bacterially expressed glutathione S-transferase fusion proteins, we found that two regions (amino acids 65-192 (the phagocytic oxidase domain) and 724-825) of PLD(2) and a single region (amino acids 243-300) of CRMP-2 are required for the direct binding of both proteins. A co-immunoprecipitation study in COS-7 cells also showed an in vivo interaction between CRMP-2 and PLD(2). Interestingly, CRMP-2 was found to potently inhibit PLD(2) activity in a concentration-dependent manner (IC(50) = 30 nm). Overexpression studies also showed that CRMP-2 is an in vivo inhibitor of PLD(2) in PC12 cells. Moreover, increasing the concentration of semaphorin 3A, one of the repulsive axon guidance cues, showed that PLD(2) activity can be inhibited in PC12 cells. Immunocytochemistry further revealed that PLD(2) is co-localized with CRMP-2 in the distal tips of neurites, its possible action site, in differentiated PC12 cells. Taken together, our results indicate that CRMP-2 may interact directly with and inhibit neuronal PLD(2), suggesting that this inhibitory mode of regulation may play a role in neuronal pathfinding during the developmental stage.     

Drosophila Plexin B is a Sema-2a receptor required for axon guidance

Plexin receptors play a crucial role in the transduction of axonal guidance events elicited by semaphorin proteins. In Drosophila, Plexin A (PlexA) is a receptor for the transmembrane semaphorin semaphorin-1a (Sema-1a) and is required for motor and central nervous system (CNS) axon guidance in the developing embryonic nervous system. However, it remains unknown how PlexB functions during neural development and which ligands serve to activate this receptor. Here, we show that plexB, like plexA, is robustly expressed in the developing CNS and is required for motor and CNS axon pathfinding. PlexB and PlexA serve both distinct and shared neuronal guidance functions. We observe a physical association between these two plexin receptors in vivo and find that they can utilize common downstream signaling mechanisms. PlexB does not directly bind to the cytosolic semaphorin signaling component MICAL (molecule that interacts with CasL), but requires MICAL for certain axonal guidance functions. Ligand binding and genetic analyses demonstrate that PlexB is a receptor for the secreted semaphorin Sema-2a, suggesting that secreted and transmembrane semaphorins in Drosophila use PlexB and PlexA, respectively, for axon pathfinding during neural development. These results establish roles for PlexB in central and peripheral axon pathfinding, define a functional ligand for PlexB, and implicate common signaling events in plexin-mediated axonal guidance.     

Discrete roles for secreted and transmembrane semaphorins in neuronal growth cone guidance in vivo

From the initial stages of axon outgrowth to the formation of a functioning synapse, neuronal growth cones continuously integrate and respond to multiple guidance cues. To investigate the role of semaphorins in the establishment of appropriate axon trajectories, we have characterized a novel secreted semaphorin in grasshopper, gSema 2a. Sema 2a is expressed in a gradient in the developing limb bud epithelium during Ti pioneer axon outgrowth. We demonstrate that Sema 2a acts as chemorepulsive guidance molecule critical for axon fasciculation and for determining both the initial direction and subsequent pathfinding events of the Ti axon projection. Interestingly, simultaneous perturbation of both secreted Sema 2a and transmembrane Sema I results in a broader range and increased incidence of abnormal Ti pioneer axon phenotypes, indicating that different semaphorin family members can provide functionally distinct guidance information to the same growth cone in vivo.     

CRMP-2 regulates polarized Numb-mediated endocytosis for axon growth

Axon growth during neural development is highly dependent on both cytoskeletal re-organization and polarized membrane trafficking. Previously, we demonstrated that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate and axon growth in cultured hippocampal neurons, possibly by interacting with tubulin heterodimers and promoting microtubule assembly. Here, we identify Numb as a CRMP-2-interacting protein. Numb has been shown to interact with alpha-adaptin and to be involved in endocytosis. We found that Numb was associated with L1, a neuronal cell adhesion molecule that is endocytosed and recycled at the growth cone, where CRMP-2 and Numb were colocalized. Furthermore, expression of dominant-negative CRMP-2 mutants or knockdown of CRMP-2 message with small-interfering (si) RNA inhibited endocytosis of L1 at axonal growth cones and suppressed axon growth. These results suggest that in addition to regulating microtubule assembly, CRMP-2 is involved in polarized Numb-mediated endocytosis of proteins such as L1.     

Semaphorin 3E/collapsin-5 inhibits growing retinal axons

During development, the formation of neural networks is reflected by the oriented extension of neurites. Using retinal ganglion cells (RGCs) as a model, we identified the yet uncharacterized chick semaphorin Sema3E/collapsin-5 as a repulsive cue for outgrowing axons. Sema3E/collapsin-5 was highly regulated during retinal histogenesis, with peak expression during the period of intraretinal axon growth. Polymerase chain reaction analysis demonstrated Sema3E/collapsin-5 mRNA in retina layers, from which RGC axons are excluded. Neither isolated RGCs nor purified retinal Müller glia cells synthesized Sema3E/collapsin-5. Sema3E/collapsin-5 receptor sites were visualized by alkaline phosphatase fusion proteins in the axon-rich optic fiber layer. Time-lapse video recording of chick in vitro cultures revealed a growth cone collapsing activity of recombinant Sema3E/collapsin-5. This effect was specific for RGCs, since dorsal root ganglia (DRG) neurons of the peripheral nervous system were not affected. Comparison with Sema3A/collapsin-1 displayed a reciprocal specificity, because Sema3A/collapsin-1 hampered exclusively DRG but not RGC growth cones. The collapsing effect was mediated by low cGMP levels, but not cAMP, as revealed by a set of agonists. In summary, the data suggest a possible role of chick Sema3E/collapsin-5 in restricting growth of retinal ganglion cell axons to the optic fiber layer.     

Tocotrienols prevent hydrogen peroxide-induced axon and dendrite degeneration in cerebellar granule cells

It is well known that reactive oxygen species (ROS) attack several living tissues and increase the risk of development and progression of serious diseases. In neuronal level, ROS induce cell death in concentration-dependent fashion. However, little is known about the mechanisms of neuronal changes by ROS prior to induction of cell death. Here we found that treatment of cerebellar granule neurons (CGCs) with 0.5 μM hydrogen peroxide induced axonal injury, but not cell death. The number of dendrites remarkably decreased in hydrogen peroxide-treated CGCs, and extensive beading was observed on survival dendrites. In addition, an abnormal band of the original collapsin response mediator protein (CRMP)-2 was detected by Western blotting in hydrogen peroxide-treated CGCs. Treatment with each tocotrienol isoform prevented axonal and dendrite degeneration and induction of the abnormal band of the original band of CRMP-2 in hydrogen peroxide-treated CGCs. These results indicate that treatment with tocotrienols may therefore be neuroprotective in the presence of hydrogen peroxide by preventing changes to the CRMP-2 that occur before neuron death.