Neuropilin Is a Receptor for the Axonal Chemorepellent Semaphorin III

AbstractExtending axons in the developing nervous system are guided to their targets through the coordinate actions of attractive and repulsive guidance cues. The semaphorin family of guidance cues comprises several members that can function as diffusible axonal chemorepellents. To begin to elucidate the mechanisms that mediate the repulsive actions of Collapsin-1/Semaphorin III/D (Sema III), we searched for Sema III–binding proteins in embryonic rat sensory neurons by expression cloning. We report that Sema III binds with high affinity to the transmembrane protein neuropilin, and that antibodies to neuropilin block the ability of Sema III to repel sensory axons and to induce collapse of their growth cones. These results provide evidence that neuropilin is a receptor or a component of a receptor complex that mediates the effects of Sema III on these axons

beta1-integrin mediates myelin-associated glycoprotein signaling in neuronal growth cones

Several myelin-associated factors that inhibit axon growth of mature neurons, including Nogo66, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), can associate with a common GPI-linked protein Nogo-66 receptor (NgR). Accumulating evidence suggests that myelin inhibitors also signal through unknown NgR-independent mechanisms. Here we show that MAG, a RGD tri-peptide containing protein, forms a complex with β1-integrin to mediate axonal growth cone turning responses of several neuronal types. Mutations that alter the RGD motif in MAG or inhibition of β1-integrin function, but not removal of NgRs, abolish these MAG-dependent events. In contrast, OMgp-induced repulsion is not affected by inhibition of b1-integrin function. We further show that MAG stimulates tyrosine phosphorylation of focal adhesion kinase (FAK), which in turn is required for MAG-induced growth cone turning. These studies identify β1-integrin as a specific mediator for MAG in growth cone turning responses, acting through FAK activation

Plexin A Is a Neuronal Semaphorin Receptor that Controls Axon Guidance

AbstractThe Semaphorins comprise a large family of secreted and transmembrane proteins, some of which function as repellents during axon guidance. Semaphorins fall into seven subclasses. Neuropilins are neuronal receptors for class III Semaphorins. In the immune system, VESPR, a member of the Plexin family, is a receptor for a viral-encoded Semaphorin. Here, we identify two Drosophila Plexins, both of which are expressed in the developing nervous system. We present evidence that Plexin A is a neuronal receptor for class I Semaphorins (Sema 1a and Sema 1b) and show that Plexin A controls motor and CNS axon guidance. Plexins, which themselves contain complete Semaphorin domains, may be both the ancestors of classical Semaphorins and binding partners for Semaphorins

Slit1 and Slit2 Cooperate to Prevent Premature Midline Crossing of Retinal Axons in the Mouse Visual System

AbstractDuring development, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. In Drosophila, the Slit protein regulates midline axon crossing through repulsion. To determine the role of Slit proteins in RGC axon guidance, we disrupted Slit1 and Slit2, two of three known mouse Slit genes. Mice defective in either gene alone exhibited few RGC axon guidance defects, but in double mutant mice a large additional chiasm developed anterior to the true chiasm, many retinal axons projected into the contralateral optic nerve, and some extended ectopically—dorsal and lateral to the chiasm. Our results indicate that Slit proteins repel retinal axons in vivo and cooperate to establish a corridor through which the axons are channeled, thereby helping define the site in the ventral diencephalon where the optic chiasm forms

NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord.

RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development

14-3-3 Proteins Regulate a Cell-Intrinsic Switch from Sonic Hedgehog-Mediated Commissural Axon Attraction to Repulsion after Midline Crossing

SummaryAxons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses

Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons

Prevalent presence of periodic actin-spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens