Ephrin-As play a rhombomere-specific role in trigeminal motor axon projections in the chick embryo

In this study, we investigate the possible role of ephrin-Eph signaling in trigeminal motor axon projections. We find that EphA receptors are expressed at higher levels by rhombomere 2 (r2) trigeminal motor neurons than by r3 trigeminal motor neurons in the chick embryo. Mapping of rhombomere-specific axon projections shows that r2 and r3 trigeminal motor neurons project to different muscle targets, including the mandibular adductor and the intermandibularis muscles respectively. Ephrin-A5 is expressed in these muscles, especially in some regions of the intermandibularis muscle, and can cause growth cone collapse of both r2 and r3 motor axons in vitro. We demonstrate that in vivo overexpression of ephrin-A5 in the intermandibularis muscle, or overexpression of dominant-negative EphA receptors in trigeminal motor neurons leads to a reduction in branching of r3-derived motor axons specifically. Overexpression of full-length EphA receptors impairs the formation of r3 projections to the intermandibularis muscle. These findings indicate that ephrins and their Eph receptors play a role in trigeminal motor axon topographic mapping and in rhombomere 3-derived projections in particular.     

Constitutively active myosin light chain kinase alters axon guidance decisions in Drosophila embryos

Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Here, evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons. Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues.     

Interactions among the SNARE proteins and complexin analyzed by a yeast four-hybrid assay

Exocytosis is one of the most crucial and ubiquitous processes in all of biology. This event is mediated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, ternary assemblies of syntaxin, SNAP23/SNAP25 (synaptosomal-associated protein of 23 or 25 kDa), and synaptobrevin. The exocytotic process can be further regulated by complexin, which interacts with the SNARE complex. Complexin is involved in a Ca²⁺-triggered exocytotic process. In eukaryotic cells, multiple isoforms of SNARE proteins are expressed and are involved in distinct types of exocytosis. To understand the underlying biochemical mechanism of various exocytotic processes mediated by different SNARE protein isoforms, we systematically analyzed the interactions among syntaxin, SNAP23/SNAP25, synaptobrevin, and complexin by employing a newly developed yeast four-hybrid interaction assay. The efficiency of SNARE complex formation and the specificity of complexin binding are regulated by the different SNARE protein isoforms. Therefore, various types of exocytosis, occurring on different time scales with different efficiencies, can be explained by the involved SNARE complexes composed of different combinations of SNARE protein isoforms.     

Dual roles of the chemorepellent axon guidance molecule RGMa in establishing pioneering axon tracts and neural fate decisions in embryonic vertebrate forebrain

Repulsive guidance molecule A (RGMa) is a glycosylphosphatidylinositol‐anchored plasma membrane protein that was originally identified based on its chemorepulsive activity during axon navigation in the developing nervous system. Knock down of RGMa has previously shown to perturb axon navigation in the developing Xenopus forebrain (Wilson and Key, 2006). In order to further understand the in vivo role of RGMa in axon guidance, we have adopted an in vivo gain‐of‐function approach. RGMa was mosaically overexpressed in the developing Xenopus embryo by the injection of mRNA into single blastomeres. Ectopic expression of RGMa affected the morphology and the topography of developing axon tracts in vivo. Pioneer axons misrouted or aberrantly projected in response to ectopic RGMa in the developing Xenopus forebrain, confirming the in vivo chemorepulsive activity of this ligand. In addition, we show here for the first time that overexpression of RGMa acts cell‐autonomously to generate ectopic neurons in the developing embryonic brain. Taken together, the current study reveals a pleiotropic role of RGMa in early vertebrate embryonic brain in the spatial organization of axon tracts, pioneer axon guidance, and neural cell differentiation. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes

Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane-directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP-2/synaptobrevin-2, syntaxin-2 and -4, nsec-1/munc-18-1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP-2, the small GTP-binding protein Rab3a, and the target SNARE syntaxin-4 were up-regulated. VAMP-2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network; in situ anti-VAMP-2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane-directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.     

Mechanisms of axon guidance: membrane dynamics and axonal transport in semaphorin signalling

The intricate geometry of neuronal networks poses many unique cell-biological problems regarding the way a growing axon responds to its environment. Several groups of ligand-receptor pairs have been identified to regulate such processes. In this study, we take class 3 semaphorins as an example and review what is known about the intracellular movements of semaphorins throughout neuronal cells, transport support structures and location of release sites. We discuss how their receptor trafficking may contribute to regulate membrane dynamics underlying growth cone motility and the physiological contribution made by class 3 semaphorins-induced acceleration of axoplasmic transport on neurite development.     

Cooperative activity of multiple upper layer proteins for thalamocortical axon growth

During development, sensory thalamocortical (TC) axons grow into the neocortex and terminate primarily in layer 4. To study the molecular mechanism that underlies lamina-specific TC axon termination, we investigated the responsiveness of TC axons to ephrin-A5, semaphorin-7A (Sema7A) and kit ligand (KL), which are expressed in the upper layers of the developing cortex. Dissociated cells of the dorsal thalamus from embryonic rat brain were cultured on dishes that were coated with preclustered Fc-tagged extracellular domains of these molecules. Each protein was found to promote TC axon growth in a dose-dependent fashion of a bell-shaped curve. Any combination of the three proteins showed a cooperative effect in lower concentrations but not in higher concentrations, suggesting that their growth-promoting activities act in a common pathway. The effect of spatial distributions of these proteins was further tested on a filter membrane, in which these proteins were printed at a size that recapitulates the scale of laminar thickness in vivo, using a novel protein-printing technique, Simple-To-mAke Micropore Protein-Printing (STAMP2) method. The results demonstrated that TC axons grew massively on the laminin-coated region but were prevented from invading the adjacent ephrin-A5-printed region, suggesting that TC axons detect relative differences in the growth effect between these regions. Moreover, the inhibitory action of ephrin-A5 was enhanced by copresence with KL and Sema7A. Together, these results suggest that the lamina-specific TC axon targeting mechanism involves growth-inhibitory activity by multiple molecules in the upper layers and detection in the molecular environments between the upper and deep layers.     

Loss of phosphatase activity in Ptp69D alleles supporting axon guidance defects

PTP69D is a receptor protein tyrosine phosphatase that was identified as a key regulator of neuromuscular axon guidance in Drosophila, and has subsequently been shown to play a similar role in the central nervous system and retina. Three Ptp69D alleles with mutations involving catalytically important residues exhibit a high degree of phenotypic variation with viability of mutant adult flies ranging from 0 to 96%, and ISNb motor nerve defects ranging from 11 to 57% [Desai and Purdy, 2003]. To determine whether mutations in Ptp69D affecting axon guidance and viability demonstrate losses of phosphatase activity and whether differences in catalytic potential underlie phenotypic variability, we expressed full-length wild-type and mutant PTP69D protein in Schneider 2 cells, and assessed phosphatase activity using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferone phosphate (DiFMUP). Detailed biochemical characterization of wild-type PTP69D, including an examination of sensitivity to various inhibitors, in vitro catalytic efficiency, and the pH-k(cat) profile of the enzyme, suggests a common tyrosine phosphatase reaction mechanism despite lack of sequence conservation in the WPD loop. Analysis of mutant proteins revealed that every mutant had less than 1% activity relative to the wild-type enzyme, and these rates did not differ significantly from one another. These results indicate that mutations in Ptp69D resulting in axon guidance defects and lethality significantly compromise catalytic activity, yet the range of biological activity exhibited by Ptp69D mutants cannot be explained by differences in catalytic activity, as gauged by their ability to hydrolyze the substrate DiFMUP.     

The phosphoinositide phosphatase Sac1 is required for midline axon guidance

Sac1 phosphoinositide (PI) phosphatases are important regulators of PtdIns(4)P turnover at the ER, Golgi, and plasma membrane (PM) and are involved in diverse cellular processes including cytoskeletal organization and vesicular trafficking. Here, we present evidence that Sac1 regulates axon guidance in the embryonic CNS of Drosophila. Sac1 is expressed on three longitudinal axon tracts that are defined by the cell adhesion molecule Fasciclin II (Fas II). Mutations in the sac1 gene cause ectopic midline crossing of Fas II-positive axon tracts. This phenotype is rescued by neuronal expression of wild-type Sac1 but not by a catalytically-inactive mutant. Finally, sac1 displays dosage-sensitive genetic interactions with mutations in the genes that encode the midline repellent Slit and its axonal receptor Robo. Taken together, our results suggest that Sac1-mediated regulation of PIs is critical for Slit/Robo-dependent axon repulsion at the CNS midline.     

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.     

The autophagy-inducing kinases,ULK1 and ULK2, regulate axon guidance in the developing mouse forebrain via a noncanonical pathway

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, Caenorhabditis elegans UNC-51, and Drosophila melanogaster Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. C. elegans UNC-51 and D. melanogaster Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking Ulk1 and Ulk2 in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in ulk1/2 double-knockout mice and central nervous system-specific (Nes-Cre) Ulk1/2-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., Atg7 or Rb1cc1 [RB1-inducible coiled-coil 1]). The brains of Ulk1/2-deficient mice did not show stem cell defects previously attributed to defective autophagy in ambra1 (autophagy/Beclin 1 regulator 1)- and Rb1cc1-deficient mice or accumulation of SQSTM1 (sequestosome 1)⁺ or ubiquitin⁺ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.     

Semaphorin 5A is a bifunctional axon guidance cue for axial motoneurons in vivo

Semaphorins are a large class of proteins that function throughout the nervous system to guide axons. It had previously been shown that Semaphorin 5A (Sema5A) was a bifunctional axon guidance cue for mammalian midbrain neurons. We found that zebrafish sema5A was expressed in myotomes during the period of motor axon outgrowth. To determine whether Sema5A functioned in motor axon guidance, we knocked down Sema5A, which resulted in two phenotypes: a delay in motor axon extension into the ventral myotome and aberrant branching of these motor axons. Both phenotypes were rescued by injection of full-length rat Sema5A mRNA. However, adding back RNA encoding the sema domain alone significantly rescued the branching phenotype in sema5A morphants. Conversely, adding back RNA encoding the thrombospondin repeat (TSR) domain alone into sema5A morphants exclusively rescued delay in ventral motor axon extension. Together, these data show that Sema5A is a bifunctional axon guidance cue for vertebrate motor axons in vivo. The TSR domain promotes growth of developing motor axons into the ventral myotome whereas the sema domain mediates repulsion and keeps these motor axons from branching into surrounding myotome regions.     

Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission

Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission.     

Robo1 and Robo2 have distinct roles in pioneer longitudinal axon guidance

Pioneer longitudinal axons grow long distances parallel to the floor plate and precisely maintain their positions using guidance molecules released from the floor plate. Two receptors, Robo1 and Robo2, are critical for longitudinal axon guidance by the Slit family of chemorepellents. Previous studies showed that Robo1^−/−;2^−/− double mutant mouse embryos have disruptions in both ventral and dorsal longitudinal tracts. However, the role of each Robo isoform remained unclear, because Robo1 or 2 single mutants have mild or no errors. Here we utilized a more sensitive genetic strategy to reduce Robo levels for determining any separate functions of the Robo1 and 2 isoforms. We found that Robo1 is the predominant receptor for guiding axons in ventral tracts and prevents midline crossing. In contrast, Robo2 is the main receptor for directing axons within dorsal tracts. Robo2 also has a distinct function in repelling neuron cell bodies from the floor plate. Therefore, while Robo1 and 2 have some genetic overlap to cooperate in guiding longitudinal axons, each isoform has distinct functions in specific longitudinal axon populations.     

Synaptic proteins and SNARE complexes are localized in lipid rafts from rat brain synaptosomes

The biochemical characterization of the SNARE proteins present in lipid microdomains, also known as "lipid rafts," has been addressed in earlier studies, with conflicting data from different laboratories. In this study, we use rat brain synaptosomes as a model with which to examine the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM), also known as 'lipid rafts.' By means of buoyancy analysis in sucrose gradients of Triton X-100-solubilized synaptosomes, we identified a pool of SNARE proteins (SNAP 25, syntaxin 1, and synaptobrevin2/VAMP2) significantly associated with DRM. Furthermore, Munc18, synaptophysin, and high amounts of the isoforms I and II of synaptotagmin were also found in DRM. In addition, SDS-resistant and temperature-dependent SNARE complexes were also detected in DRM. Treatment of synaptosomes with methyl-beta-cyclodextrin resulted in persistence of the proteins present in the DRM isolated using Triton X-100, whilst strongly impairing calcium-dependent glutamate release. The results from the present work show that lipid microdomains are sites where SNARE proteins and complexes are actually present, as well as important elements in the control of regulated exocytosis.     

Sec1p binds to SNARE complexes and concentrates at sites of secretion.

Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.