The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and,in parallel,Rac and UNC-115/AbLIM

Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, we generated a gain-of-function UNC-40 molecule, MYR::UNC-40. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. Our results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton.     

Localization mechanisms of the axon guidance molecule UNC-6/Netrin and its receptors, UNC-5 and UNC-40, in Caenorhabditis elegans

Netrin is an evolutionarily conserved, secretory axon guidance molecule. Netrin's receptors, UNC-5 and UNC-40/DCC, are single trans-membrane proteins with immunoglobulin domains at their extra-cellular regions. Netrin is thought to provide its positional information by establishing a concentration gradient. UNC-5 and UNC-40 act at growth cones, which are specialized axonal tip structures that are generally located at a long distance from the neural cell body. Thus, the proper localization of both Netrin and its receptors is critical for their function. This review addresses the localization mechanisms of UNC-6/Netrin and its receptors in Caenorhabditis elegans, focusing on our recent reports. These findings include novel insights on cytoplasmic proteins that function upstream of the receptors.     

CYY-1/cyclin Y and CDK-5 differentially regulate synapse elimination and formation for rewiring neural circuits

The assembly and maturation of neural circuits require a delicate balance between synapse formation and elimination. The cellular and molecular mechanisms that coordinate synaptogenesis and synapse elimination are poorly understood. In C. elegans, DD motoneurons respecify their synaptic connectivity during development by completely eliminating existing synapses and forming new synapses without changing cell morphology. Using loss- and gain-of-function genetic approaches, we demonstrate that CYY-1, a cyclin box-containing protein, drives synapse removal in this process. In addition, cyclin-dependent kinase-5 (CDK-5) facilitates new synapse formation by regulating the transport of synaptic vesicles to the sites of synaptogenesis. Furthermore, we show that coordinated activation of UNC-104/Kinesin3 and Dynein is required for patterning newly formed synapses. During the remodeling process, presynaptic components from eliminated synapses are recycled to new synapses, suggesting that signaling mechanisms and molecular motors link the deconstruction of existing synapses and the assembly of new synapses during structural synaptic plasticity.     

UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph Polarize F-Actin during Embryonic Morphogenesis by Regulating the WAVE/SCAR Actin Nucleation Complex

Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton. However, how guidance cues regulate the actin cytoskeleton is not well understood. We report here that UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph differentially regulate the abundance and subcellular localization of the WAVE/SCAR actin nucleation complex and its activator, Rac1/CED-10, in the Caenorhabditis elegans embryonic epidermis. Loss of any of these three pathways results in embryos that fail embryonic morphogenesis. Similar defects in epidermal enclosure have been observed when CED-10/Rac1 or the WAVE/SCAR actin nucleation complex are missing during embryonic development in C. elegans. Genetic and molecular experiments demonstrate that in fact, these three axonal guidance proteins differentially regulate the levels and membrane enrichment of the WAVE/SCAR complex and its activator, Rac1/CED-10, in the epidermis. Live imaging of filamentous actin (F-actin) in embryos developing in the absence of individual guidance receptors shows that high levels of F-actin are not essential for polarized cell migrations, but that properly polarized distribution of F-actin is essential. These results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR by signals at the plasma membrane result in polarized F-actin that permits directed movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.     

The dual role of the ligand UNC-6/Netrin in both axon guidance and synaptogenesis in C. elegans

The extracellular cue UNC-6/Netrin is a well-known axon guidance molecule and recently it has also been shown to be involved with localization of pre-synaptic complexes. Working through the UNC-40/DCC/Fra receptor, UNC-6/Netrin promotes the formation of pre-synaptic terminals between the pre-synaptic AIY interneuron and its post-synaptic partner, the RIA interneuron. In the DA9 motor neuron, UNC-6/Netrin has an alternate role promoting the exclusion of pre-synaptic components from the dendrite via its UNC-5-receptor. Surprisingly, the requirement for UNC-5 persists even after DA9 axon migration is complete, because synapses become mis-localized after it is depleted. This observation provides at least a partial explanation for the persistence of UNC-6/Netrin and UNC-5 in the adult nervous system. These activities parallel the previously known bi-functional axon guidance effects of UNC-6/Netrin, since it can attract cells and axons expressing UNC-40/DCC/Fra and repel those expressing UNC-5 alone or in combination with UNC-40. UNC-6/Netrin cooperates with the Wnt family members to exclude synapses from compartments within the DA9 axon, so that they only occur in regions free of the influence of both UNC-6/Netrin and the Wnts. Regulation of both axon guidance and synapse formation by axon guidance cues permits coordination in circuit assembly between pre- and post-synaptic cells.Key words: nervous system development, axon guidance, synaptogenesis, Netrin/UNC-6, UNC-40/DCC/Fra, UNC-5, LIN-44/Wnt, EGL-20/Wnt, LIN-17/FrizzledDuring development of the nervous system, differentiated pro-neural cells become polarized and send out processes from the cell body that later become dendrites and axons. The pro-neural cells themselves and later their axons, often migrate long distances to their eventual targets using guidance cues.¹ Once the destination is reached, the axon usually selects among several available targets and establishes synapses with the correct post-synaptic partner. The synapse is the site of communication between the pre- and post-synaptic cell and many of the molecules involved in synapse formation are known.² Development of both the pre- and post-synaptic cells needs to be orchestrated to ensure that they are available to form synapses with each other and this process can be directed by guidepost cells. In organisms such as vertebrates, the guidepost cells are often glia, which guide two neurons to ensure that the correct synapse is formed.³ A case is presented here where glial cells secrete a cue to control the localization of pre-synaptic complexes in C. elegans. One notable aspect of this process is that the glial-secreted cue is a well-known axon guidance molecule, namely Netrin/UNC-6, but here it plays an additional and surprising role in selecting the site for the construction of a pre-synaptic complex.⁴UNC-6/Netrin, is a well-known bi-functional axon guidance cue that can attract some axons and repel others. It is a laminin-related molecule, originally isolated from C. elegans, with homologues in higher organisms.^5–7 UNC-6 has two receptors in C. elegans: UNC-40 and UNC-5.^8,9 Both have homologues in higher organisms: UNC-40/DCC/Fra (Deleted in Colorectal Cancer in vertebrates/Frazzled in Drosophila) and UNC-5/Unc5.^6,7,10 UNC-6/Netrin is expressed by cells mostly located in the ventral regions of C. elegans where it attracts many cells and axons expressing the receptor UNC-40.^8,11 Conversely, UNC-6/Netrin repulses axons and cells expressing UNC-5 alone, or in combination with UNC-40.^9,12 One aspect of this developmental process, however, that has been somewhat puzzling has been the observation that expression of both UNC-6 and UNC-5 persist into adulthood.^11,13 A partial explanation for the persistence of UNC-6 and UNC-5 is provided by Poon et al.¹⁴ who found that UNC-5 is required for both the initial polarized localization and maintenance of the pre-synaptic complexes in the DA9 motor neuron axon in C. elegans. The mechanisms used by the proteins in these new roles have not been established, but the localization of both UNC-5 and UNC-40 in the axons is controlled by their normal ligand, UNC-6/Netrin.UNC-40/DCC/Fra plays two independent roles in establishing the connection between the pre-synaptic AIY amphid inter-neuron and its post-synaptic partner, the RIA inter-neuron in the nerve ring of C. elegans, since it is involved in both axon guidance of RIA and synapse localization in AIY.⁴ The two neurons are located in the head region, close to the nerve ring and can be visualized using cell-specific markers (Fig. 1). UNC-6/Netrin plays a conventional guidance role in directing migration of the post-synaptic inter-neuron RIA, since the ventral trajectory of its axon is altered in the absence of UNC-40. The axon of the AIY inter-neuron migrates anteriorly from its cell body, then dorsally and synapses onto three other interneurons: RIA, AIZ and RIB. The AIY axon usually migrates normally without the UNC-40 receptor, which is not surprising as it does not make a ventral migration.Open in a separate windowFigure 1A schematic of the region close to the head of C. elegans is shown where the synapses between the pre-synaptic AIY interneuron (red) and the post-synaptic RIA interneuron (blue) occur. The pre-synaptic regions are shown as black dots. The glial cell (sheath cell) that is the source of UNC-6/Netrin is shown in green. The insert below the schematic shows the regions of the AIY axon divided into zones 1, 2 and 3 that are discussed in the text. Redrawn from Colon-Ramos et al.⁴ and WormAtlas²⁴ (with permission).Pre-synaptic complexes in AIY were detected by expression of fluorescently-tagged synaptic vesicle associated RAB-3.⁴ They were found mainly in the “elbow” region (Fig. 1, zone 2) and about eight more complex-containing areas were found in the region most distant from the cell body within the nerve ring in wild-type animals (Fig. 1, zone 3). A hypomorphic allele of unc-40, wy81, was found in a genetic screen for mutants exhibiting altered localization of pre-synaptic complexes. In the absence of fully functional UNC-40, the pre-synaptic markers were not observed in zone 2, but were present in the more distal region, zone 3. In addition, the pre-synaptic region (zone 2) of AIY did not have an expanded diameter in the manner characteristic of this particular synapse, as detected by electron microscopy. The synapses between AIY and RIA in the absence of UNC-40 were abnormal in several other respects. There was a severe reduction in the active zone proteins ELKS-1/ERC/CAST and SYD-2/α Liprin, suggesting a defect in the pre-synaptic differentiation of AIY. Pre-synaptic defects in AIY caused by absence of UNC-40 could only be rescued by cell-autonomous expression of the receptor.Localization of UNC-40/DCC/Fra in the AIY interneuron is controlled by UNC-6/Netrin emanating from a pair of glial cells called the ventral cephalic sheath cells (VCSCs), which are similar to astrocytes.⁴ Wadsworth et al.¹¹ have previously shown that the VCSCs at the nerve ring express UNC-6/Netrin during neurulation. Colon-Ramos et al.⁴ found the VCSCs project deeply invaginated end-feet that form membranous lamellae, thereby ensheathing the region of AIY-RIA synapses. There is thus a very tight association between the glial cell and the two interneurons in the region of the synapses in zone 2. UNC-40 localizes to the pre-synaptic zones 2 and 3. In the absence of UNC-6, UNC-40 is more diffuse and is present along the entire neuron.The anatomical relationship between the sheath cells and synapses is instructive in mediating AIY:RIA innervations. Sheath cell morphology was altered by the absence of UNC-34/Enabled such that the glial end-feet now migrated further posteriorly to include zone 1.⁴ There was a concomitant appearance of both ectopic pre-synaptic complexes and UNC-40 localization in zone 1 due to an alteration in the source of UNC-6. In UNC-34/Enabled mutants, the trajectory of the RIA interneuron was also altered, such that it had migrated towards the new site of the synapses. Therefore, in this study UNC-40 is playing two independent roles, one in axon path-finding of the RIA axon and a second in positioning the synapses in the AIY pre-synaptic cell. Both of these activities are under the control of UNC-40''s normal ligand, UNC-6/Netrin, that is expressed by glial cells that ensheath the region of the synapses. Regulation of both processes by a single molecule allows co-ordination in circuit assembly.In contrast to the work described above, UNC-6/Netrin and its receptor UNC-5 have recently been reported to exclude synaptic vesicle and active zone components from the dendrite of the DA9 motor neuron in C. elegans (Fig. 2).¹⁴ The DA9 neuron can be divided into five zones (see insert in Fig. 2). It synapses en passant with the VD/DD motor neurons and the body wall muscles along the dorsal cord. In wild-type animals, the synapses of the DA9 neuron were detected using a fluorescently labelled RAB-3, a synaptic vesicle associated protein, and they were found mainly in the region most distant from the cell body (zone 5 of DA9 in Fig. 2). Synapses were entirely excluded from the dendrite (zone 1) and the remainder of the axon (zones 2, 3 and 4) in wild-type animals. Interestingly, UNC-5 had a somewhat complementary distribution to the pre-synaptic complexes since it was expressed mainly in the dendrite of DA9 and the ventral region of the axon (zones 1 and 2 in Fig. 2), suggesting that the presence of UNC-5 can exclude synapses.Open in a separate windowFigure 2The tail region of C. elegans is shown with the DA9 motor neuron. The DA9 axon is in blue and the dendrite in orange. The pre-synaptic structures are in black. The sources of the LIN-44/Wnt, EGL-20/Wnt and UNC-6/Netrin are shown. The insert below shows the various zones of the DA9 neuron described in the text. Redrawn from Poon et al.¹⁴ and WormAtlas²⁴ (with permission).The correct location of the pre-synaptic complexes in DA9 is dependent on both the ligand UNC-6/Netrin and the receptor UNC-5. RAB-3 was found ectopically in the dendrites of either unc-6(ev400), or unc-5(e53), both considered to be null mutants.^5,9,15 Other pre-synaptic vesicle proteins tested, including SNB-1/Synaptobrevin and SNG-1/Synaptogyrin, as well as CCB-1, an L-type voltage-gated calcium channel β subunit and the active zone protein SYD-2/α-liprin, were also mis-localized in the dendrite in the absence of either UNC-6 or UNC-5.¹⁴ UNC-5 functions cell autonomously for the exclusion of pre-synaptic complexes. Interestingly, deletion of either one of the immunoglobulin domains or one of the thrombospondin domains from the extracellular regions of an UNC-5 protein was previously shown to alter the sub-cellular localization of the protein so that is was more localized to the cell body than wild-type UNC-5.¹⁵ This suggests that the extracellular region of UNC-5 is responsible for its localization in the neuron and it would be interesting to see if synapse localization is affected in the absence of the extracellular domains.In addition to its roles in axon guidance and localizing pre-synaptic complexes, an ongoing supply of UNC-5 is required in DA9 to maintain the position of the synapses. This has been demonstrated by the use of a temperature-sensitive silencing intron construct that allowed UNC-5 expression at a permissive temperature of 25°C but not at the restrictive temperature of 16°C.¹⁴ Temperature shift experiments from the permissive temperature to the restrictive temperature at the L4 stage, after the axon was already fully developed, caused synapse mis-localization similar to that observed in the absence of UNC-5. Initial synapse mislocalization was irreversible as the reverse shift from the restrictive to the permissive temperature at L4 failed to rescue the defect. The exclusion of pre-synaptic complexes from all the compartments of DA9 except for the most distal regions (zones 4 and 5) was not simply a consequence of axon misguidance, since axons that were not misguided due to the absence of UNC-5, still exhibited altered RAB-3 localization. Additionally, animals lacking another axon guidance cue, UNC-129/TGFβ exhibited misguidance of DA9 but not mis-localization of pre-synaptic components. Dendritic localization of the pre-synaptic proteins was also not just a reversal of the axons and dendrites in DA9, since four different dendritic proteins were correctly localized in the absence of both UNC-6/Netrin and UNC-5. The need for an ongoing supply of UNC-5 accounts for the observation that both UNC-5 and UNC-6 persist into adulthood, long after axon guidance or synapse formation in worms.^11,13 The finding that UNC-5 must be present on an ongoing basis to maintain localization of pre-synaptic complexes suggests a novel role for UNC-5 in maintaining the polarized localization of the pre-synaptic complexes in a manner independent of axon guidance or initial synaptic polarization. This is an intriguing finding and one that deserves investigation for other neurons and axon guidance molecules.Two Wnt cues also control synapse localization in the DA9 neuron but in different regions than UNC-6/Netrin.¹⁶ LIN-44/Wnt emanating from the tail region (light pink patch in Fig. 2) causes exclusion of synapses from the more posterior section of the DA9 axon located in the dorsal cord (zone 4 in Fig. 2). A second Wnt, EGL-20 is also produced by tail cells (deeper pink region in Fig. 2), and it excludes synapses from the region of the axon in the ventral cord (zone 2 in Fig. 2). Both Wnts cooperate to exclude synapses from zone 3. There is a strict correlation between the presence of the LIN-44/Wnt receptor, LIN-17/Fz, in zones 2, 3 and 4 and the absence of synapses in these regions. LIN-17/Fz is required cell-autonomously in DA9 to rescue synaptic localization defects. In the absence of LIN-44/Wnt, both the receptor LIN-17/Fz and the pre-synaptic complexes were mis-localized since they were now found in both zone 4 and 5 of the axon. Therefore, LIN-44/Wnt is instructive in regulating the location of the synapses in the axon of the DA9 neuron. Both LIN-44/Wnt and EGL-20/Wnt normally work cooperatively to exclude synapses, since animals lacking both had synapses in zones 3, 4 and 5 of DA9.UNC-6/Netrin cooperates with the Wnt family members to exclude synapses from particular regions of the DA9 axon and only allow them to occur in regions free of the influence of both UNC-6/Netrin and the Wnts. Ectopic expression of UNC-6/Netrin and LIN-44/Wnt in various cells and genetic backgrounds was used to show that UNC-6/Netrin and LIN-44/Wnt could function interchangeably in excluding synapses in the DA9 neuron.¹⁴ Ectopic expression of UNC-6 in a posterior to anterior gradient close to DA9 caused RAB-3 to be localized more posteriorly in zone 5, rather than in both zone 4 and 5. The mis-localization was UNC-5 dependent and was seen regardless of whether or not DA9 was misguided. Ectopic UNC-6 could also rescue mis-localization defects in the absence of either LIN-44/Wnt or its receptor LIN-17/Frizzled. These observations suggest that UNC-6/Netrin and LIN-44/Wnt both exclude synapses and can function together to control both axon guidance and pre-synaptic complex localization. Therefore, EGL-20/Wnt and LIN-44/Wnt work cooperatively with the UNC-6/Netrin ligand to inhibit the assembly of pre-synaptic complexes from inappropriate neuronal compartments. Synapses are excluded from the dendrite (zone 1) by UNC-6/Netrin, the region of the axon proximal to the cell body (zone 2) by EGL-20/Wnt, the commissures (zone 3) by EGL-44/Wnt and EGL-20/Wnt, and the distal portion of the axon (zone 4) by LIN-44/Wnt.¹⁴It remains to be seen whether UNC-6/Netrin and its receptors are usually involved in synapse localization in C. elegans itself and in other organisms, beyond the highly specific cell contexts discussed. The involvement of these molecules in both axon guidance and synaptogenesis is likely to be a general phenomenon, as the Netrins are expressed in the adult nervous systems of vertebrates including neurons and oligodendrocytes in the adult rat.¹⁷ DCC is expressed in the adult rat forebrain.¹⁸ UNC-5 is expressed in the heart and brain of adult vertebrates.¹⁹ Ephrins have also been shown to be involved in both axon guidance and synapse formation.²⁰ Wnts have been found to play roles in regulating neuronal connectivity by controlling axon pathfinding, axon remodelling, dendrite morphogenesis and synapse formation in invertebrates and mammals.²¹ Recently, it was shown that pro- and anti-synaptogenic effects of Wnt proteins are associated with the activation of canonical and non-canonical Wnt signaling pathways in Drosophila and mouse.^22,23 It is anticipated that many more instances of axon guidance molecules involved in synapse formation will be described. For instance, in the case of the synapse between the AIY and the RIA interneurons just discussed, AIY also synapses with two other interneurons, the AIZ and RIB but these synapses are not altered significantly in the absence of UNC-40/DCC/Fra. Presumably, these synapses require other molecules to guide synapse formation. Although the two receptors UNC-40 and UNC-5 are functioning with their normal ligand UNC-6/Netrin, it is not clear whether the remainder of the signaling pathways are conserved, and this question will be an interesting topic for future work on synapse formation.     

CED-10/Rac1 mediates axon guidance by regulating the asymmetric distribution of MIG-10/lamellipodin

Axon migrations are guided by extracellular cues that induce asymmetric outgrowth activity in the growth cone. Several intracellular signaling proteins have been implicated in the guidance response. However, how these proteins interact to generate asymmetric outgrowth activity is unknown. Here, we present evidence that in C. elegans, the CED-10/Rac1 GTPase binds to and causes asymmetric localization of MIG-10/lamellipodin, a protein that regulates actin polymerization and has outgrowth-promoting activity in neurons. Genetic analysis indicates that mig-10 and ced-10 function together to orient axon outgrowth. The RAPH domain of MIG-10 binds to activated CED-10/Rac1, and ced-10 function is required for the asymmetric MIG-10 localization that occurs in response to the UNC-6/netrin guidance cue. We also show that asymmetric localization of MIG-10 in growth cones is associated with asymmetric concentrations of f-actin and microtubules. These results suggest that CED-10/Rac1 is asymmetrically activated in response to the UNC-6/netrin signal and thereby causes asymmetric recruitment of MIG-10/lamellipodin. We propose that the interaction between activated CED-10/Rac1 and MIG-10/lamellipodin triggers local cytoskeletal assembly and polarizes outgrowth activity in response to UNC-6/netrin.     

The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans

UNC-51 and UNC-14 are required for the axon guidance of many neurons in Caenorhabditis elegans. UNC-51 is a serine/threonine kinase homologous to yeast Atg1, which is required for autophagy. The binding partner of UNC-51, UNC-14, contains a RUN domain that is predicted to play an important role in multiple Ras-like GTPase signaling pathways. How these molecules function in axon guidance is largely unknown. Here we observed that, in unc-51 and unc-14 mutants, UNC-5, the receptor for axon-guidance protein Netrin/UNC-6, abnormally localized in neuronal cell bodies. By contrast, the localization of many other proteins required for axon guidance was undisturbed. Moreover, UNC-5 localization was normal in animals with mutations in the genes for axon guidance proteins, several motor proteins, vesicle components and autophagy-related proteins. We also found that unc-5 and unc-6 interacted genetically with unc-51 and unc-14 to affect axon guidance, and that UNC-5 co-localized with UNC-51 and UNC-14 in neurons. These results suggest that UNC-51 and UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5, and that UNC-5 uses a unique mechanism for its localization; the functionality of UNC-5 is probably regulated by this localization.     

Synapses are regulated by the cytoplasmic tyrosine kinase Fer in a pathway mediated by p120catenin,Fer, SHP-2, and β-catenin

Localization of presynaptic components to synaptic sites is critical for hippocampal synapse formation. Cell adhesion–regulated signaling is important for synaptic development and function, but little is known about differentiation of the presynaptic compartment. In this study, we describe a pathway that promotes presynaptic development involving p120catenin (p120ctn), the cytoplasmic tyrosine kinase Fer, the protein phosphatase SHP-2, and β-catenin. Presynaptic Fer depletion prevents localization of active zone constituents and synaptic vesicles and inhibits excitatory synapse formation and synaptic transmission. Depletion of p120ctn or SHP-2 similarly disrupts synaptic vesicle localization with active SHP-2, restoring synapse formation in the absence of Fer. Fer or SHP-2 depletion results in elevated tyrosine phosphorylation of β-catenin. β-Catenin overexpression restores normal synaptic vesicle localization in the absence of Fer or SHP-2. Our results indicate that a presynaptic signaling pathway through p120ctn, Fer, SHP-2, and β-catenin promotes excitatory synapse development and function.     

The Rac GTP exchange factor TIAM-1 acts with CDC-42 and the guidance receptor UNC-40/DCC in neuronal protrusion and axon guidance

The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions.     

Rab2 regulates presynaptic precursor vesicle biogenesis at the trans-Golgi

Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi.     

Regulation of retrograde signaling at neuromuscular junctions by the novel C2 domain protein AEX-1.

Retrograde signaling from postsynaptic cells to presynaptic neurons is essential for regulation of synaptic development, maintenance, and plasticity. Here we report that the novel protein AEX-1 controls retrograde signaling at neuromuscular junctions in C. elegans. aex-1 mutants show neural defects including reduced presynaptic activity and abnormal localization of the synaptic vesicle fusion protein UNC-13. Muscle-specific AEX-1 expression rescues these defects but neuron-specific expression does not. AEX-1 has an UNC-13 homologous domain and appears to regulate exocytosis in muscles. This retrograde signaling requires prohormone-convertase function in muscles, suggesting that a peptide is the retrograde signal. This signal regulates synaptic vesicle release via the EGL-30 Gq(alpha) protein at presynaptic terminals.     

Scribble Interacts with β-Catenin to Localize Synaptic Vesicles to Synapses

An understanding of how synaptic vesicles are recruited to and maintained at presynaptic compartments is required to discern the molecular mechanisms underlying presynaptic assembly and plasticity. We have previously demonstrated that cadherin–β-catenin complexes cluster synaptic vesicles at presynaptic sites. Here we show that scribble interacts with the cadherin–β-catenin complex to coordinate vesicle localization. Scribble and β-catenin are colocalized at synapses and can be coimmunoprecipitated from neuronal lysates, indicating an interaction between scribble and β-catenin at the synapse. Using an RNA interference approach, we demonstrate that scribble is important for the clustering of synaptic vesicles at synapses. Indeed, in scribble knockdown cells, there is a diffuse distribution of synaptic vesicles along the axon, and a deficit in vesicle recycling. Despite this, synapse number and the distribution of the presynaptic active zone protein, bassoon, remain unchanged. These effects largely phenocopy those observed after ablation of β-catenin. In addition, we show that loss of β-catenin disrupts scribble localization in primary neurons but that the localization of β-catenin is not dependent on scribble. Our data supports a model by which scribble functions downstream of β-catenin to cluster synaptic vesicles at developing synapses.     

Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-alpha as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-alpha complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.     

The actin cytoskeleton in presynaptic assembly

Dramatic morphogenetic processes underpin nearly every step of nervous system development, from initial neuronal migration and axon guidance to synaptogenesis. Underlying this morphogenesis are dynamic rearrangements of cytoskeletal architecture. Here we discuss the roles of the actin cytoskeleton in the development of presynaptic terminals, from the elaboration of terminal arbors to the recruitment of presynaptic vesicles and active zone components. The studies discussed here underscore the importance of actin regulation at every step in neuronal circuit assembly.     

Synapse development in health and disease

Recent insights into the genetic basis of neurological disease have led to the hypothesis that molecular pathways involved in synaptic growth, development, and stability are perturbed in a variety of mental disorders. Formation of a functional synapse is a complex process requiring stabilization of initial synaptic contacts by adhesive protein interactions, organization of presynaptic and postsynaptic specializations by scaffolding proteins, regulation of growth by intercellular signaling pathways, reorganization of the actin cytoskeleton, and proper endosomal trafficking of synaptic growth signaling complexes. Many neuropsychiatric disorders, including autism, schizophrenia, and intellectual disability, have been linked to inherited mutations which perturb these processes. Our understanding of the basic biology of synaptogenesis is therefore critical to unraveling the pathogenesis of neuropsychiatric disorders.     

Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment

Time-lapse microscopy, retrospective immunohistochemistry, and cultured hippocampal neurons were used to determine the time frame of individual glutamatergic synapse assembly and the temporal order in which specific molecules accumulate at new synaptic junctions. New presynaptic boutons capable of activity-evoked vesicle recycling were observed to form within 30 min of initial axodendritic contact. Clusters of the presynaptic active zone protein Bassoon were present in all new boutons. Conversely, clusters of the postsynaptic molecule SAP90/PSD-95 and glutamate receptors were found on average only approximately 45 min after such boutons were first detected. AMPA- and NMDA-type glutamate receptors displayed similar clustering kinetics. These findings suggest that glutamatergic synapse assembly can occur within 1-2 hr after initial contact and that presynaptic differentiation may precede postsynaptic differentiation.     

The function of mitochondria in presynaptic development at the neuromuscular junction

Mitochondria with high membrane potential (ΔΨ_m) are enriched in the presynaptic nerve terminal at vertebrate neuromuscular junctions, but the exact function of these localized synaptic mitochondria remains unclear. Here, we investigated the correlation between mitochondrial ΔΨ_m and the development of synaptic specializations. Using mitochondrial ΔΨ_m-sensitive probe JC-1, we found that ΔΨ_m in Xenopus spinal neurons could be reversibly elevated by creatine and suppressed by FCCP. Along naïve neurites, preexisting synaptic vesicle (SV) clusters were positively correlated with mitochondrial ΔΨ_m, suggesting a potential regulatory role of mitochondrial activity in synaptogenesis. Indicating a specific role of mitochondrial activity in presynaptic development, mitochondrial ATP synthase inhibitor oligomycin, but not mitochondrial Na⁺/Ca²⁺ exchanger inhibitor CGP-37157, inhibited the clustering of SVs induced by growth factor–coated beads. Local F-actin assembly induced along spinal neurites by beads was suppressed by FCCP or oligomycin. Our results suggest that a key role of presynaptic mitochondria is to provide ATP for the assembly of actin cytoskeleton involved in the assembly of the presynaptic specialization including the clustering of SVs and mitochondria themselves.     

Drosophila cyfip Regulates Synaptic Development and Endocytosis by Suppressing Filamentous Actin Assembly

The formation of synapses and the proper construction of neural circuits depend on signaling pathways that regulate cytoskeletal structure and dynamics. After the mutual recognition of a growing axon and its target, multiple signaling pathways are activated that regulate cytoskeletal dynamics to determine the morphology and strength of the connection. By analyzing Drosophila mutations in the cytoplasmic FMRP interacting protein Cyfip, we demonstrate that this component of the WAVE complex inhibits the assembly of filamentous actin (F-actin) and thereby regulates key aspects of synaptogenesis. Cyfip regulates the distribution of F-actin filaments in presynaptic neuromuscular junction (NMJ) terminals. At cyfip mutant NMJs, F-actin assembly was accelerated, resulting in shorter NMJs, more numerous satellite boutons, and reduced quantal content. Increased synaptic vesicle size and failure to maintain excitatory junctional potential amplitudes under high-frequency stimulation in cyfip mutants indicated an endocytic defect. cyfip mutants exhibited upregulated bone morphogenetic protein (BMP) signaling, a major growth-promoting pathway known to be attenuated by endocytosis at the Drosophila NMJ. We propose that Cyfip regulates synapse development and endocytosis by inhibiting actin assembly.     

Transsynaptic channelosomes: non-conducting roles of ion channels in synapse formation

Recent findings demonstrate that synaptic channels are directly involved in the formation and maintenance of synapses by interacting with synapse organizers. The synaptic channels on the pre- and postsynaptic membranes possess non-conducting roles in addition to their functional roles as ion-conducting channels required for synaptic transmission. For example, presynaptic voltage-dependent calcium channels link the target-derived synapse organizer laminin β2 to cytomatrix of the active zone and function as scaffolding proteins to organize the presynaptic active zones. Furthermore, postsynaptic δ2-type glutamate receptors organize the synapses by forming transsynaptic protein complexes with presynaptic neurexins through synapse organizer cerebellin 1 precursor proteins. Interestingly, the synaptic clustering of AMPA receptors is regulated by neuronal activity-regulated pentraxins, while postsynaptic differentiation is induced by the interaction of postsynaptic calcium channels and thrombospondins. This review will focus on the non-conducting functions of ion-channels that contribute to the synapse formation in concert with synapse organizers and active-zone-specific proteins.     

Transsynaptic channelosomes

Recent findings demonstrate that synaptic channels are directly involved in the formation and maintenance of synapses by interacting with synapse organizers. The synaptic channels on the pre- and postsynaptic membranes possess non-conducting roles in addition to their functional roles as ion-conducting channels required for synaptic transmission. For example, presynaptic voltage-dependent calcium channels link the target-derived synapse organizer laminin β2 to cytomatrix of the active zone and function as scaffolding proteins to organize the presynaptic active zones. Furthermore, postsynaptic δ2-type glutamate receptors organize the synapses by forming transsynaptic protein complexes with presynaptic neurexins through synapse organizer cerebellin 1 precursor proteins. Interestingly, the synaptic clustering of AMPA receptors is regulated by neuronal activity-regulated pentraxins, while postsynaptic differentiation is induced by the interaction of postsynaptic calcium channels and thrombospondins. This review will focus on the non-conducting functions of ion-channels that contribute to the synapse formation in concert with synapse organizers and active-zone-specific proteins.