Presynaptic UNC-31 (CAPS) is required to activate the G alpha(s) pathway of the Caenorhabditis elegans synaptic signaling network

C. elegans mutants lacking the dense-core vesicle priming protein UNC-31 (CAPS) share highly similar phenotypes with mutants lacking a neuronal G alpha(s) pathway, including strong paralysis despite exhibiting near normal levels of steady-state acetylcholine release as indicated by drug sensitivity assays. Our genetic analysis shows that UNC-31 and neuronal G alpha(s) are different parts of the same pathway and that the UNC-31/G alpha(s) pathway is functionally distinct from the presynaptic G alpha(q) pathway with which it interacts. UNC-31 acts upstream of G alpha(s) because mutations that activate the G alpha(s) pathway confer similar levels of strongly hyperactive, coordinated locomotion in both unc-31 null and (+) backgrounds. Using cell-specific promoters, we show that both UNC-31 and the G alpha(s) pathway function in cholinergic motor neurons to regulate locomotion rate. Using immunostaining we show that UNC-31 is often concentrated at or near active zones of cholinergic motor neuron synapses. Our data suggest that presynaptic UNC-31 activity, likely acting via dense-core vesicle exocytosis, is required to locally activate the neuronal G alpha(s) pathway near synaptic active zones.     

Mutations that rescue the paralysis of Caenorhabditis elegans ric-8 (synembryn) mutants activate the G alpha(s) pathway and define a third major branch of the synaptic signaling network

To identify hypothesized missing components of the synaptic G alpha(o)-G alpha(q) signaling network, which tightly regulates neurotransmitter release, we undertook two large forward genetic screens in the model organism C. elegans and focused first on mutations that strongly rescue the paralysis of ric-8(md303) reduction-of-function mutants, previously shown to be defective in G alpha(q) pathway activation. Through high-resolution mapping followed by sequence analysis, we show that these mutations affect four genes. Two activate the G alpha(q) pathway through gain-of-function mutations in G alpha(q); however, all of the remaining mutations activate components of the G alpha(s) pathway, including G alpha(s), adenylyl cyclase, and protein kinase A. Pharmacological assays suggest that the G alpha(s) pathway-activating mutations increase steady-state neurotransmitter release, and the strongly impaired neurotransmitter release of ric-8(md303) mutants is rescued to greater than wild-type levels by the strongest G alpha(s) pathway activating mutations. Using transgene induction studies, we show that activating the G alpha(s) pathway in adult animals rapidly induces hyperactive locomotion and rapidly rescues the paralysis of the ric-8 mutant. Using cell-specific promoters we show that neuronal, but not muscle, G alpha(s) pathway activation is sufficient to rescue ric-8(md303)'s paralysis. Our results appear to link RIC-8 (synembryn) and a third major G alpha pathway, the G alpha(s) pathway, with the previously discovered G alpha(o) and G alpha(q) pathways of the synaptic signaling network.     

Latrotoxin receptor signaling engages the UNC-13-dependent vesicle-priming pathway in C. elegans

alpha-latrotoxin (LTX), a 120 kDa protein in black widow spider venom, triggers massive neurotransmitter exocytosis. Previous studies have highlighted a role for both intrinsic pore-forming activity and receptor binding in the action of this toxin. Intriguingly, activation of a presynaptic G protein-coupled receptor, latrophilin, may trigger release independent of pore-formation. Here we have utilized a previously identified ligand of nematode latrophilin, emodepside, to define a latrophilin-dependent pathway for neurotransmitter release in C. elegans. In the pharyngeal nervous system of this animal, emodepside (100 nM) stimulates exocytosis and elicits pharyngeal paralysis. The pharynxes of animals with latrophilin (lat-1) gene knockouts are resistant to emodepside, indicating that emodepside exerts its high-affinity paralytic effect through LAT-1. The expression pattern of lat-1 supports the hypothesis that emodepside exerts its effect on the pharynx primarily via neuronal latrophilin. We build on these observations to show that pharynxes from animals with either reduction or loss of function mutations in Gq, phospholipaseC-beta, and UNC-13 are resistant to emodepside. The latter is a key priming molecule essential for synaptic vesicle-mediated release of neurotransmitter. We conclude that the small molecule ligand emodepside triggers latrophilin-mediated exocytosis via a pathway that engages UNC-13-dependent vesicle priming.     

New roles for Galpha and RGS proteins: communication continues despite pulling sisters apart

Large G protein alpha subunits and their attendant regulators of G-protein signaling (RGS) proteins control both intercellular signaling and asymmetric cell divisions by distinct pathways. The classical pathway, found throughout higher eukaryotic organisms, mediates intercellular communication via hormone binding to G-protein-coupled receptors (GPCRs). Recent studies have led to the discovery of GPCR-independent activation of Galpha subunits by the guanine nucleotide exchange factor RIC-8 in both asymmetric cell division and synaptic vesicle priming in metazoan organisms. Protein-protein interactions and protein function in each pathway are driven through the cycle of GTP binding and hydrolysis by the Galpha subunit. This review builds a conceptual framework for understanding RIC-8-mediated pathways by comparison with the mechanism of classical G-protein activation and inhibition in GPCR signaling.     

Synaptic drosophila UNC‐13 is regulated by antagonistic G‐protein pathways via a proteasome‐dependent degradation mechanism

UNC‐13 is a highly conserved plasma membrane‐associated synaptic protein implicated in the regulation of neurotransmitter release through the direct modulation of the SNARE exocytosis complex. Previously, we characterized the Drosophila homologue (DUNC‐13) and showed it to be essential for neurotransmitter release immediately upstream of vesicular fusion (“priming”) at the neuromuscular junction (NMJ). Here, we show that the abundance of DUNC‐13 in NMJ synaptic boutons is regulated downstream of GαS and Gαq pathways, which have inhibitory and facilitatory roles, respectively. Both cAMP modulation and PKA function are required for DUNC‐13 synaptic up‐regulation, suggesting that the cAMP pathway enhances synaptic efficacy via DUNC‐13. Similarly, PLC function and DAG modulation also regulate the synaptic levels of DUNC‐13, through a mechanism that appears independent of PKC. Our results suggest that proteasome‐mediated protein degradation is the primary mechanism regulating DUNC‐13 levels at the synapse. Both PLC‐ and PKA‐mediated pathways appear to regulate synaptic levels of DUNC‐13 through controlling the rate of proteasome‐dependent DUNC‐13 degradation. We conclude that the functional abundance of DUNC‐13 at the synapse, a key determinant of synaptic vesicle priming and neurotransmitter release probability, is primarily regulated by the rate of protein degradation, rather than translocation or transport, convergently controlled via both cAMP and DAG signal transduction pathways. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 417–438, 2003     

Role of heterotrimeric G-proteins in lysophosphatidic acid-mediated neurite retraction by RhoA-dependent and -independent mechanisms in N1E-115 cells

In neuronal cells, current evidence suggests that G(13)alpha and RhoA play significant roles in LPA-mediated neurite retraction; however, the contribution of other G-proteins to this process is less well-understood. We provide evidence that LPA activation of G(13), G(q) and G(i) occurs rapidly in neuroblastoma cells, but that stimulation of RhoA is transient whereas the activation of G(q)- and G(i)-mediated pathways is sustained. In addition to G(13)alpha, we demonstrate that G(q)alpha is capable of promoting neurite retraction. G(q)-mediated retraction is RhoA-independent and is likely mediated via a mechanism involving protein kinase C and calcium flux. Additionally, we provide evidence that activation of adenylyl cyclase via G(s) inhibits RhoA-mediated neurite retraction via protein kinase A-mediated inhibition of RhoA action. Taken together, we hypothesize that LPA promotes neurite retraction via RhoA-dependent and -independent pathways involving G(13) and G(q), respectively, and that agonists that activate G(s) inhibit the RhoA-dependent pathway.     

Disabled is a bona fide component of the Abl signaling network

Abl is an essential regulator of cell migration and morphogenesis in both vertebrates and invertebrates. It has long been speculated that the adaptor protein Disabled (Dab), which is a key regulator of neuronal migration in the vertebrate brain, might be a component of this signaling pathway, but this idea has been controversial. We now demonstrate that null mutations of Drosophila Dab result in phenotypes that mimic Abl mutant phenotypes, both in axon guidance and epithelial morphogenesis. The Dab mutant interacts genetically with mutations in Abl, and with mutations in the Abl accessory factors trio and enabled (ena). Genetic epistasis tests show that Dab functions upstream of Abl and ena, and, consistent with this, we show that Dab is required for the subcellular localization of these two proteins. We therefore infer that Dab is a bona fide component of the core Abl signaling pathway in Drosophila.     

Calcium/calmodulin-dependent protein kinase II regulates Caenorhabditis elegans locomotion in concert with a G(o)/G(q) signaling network

Caenorhabditis elegans locomotion is a complex behavior generated by a defined set of motor neurons and interneurons. Genetic analysis shows that UNC-43, the C. elegans Ca(2+)/calmodulin protein kinase II (CaMKII), controls locomotion rate. Elevated UNC-43 activity, from a gain-of-function mutation, causes severely lethargic locomotion, presumably by inappropriate phosphorylation of targets. In a genetic screen for suppressors of this phenotype, we identified multiple alleles of four genes in a G(o)/G(q) G-protein signaling network, which has been shown to regulate synaptic activity via diacylglycerol. Mutations in goa-1, dgk-1, eat-16, or eat-11 strongly or completely suppressed unc-43(gf) lethargy, but affected other mutants with reduced locomotion only weakly. We conclude that CaMKII and G(o)/G(q) pathways act in concert to regulate synaptic activity, perhaps through a direct interaction between CaMKII and G(o).     

Molecular dissection of G protein preference using Gsalpha chimeras reveals novel ligand signaling of GPCRs

Although only 16 genes have been identified in mammals, several Galpha subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective Galpha activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by G(s)alpha and the carboxyl termini of the various Galpha proteins determine their receptor coupling specificity, we proposed a set of chimeric G(s)alpha where the COOH-terminal five amino acids are replaced by those of other Galpha proteins and used these to dissect the potential Galpha linked to a given GPCR. Unlike G(q)alpha, G(12)alpha, and G(i)alpha outputs, compounding the signals from several Galpha members, the chimeric G(s)alpha proteins provide a superior molecular approach that reflects the previously uncharacterized pathways of GPCRs under the same cAMP platform. This is, to our knowledge, the first time allowing verification of the whole spectrum of Galpha coupling preference of adenosine A1 receptor, reported to couple to multiple G proteins and modulate many physiological processes. Furthermore, we were able to distinguish the uncharacterized pathways between the two neuromedin U receptors (NMURs), which distribute differently but are stimulated by a common agonist. In contrast to the G(q) signals mainly conducted by NMUR1, NMUR2 routed preferentially to the G(i) pathways. Dissecting the potential Galpha coupling to these GPCRs will promote an understanding of their physiological roles and benefit the pharmaceutical development of agonists/antagonists by exploiting the selective affinity toward a certain Galpha subclass.     

Molecular machines in the synapse: overlapping protein sets control distinct steps in neurosecretion

Activity regulated neurotransmission shapes the computational properties of a neuron and involves the concerted action of many proteins. Classical, intuitive working models often assign specific proteins to specific steps in such complex cellular processes, whereas modern systems theories emphasize more integrated functions of proteins. To test how often synaptic proteins participate in multiple steps in neurotransmission we present a novel probabilistic method to analyze complex functional data from genetic perturbation studies on neuronal secretion. Our method uses a mixture of probabilistic principal component analyzers to cluster genetic perturbations on two distinct steps in synaptic secretion, vesicle priming and fusion, and accounts for the poor standardization between different studies. Clustering data from 121 perturbations revealed that different perturbations of a given protein are often assigned to different steps in the release process. Furthermore, vesicle priming and fusion are inversely correlated for most of those perturbations where a specific protein domain was mutated to create a gain-of-function variant. Finally, two different modes of vesicle release, spontaneous and action potential evoked release, were affected similarly by most perturbations. This data suggests that the presynaptic protein network has evolved as a highly integrated supramolecular machine, which is responsible for both spontaneous and activity induced release, with a group of core proteins using different domains to act on multiple steps in the release process.     

The Dunce cAMP phosphodiesterase PDE-4 negatively regulates G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the Caenorhabditis elegans synaptic signaling network

Forward genetic screens for mutations that rescue the paralysis of ric-8 (Synembryn) reduction-of-function mutations frequently reveal mutations that cause hyperactivation of one or more components of the G alpha(s) pathway. Here, we report that one of these mutations strongly reduces the function of the Dunce cAMP phosphodiesterase PDE-4 by disrupting a conserved active site residue. Loss of function and neural overexpression of PDE-4 have profound and opposite effects on locomotion rate, but drug-response assays suggest that loss of PDE-4 function does not affect steady-state acetylcholine release or reception. Our genetic analysis suggests that PDE-4 regulates both G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the neurons controlling locomotion rate. By immunostaining, PDE-4 is strongly expressed throughout the nervous system, where it localizes to small regions at the outside boundaries of synaptic vesicle clusters as well as intersynaptic regions. The synaptic subregions containing PDE-4 are distinct from those containing active zones, as indicated by costaining with an antibody against the long form of UNC-13. This highly focal subsynaptic localization suggests that PDE-4 may exert its effects by spatially regulating intrasynaptic cAMP pools.     

RGS2 determines short-term synaptic plasticity in hippocampal neurons by regulating Gi/o-mediated inhibition of presynaptic Ca2+ channels

RGS2, one of the small members of the regulator of G protein signaling (RGS) family, is highly expressed in brain and regulates G(i/o) as well as G(q)-coupled receptor pathways. RGS2 modulates anxiety, aggression, and blood pressure in mice, suggesting that RGS2 regulates synaptic circuits underlying animal physiology and behavior. How RGS2 in brain influences synaptic activity is unknown. We therefore analyzed the synaptic function of RGS2 in hippocampal neurons by comparing electrophysiological recordings from RGS2 knockout and wild-type mice. Our study provides a general mechanism of the action of the RGS family containing RGS2 by demonstrating that RGS2 increases synaptic vesicle release by downregulating the G(i/o)-mediated presynaptic Ca(2+) channel inhibition and therefore provides an explanation of how regulation of RGS2 expression can modulate the function of neuronal circuits underlying behavior.     

Interdependence of PKC-dependent and PKC-independent pathways for presynaptic plasticity

Diacylglycerol (DAG) is a prominent endogenous modulator of synaptic transmission. Recent studies proposed two apparently incompatible pathways, via protein kinase C (PKC) and via Munc13. Here we show how these two pathways converge. First, we confirm that DAG analogs indeed continue to potentiate transmission after PKC inhibition (the Munc13 pathway), but only in neurons that previously experienced DAG analogs, before PKC inhibition started. Second, we identify an essential PKC pathway by expressing a PKC-insensitive Munc18-1 mutant in munc18-1 null mutant neurons. This mutant supported basic transmission, but not DAG-induced potentiation and vesicle redistribution. Moreover, synaptic depression was increased, but not Ca2+-independent release evoked by hypertonic solutions. These data show that activation of both PKC-dependent and -independent pathways (via Munc13) are required for DAG-induced potentiation. Munc18-1 is an essential downstream target in the PKC pathway. This pathway is of general importance for presynaptic plasticity.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

Synaptic Drosophila UNC-13 is regulated by antagonistic G-protein pathways via a proteasome-dependent degradation mechanism

UNC-13 is a highly conserved plasma membrane-associated synaptic protein implicated in the regulation of neurotransmitter release through the direct modulation of the SNARE exocytosis complex. Previously, we characterized the Drosophila homologue (DUNC-13) and showed it to be essential for neurotransmitter release immediately upstream of vesicular fusion ("priming") at the neuromuscular junction (NMJ). Here, we show that the abundance of DUNC-13 in NMJ synaptic boutons is regulated downstream of GalphaS and Galphaq pathways, which have inhibitory and facilitatory roles, respectively. Both cAMP modulation and PKA function are required for DUNC-13 synaptic up-regulation, suggesting that the cAMP pathway enhances synaptic efficacy via DUNC-13. Similarly, PLC function and DAG modulation also regulate the synaptic levels of DUNC-13, through a mechanism that appears independent of PKC. Our results suggest that proteasome-mediated protein degradation is the primary mechanism regulating DUNC-13 levels at the synapse. Both PLC- and PKA-mediated pathways appear to regulate synaptic levels of DUNC-13 through controlling the rate of proteasome-dependent DUNC-13 degradation. We conclude that the functional abundance of DUNC-13 at the synapse, a key determinant of synaptic vesicle priming and neurotransmitter release probability, is primarily regulated by the rate of protein degradation, rather than translocation or transport, convergently controlled via both cAMP and DAG signal transduction pathways.     

Genetic modifiers of Drosophila palmitoyl-protein thioesterase 1-induced degeneration

Infantile neuronal ceroid lipofuscinosis (INCL) is a pediatric neurodegenerative disease caused by mutations in the human CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase 1 (PPT1), suggesting an important role for the regulation of palmitoylation in normal neuronal function. To further elucidate Ppt1 function, we performed a gain-of-function modifier screen in Drosophila using a collection of enhancer-promoter transgenic lines to suppress or enhance the degeneration produced by overexpression of Ppt1 in the adult visual system. Modifier genes identified in our screen connect Ppt1 function to synaptic vesicle cycling, endo-lysosomal trafficking, synaptic development, and activity-dependent remodeling of the synapse. Furthermore, several homologs of the modifying genes are known to be regulated by palmitoylation in other systems and may be in vivo substrates for Ppt1. Our results complement recent work on mouse Ppt1(-/-) cells that shows a reduction in synaptic vesicle pools in primary neuronal cultures and defects in endosomal trafficking in human fibroblasts. The pathways and processes implicated by our modifier loci shed light on the normal cellular function of Ppt1. A greater understanding of Ppt1 function in these cellular processes will provide valuable insight into the molecular etiology of the neuronal dysfunction underlying the disease.     

Opposing functions of calcineurin and CaMKII regulate G-protein signaling in egg-laying behavior of C.elegans

Ca(2+)/calmodulin-dependent calcineurin has been shown to have important roles in various Ca(2+) signaling pathways. We have previously reported that cnb-1(jh103) mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in Caenorhabditis elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1(null) mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C.elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10, which are known to be involved in G-protein signaling pathways suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goalpha) and egl-30(Gqalpha). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C.elegans.     

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor

G protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT(4b) receptor, a GPCR with high constitutive G(s) signaling and strong ligand-induced G-protein activation of the G(s) and G(s/q) pathways. The first receptor in this series, 5-HT(4)-D(100)A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced G(s) signaling, but only a few (e.g., zacopride) also induced signaling via the G(q) pathway. Zacopride-induced G(q) signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT(2C) receptor. Additional point mutations (D(66)A and D(66)N) blocked constitutive G(s) signaling and lowered ligand-induced G(q) signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT(1A) conferred ligand-mediated G(i) signaling. This G(i)-coupled RASSL, Rs1.3, exhibited no measurable signaling to the G(s) or G(q) pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.     

ITSN-1 controls vesicle recycling at the neuromuscular junction and functions in parallel with DAB-1

Intersectins (Itsn) are conserved EH and SH3 domain containing adaptor proteins. In Drosophila melanogaster, ITSN is required to regulate synaptic morphology, to facilitate efficient synaptic vesicle recycling and for viability. Here, we report our genetic analysis of Caenorhabditis elegans intersectin. In contrast to Drosophila , C. elegans itsn-1 protein null mutants are viable and display grossly normal locomotion and development. However, motor neurons in these mutants show a dramatic increase in large irregular vesicles and accumulate membrane-associated vesicles at putative endocytic hotspots, approximately 300 nm from the presynaptic density. This defect occurs precisely where endogenous ITSN-1 protein localizes in wild-type animals and is associated with a significant reduction in synaptic vesicle number and reduced frequency of endogenous synaptic events at neuromuscular junctions (NMJs). ITSN-1 forms a stable complex with EHS-1 (Eps15) and is expressed at reduced levels in ehs-1 mutants. Thus, ITSN-1 together with EHS-1, coordinate vesicle recycling at C. elegans NMJs. We also found that both itsn-1 and ehs-1 mutants show poor viability and growth in a Disabled (dab-1) null mutant background. These results show for the first time that intersectin and Eps15 proteins function in the same genetic pathway, and appear to function synergistically with the clathrin-coat-associated sorting protein, Disabled, for viability.