Presynaptic neurons self-tune by inversely coupling neurotransmitter release with the abundance of CaV2 voltage-gated Ca2+ channels.

The abundance of CaV2 voltage-gated calcium channels is linked to presynaptic homeostatic plasticity (PHP), a process that recalibrates synaptic strength to maintain the stability of neural circuits. However, the molecular and cellular mechanisms governing PHP and CaV2 channels are not completely understood. Here, we uncover a previously not described form of PHP in Caenorhabditis elegans, revealing an inverse regulatory relationship between the efficiency of neurotransmitter release and the abundance of UNC-2/CaV2 channels. Gain-of-function unc-2SL(S240L) mutants, which carry a mutation analogous to the one causing familial hemiplegic migraine type 1 in humans, showed markedly reduced channel abundance despite increased channel functionality. Reducing synaptic release in these unc-2SL(S240L) mutants restored channel levels to those observed in wild-type animals. Conversely, loss-of-function unc-2DA(D726A) mutants, which harbor the D726A mutation in the channel pore, exhibited a marked increase in channel abundance. Enhancing synaptic release in unc-2DA mutants reversed this increase in channel levels. Importantly, this homeostatic regulation of UNC-2 channel levels is accompanied by the structural remodeling of the active zone (AZ); specifically, unc-2DA mutants, which exhibit increased channel abundance, showed parallel increases in select AZ proteins. Finally, our forward genetic screen revealed that WWP-1, a HECT family E3 ubiquitin ligase, is a key homeostatic mediator that removes UNC-2 from synapses. These findings highlight a self-tuning PHP regulating UNC-2/CaV2 channel abundance along with AZ reorganization, ensuring synaptic strength and stability.

C. elegans Clarinet/CLA-1 recruits RIMB-1/RIM-binding protein and UNC-13 to orchestrate presynaptic neurotransmitter release.

Synaptic transmission requires the coordinated activity of multiple synaptic proteins that are localized at the active zone (AZ). We previously identified a Caenorhabditis elegans protein named Clarinet (CLA-1) based on homology to the AZ proteins Piccolo, Rab3-interactingmolecule (RIM)/UNC-10 and Fife. At the neuromuscular junction (NMJ), cla-1 null mutants exhibit release defects that are greatly exacerbated in cla-1;unc-10 double mutants. To gain insights into the coordinated roles of CLA-1 and UNC-10, we examined the relative contributions of each to the function and organization of the AZ. Using a combination of electrophysiology, electron microscopy, and quantitative fluorescence imaging we explored the functional relationship of CLA-1 to other key AZ proteins including: RIM1, Cav2.1 channels, RIM1-binding protein, and Munc13 (C. elegans UNC-10, UNC-2, RIMB-1 and UNC-13, respectively). Our analyses show that CLA-1 acts in concert with UNC-10 to regulate UNC-2 calcium channel levels at the synapse via recruitment of RIMB-1. In addition, CLA-1 exerts a RIMB-1-independent role in the localization of the priming factor UNC-13. Thus C. elegans CLA-1/UNC-10 exhibit combinatorial effects that have overlapping design principles with other model organisms: RIM/RBP and RIM/ELKS in mouse and Fife/RIM and BRP/RBP in Drosophila. These data support a semiconserved arrangement of AZ scaffolding proteins that are necessary for the localization and activation of the fusion machinery within nanodomains for precise coupling to Ca2+ channels.

Active Zone Trafficking of CaV2/UNC-2 Channels Is Independent of ß/CCB-1 and α2δ/UNC-36 Subunits.

The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α1 subunit and two auxiliary ß and α2δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/α1 localizes to AZs even in the absence of CCB-1/ß or UNC-36/α2δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes.SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α1 subunit and two auxiliary ß and α2δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α1 and aid trafficking, they are recruited to α1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels.

Interference with glutamate antiporter system xc - enables post-hypoxic long-term potentiation in hippocampus.

Our group previously showed that genetic or pharmacological inhibition of the cystine/glutamate antiporter, system xc -, mitigates excitotoxicity after anoxia by increasing latency to anoxic depolarization, thus attenuating the ischaemic core. Hypoxia, however, which prevails in the ischaemic penumbra, is a condition where neurotransmission is altered, but excitotoxicity is not triggered. The present study employed mild hypoxia to further probe ischaemia-induced changes in neuronal responsiveness from wild-type and xCT KO (xCT-/-) mice. Synaptic transmission was monitored in hippocampal slices from both genotypes before, during and after a hypoxic episode. Although wild-type and xCT-/- slices showed equal suppression of synaptic transmission during hypoxia, mutant slices exhibited a persistent potentiation upon re-oxygenation, an effect we termed 'post-hypoxic long-term potentiation (LTP)'. Blocking synaptic suppression during hypoxia by antagonizing adenosine A1 receptors did not preclude post-hypoxic LTP. Further examination of the induction and expression mechanisms of this plasticity revealed that post-hypoxic LTP was driven by NMDA receptor activation, as well as increased calcium influx, with no change in paired-pulse facilitation. Hence, the observed phenomenon engaged similar mechanisms as classical LTP. This was a remarkable finding as theta-burst stimulation-induced LTP was equivalent between genotypes. Importantly, post-hypoxic LTP was generated in wild-type slices pretreated with system xc - inhibitor, S-4-carboxyphenylglycine, thereby confirming the antiporter's role in this phenomenon. Collectively, these data indicate that system xc - interference enables neuroplasticity in response to mild hypoxia, and, together with its regulation of cellular damage in the ischaemic core, suggest a role for the antiporter in post-ischaemic recovery of the penumbra.

BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor.

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.

UNC-2 CaV2 Channel Localization at Presynaptic Active Zones Depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans.

Presynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel localization by active zone proteins is not completely understood. In a Caenorhabditis elegans (C. elegans) forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic localization of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel puncta at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel localization, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that although SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 localization by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel localization. In addition, we find that core active zone proteins are unequal in their abundance. Although the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel localization in redundant yet distinct manners.SIGNIFICANCE STATEMENT Precise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM, our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, a core active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

Transcriptional control of parallel-acting pathways that remove specific presynaptic proteins in remodeling neurons.

Synapses are actively dismantled to mediate circuit refinement, but the developmental pathways that regulate synaptic disassembly are largely unknown. We have previously shown that the epithelial sodium channel ENaC/UNC-8 triggers an activity-dependent mechanism that drives the removal of presynaptic proteins liprin-α/SYD-2, Synaptobrevin/SNB-1, RAB-3 and Endophilin/UNC-57 in remodeling GABAergic neurons in C. elegans (Miller-Fleming et al., 2016). Here, we report that the conserved transcription factor Iroquois/IRX-1 regulates UNC-8 expression as well as an additional pathway, independent of UNC-8, that functions in parallel to dismantle functional presynaptic terminals. We show that the additional IRX-1-regulated pathway is selectively required for the removal of the presynaptic proteins, Munc13/UNC-13 and ELKS, which normally mediate synaptic vesicle fusion and neurotransmitter release. Our findings are notable because they highlight the key role of transcriptional regulation in synapse elimination during development and reveal parallel-acting pathways that coordinate synaptic disassembly by removing specific active zone proteins.SIGNIFICANT STATEMENT:Synaptic pruning is a conserved feature of developing neural circuits but the mechanisms that dismantle the presynaptic apparatus are largely unknown. We have determined that synaptic disassembly is orchestrated by parallel-acting mechanisms that target distinct components of the active zone. Thus, our finding suggests that synaptic disassembly is not accomplished by en masse destruction but depends on mechanisms that dismantle the structure in an organized process.

Alcohol induces mitochondrial fragmentation and stress responses to maintain normal muscle function in Caenorhabditis elegans.

Chronic excessive ethanol consumption has distinct toxic and adverse effects on a variety of tissues. In skeletal muscle, ethanol causes alcoholic myopathy, which is characterized by myofiber atrophy and the loss of muscle strength. Alcoholic myopathy is more prevalent than all inherited muscle diseases combined. Current evidence indicates that ethanol directly impairs muscle organization and function. However, the underlying mechanism by which ethanol causes toxicity in muscle is poorly understood. Here, we show that the nematode Caenorhabditis elegans exhibits the key features of alcoholic myopathy when exposed to ethanol. As in mammals, ethanol exposure impairs muscle strength and induces the expression of protective genes, including oxidative stress response genes. In addition, ethanol exposure causes the fragmentation of mitochondrial networks aligned with myofibril lattices. This ethanol-induced mitochondrial fragmentation is dependent on the mitochondrial fission factor DRP-1 (dynamin-related protein 1) and its receptor proteins on the outer mitochondrial membrane. Our data indicate that this fragmentation contributes to the activation of the mitochondrial unfolded protein response (UPR). We also found that robust, perpetual mitochondrial UPR activation effectively reduces muscle weakness caused by ethanol exposure. Our results strongly suggest that the modulation of mitochondrial stress responses may provide a method to ameliorate alcohol toxicity and damage to muscle.

C. elegans Punctin specifies cholinergic versus GABAergic identity of postsynaptic domains.

Because most neurons receive thousands of synaptic inputs, the neuronal membrane is a mosaic of specialized microdomains where neurotransmitter receptors cluster in register with the corresponding presynaptic neurotransmitter release sites. In many cases the coordinated differentiation of presynaptic and postsynaptic domains implicates trans-synaptic interactions between membrane-associated proteins such as neurexins and neuroligins. The Caenorhabditis elegans neuromuscular junction (NMJ) provides a genetically tractable system in which to analyse the segregation of neurotransmitter receptors, because muscle cells receive excitatory innervation from cholinergic neurons and inhibitory innervation from GABAergic neurons. Here we show that Ce-Punctin/madd-4 (ref. 5), the C. elegans orthologue of mammalian punctin-1 and punctin-2, encodes neurally secreted isoforms that specify the excitatory or inhibitory identity of postsynaptic NMJ domains. These proteins belong to the ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats)-like family, a class of extracellular matrix proteins related to the ADAM proteases but devoid of proteolytic activity. Ce-Punctin deletion causes the redistribution of synaptic acetylcholine and GABAA (γ-aminobutyric acid type A) receptors into extrasynaptic clusters, whereas neuronal presynaptic boutons remain unaltered. Alternative promoters generate different Ce-Punctin isoforms with distinct functions. A short isoform is expressed by cholinergic and GABAergic motoneurons and localizes to excitatory and inhibitory NMJs, whereas long isoforms are expressed exclusively by cholinergic motoneurons and are confined to cholinergic NMJs. The differential expression of these isoforms controls the congruence between presynaptic and postsynaptic domains: specific disruption of the short isoform relocalizes GABAA receptors from GABAergic to cholinergic synapses, whereas expression of a long isoform in GABAergic neurons recruits acetylcholine receptors to GABAergic NMJs. These results identify Ce-Punctin as a previously unknown synaptic organizer and show that presynaptic and postsynaptic domain identities can be genetically uncoupled in vivo. Because human punctin-2 was identified as a candidate gene for schizophrenia, ADAMTS-like proteins may also control synapse organization in the mammalian central nervous system.

Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex.

The number of nicotinic acetylcholine receptors (AChRs) present in the plasma membrane of muscle and neuronal cells is limited by the assembly of individual subunits into mature pentameric receptors. This process is usually inefficient, and a large number of the synthesized subunits are degraded by endoplasmic reticulum (ER)-associated degradation. To identify cellular factors required for the synthesis of AChRs, we performed a genetic screen in the nematode Caenorhabditis elegans for mutants with decreased sensitivity to the cholinergic agonist levamisole. We isolated a partial loss-of-function allele of ER membrane protein complex-6 (emc-6), a previously uncharacterized gene in C. elegans. emc-6 encodes an evolutionarily conserved 111-aa protein with two predicted transmembrane domains. EMC-6 is ubiquitously expressed and localizes to the ER. Partial inhibition of EMC-6 caused decreased expression of heteromeric levamisole-sensitive AChRs by destabilizing unassembled subunits in the ER. Inhibition of emc-6 also reduced the expression of homomeric nicotine-sensitive AChRs and GABAA receptors in C. elegans muscle cells. emc-6 is orthologous to the yeast and human EMC6 genes that code for a component of the recently identified ER membrane complex (EMC). Our data suggest this complex is required for protein folding and is connected to ER-associated degradation. We demonstrated that inactivation of additional EMC members in C. elegans also impaired AChR synthesis and induced the unfolded protein response. These results suggest that the EMC is a component of the ER folding machinery. AChRs might provide a valuable proxy to decipher the function of the EMC further.

A secreted complement-control-related protein ensures acetylcholine receptor clustering.

Efficient neurotransmission at chemical synapses relies on spatial congruence between the presynaptic active zone, where synaptic vesicles fuse, and the postsynaptic differentiation, where neurotransmitter receptors concentrate. Diverse molecular systems have evolved to localize receptors at synapses, but in most cases, they rely on scaffolding proteins localized below the plasma membrane. A few systems have been suggested to control the synaptic localization of neurotransmitter receptors through extracellular interactions, such as the pentraxins that bind AMPA receptors and trigger their aggregation. However, it is not yet clear whether these systems have a central role in the organization of postsynaptic domains in vivo or rather provide modulatory functions. Here we describe an extracellular scaffold that is necessary to cluster acetylcholine receptors at neuromuscular junctions in the nematode Caenorhabditis elegans. It involves the ectodomain of the previously identified transmembrane protein LEV-10 (ref. 6) and a novel extracellular protein, LEV-9. LEV-9 is secreted by the muscle cells and localizes at cholinergic neuromuscular junctions. Acetylcholine receptors, LEV-9 and LEV-10 are interdependent for proper synaptic localization and physically interact based on biochemical evidence. Notably, the function of LEV-9 relies on eight complement control protein (CCP) domains. These domains, also called 'sushi domains', are usually found in proteins regulating complement activity in the vertebrate immune system. Because the complement system does not exist in protostomes, our results suggest that some of the numerous uncharacterized CCP proteins expressed in the mammalian brain might be directly involved in the organization of the synapse, independently from immune functions.

Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory.

Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The tomosyn-SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting tomosyn could be an important effector in PKA-dependent synaptic plasticity. We tested this hypothesis in Drosophila, in which the role of the PKA pathway in associative learning has been well established. We first determined that panneuronal tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. We next assayed memory performance 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that tomosyn is involved in a specific component of late associative memory.

Rebalancing the motor circuit restores movement in a Caenorhabditis elegans model for TDP-43 toxicity.

Amyotrophic lateral sclerosis can be caused by abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm of neurons. Here, we use a C. elegans model for TDP-43-induced toxicity to identify the biological mechanisms that lead to disease-related phenotypes. By applying deep behavioral phenotyping and subsequent dissection of the neuromuscular circuit, we show that TDP-43 worms have profound defects in GABA neurons. Moreover, acetylcholine neurons appear functionally silenced. Enhancing functional output of repressed acetylcholine neurons at the level of, among others, G-protein-coupled receptors restores neurotransmission, but inefficiently rescues locomotion. Rebalancing the excitatory-to-inhibitory ratio in the neuromuscular system by simultaneous stimulation of the affected GABA- and acetylcholine neurons, however, not only synergizes the effects of boosting individual neurotransmitter systems, but instantaneously improves movement. Our results suggest that interventions accounting for the altered connectome may be more efficient in restoring motor function than those solely focusing on diseased neuron populations.

An alpha-catulin homologue controls neuromuscular function through localization of the dystrophin complex and BK channels in Caenorhabditis elegans.

The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 Mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.

The epithelial Na+ channel UNC-8 promotes an endocytic mechanism that recycles presynaptic components to new boutons in remodeling neurons.

Circuit refinement involves the formation of new presynaptic boutons as others are dismantled. Nascent presynaptic sites can incorporate material from recently eliminated synapses, but the recycling mechanisms remain elusive. In early-stage C. elegans larvae, the presynaptic boutons of GABAergic DD neurons are removed and new outputs established at alternative sites. Here, we show that developmentally regulated expression of the epithelial Na+ channel (ENaC) UNC-8 in remodeling DD neurons promotes a Ca2+ and actin-dependent mechanism, involving activity-dependent bulk endocytosis (ADBE), that recycles presynaptic material for reassembly at nascent DD synapses. ADBE normally functions in highly active neurons to accelerate local recycling of synaptic vesicles. In contrast, we find that an ADBE-like mechanism results in the distal recycling of synaptic material from old to new synapses. Thus, our findings suggest that a native mechanism (ADBE) can be repurposed to dismantle presynaptic terminals for reassembly at new, distant locations.

A transcriptional program promotes remodeling of GABAergic synapses in Caenorhabditis elegans.

Although transcription factors are known to regulate synaptic plasticity, downstream genes that contribute to neural circuit remodeling are largely undefined. In Caenorhabditis elegans, GABAergic Dorsal D (DD) motor neuron synapses are relocated to new sites during larval development. This remodeling program is blocked in Ventral D (VD) GABAergic motor neurons by the COUP-TF (chicken ovalbumin upstream promoter transcription factor) homolog, UNC-55. We exploited this UNC-55 function to identify downstream synaptic remodeling genes that encode a diverse array of protein types including ion channels, cytoskeletal components, and transcription factors. We show that one of these targets, the Iroquois-like homeodomain protein, IRX-1, functions as a key regulator of remodeling in DD neurons. Our discovery of irx-1 as an unc-55-regulated target defines a transcriptional pathway that orchestrates an intricate synaptic remodeling program. Moreover, the well established roles of these conserved transcription factors in mammalian neural development suggest that a similar cascade may also control synaptic plasticity in more complex nervous systems.

Membrane penetration by synaptotagmin is required for coupling calcium binding to vesicle fusion in vivo.

The vesicle protein synaptotagmin I is the Ca(2+) sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca(2+) binding by the C(2)B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca(2+) binding results in vesicle fusion remains controversial. Ca(2+)-dependent interactions between synaptotagmin and SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effector-interaction mutations to date impact synaptic transmission as severely as mutation of the C(2)B Ca(2+)-binding motif, suggesting that these interactions are facilitatory rather than essential. Here we use Drosophila to show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca(2+)-binding pockets of synaptotagmin. Mutation of this residue in the C(2)A domain (F286) resulted in a ∼50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C(2)B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that in syt(null) mutants, which is more severe than the phenotype of C(2)B Ca(2+)-binding mutants. Thus, mutation of a single, C(2)B hydrophobic residue required for Ca(2+)-dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin function in vivo to date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C(2)B domain of synaptotagmin, is the critical effector interaction for coupling Ca(2+) binding with vesicle fusion.

The dystrophin-associated protein complex maintains muscle excitability by regulating Ca(2+)-dependent K(+) (BK) channel localization.

The dystrophin-associated protein complex (DAPC) consists of several transmembrane and intracellular scaffolding elements that have been implicated in maintaining the structure and morphology of the vertebrate neuromuscular junction (NMJ). Genetic linkage analysis has identified loss-of-function mutations in DAPC genes that give rise to degenerative muscular dystrophies. Although much is known about the involvement of the DAPC in maintaining muscle integrity, less is known about the precise contribution of the DAPC in cell signaling events. To better characterize the functional role of the DAPC at the NMJ, we used electrophysiology, immunohistochemistry, and fluorescent labeling to directly assess cholinergic synaptic transmission, ion channel localization, and muscle excitability in loss-of-function (lf) mutants of Caenorhabditis elegans DAPC homologues. We found that all DAPC mutants consistently display mislocalization of the Ca(2+)-gated K(+) channel, SLO-1, in muscle cells, while ionotropic acetylcholine receptor (AChR) expression and localization at the NMJ remained unaltered. Synaptic cholinergic signaling was also not significantly impacted across DAPC(lf) mutants. Consistent with these findings and the postsynaptic mislocalization of SLO-1, we observed an increase in muscle excitability downstream of cholinergic signaling. Based on our results, we conclude that the DAPC is not involved in regulating AChR architecture at the NMJ, but rather functions to control muscle excitability, in an activity-dependent manner, through the proper localization of SLO-1 channels.

A role for the Erk MAPK pathway in modulating SAX-7/L1CAM-dependent locomotion in Caenorhabditis elegans.

L1CAMs are immunoglobulin cell adhesion molecules that function in nervous system development and function. Besides being associated with autism and schizophrenia spectrum disorders, impaired L1CAM function also underlies the X-linked L1 syndrome, which encompasses a group of neurological conditions, including spastic paraplegia and congenital hydrocephalus. Studies on vertebrate and invertebrate L1CAMs established conserved roles that include axon guidance, dendrite morphogenesis, synapse development, and maintenance of neural architecture. We previously identified a genetic interaction between the Caenorhabditis elegans L1CAM encoded by the sax-7 gene and RAB-3, a GTPase that functions in synaptic neurotransmission; rab-3; sax-7 mutant animals exhibit synthetic locomotion abnormalities and neuronal dysfunction. Here, we show that this synergism also occurs when loss of SAX-7 is combined with mutants of other genes encoding key players of the synaptic vesicle (SV) cycle. In contrast, sax-7 does not interact with genes that function in synaptogenesis. These findings suggest a postdevelopmental role for sax-7 in the regulation of synaptic activity. To assess this possibility, we conducted electrophysiological recordings and ultrastructural analyses at neuromuscular junctions; these analyses did not reveal obvious synaptic abnormalities. Lastly, based on a forward genetic screen for suppressors of the rab-3; sax-7 synthetic phenotypes, we determined that mutants in the ERK Mitogen-activated Protein Kinase (MAPK) pathway can suppress the rab-3; sax-7 locomotion defects. Moreover, we established that Erk signaling acts in a subset of cholinergic neurons in the head to promote coordinated locomotion. In combination, these results suggest a modulatory role for Erk MAPK in L1CAM-dependent locomotion in C. elegans.

Regulated lysosomal trafficking as a mechanism for regulating GABAA receptor abundance at synapses in Caenorhabditis elegans.

GABA(A) receptor plasticity is important for both normal brain function and disease progression. We are studying GABA(A) receptor plasticity in Caenorhabditis elegans using a genetic approach. Acute exposure of worms to the GABA(A) agonist muscimol hyperpolarizes postsynaptic cells, causing paralysis. Worms adapt after several hours, but show uncoordinated locomotion consistent with decreased GABA signaling. Using patch-clamp and immunofluorescence approaches, we show that GABA(A) receptors are selectively removed from synapses during adaptation. Subunit mRNA levels were unchanged, suggesting a post-transcriptional mechanism. Mutants with defective lysosome function (cup-5) show elevated GABA(A) receptor levels at synapses prior to muscimol exposure. During adaptation, these receptors are removed more slowly, and accumulate in intracellular organelles positive for the late endosome marker GFP-RAB-7. These findings suggest that chronic agonist exposure increases endocytosis and lysosomal trafficking of GABA(A) receptors, leading to reduced levels of synaptic GABA(A) receptors and reduced postsynaptic GABA sensitivity.