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Neuropeptides in the brain have both excitatory and inhibitory effects on neuronal activity through paracrine signaling to local circuits. Here, we show that neuropeptides can activate and inhibit calcium responses in a single target cell of previously unknown function, hmc, in C. elegans. We show that hmc controls a discrete muscle contraction during a rhythmic behavior. Live imaging shows that hmc is activated by the neuropeptide like protein FLP-22 in phase with the pacemaker every 50 seconds. FLP-22 is released from a bifunctional motor neuron AVL and activates the frpr-17 G protein-coupled receptor (GPCR), which leads to the activation of a G alpha s-protein kinase A (PKA) signaling pathway in hmc. PKA signaling is not required for hmc activation but instead potentiates hmc activation. hmc activity is inhibited by signaling from the neuropeptide FLP-9, which functions through the GPCR frpr-21 in hmc. Behavioral, calcium imaging, and genetic studies suggest that hmc itself is not contractile but is functionally coupled to muscles through gap junctions composed of UNC-9/innexin. These results indicate that neuropeptides can function as volume neurotransmitters to control the activity state of a target cell and reveal a function for hmc that may be similar to that of endothelial cells.

Electrical synapses are specialized structures that mediate the flow of electrical currents between neurons and have well known roles in synchronizing the activities of neuronal populations, both by mediating the current transfer from more active to less active neurons and by shunting currents from active neurons to their less active neighbors. However, how these positive and negative functions of electrical synapses are coordinated to shape rhythmic synaptic outputs and behavior is not well understood. Here, using a combination of genetics, behavioral analysis, and live calcium imaging in Caenorhabditis elegans, we show that electrical synapses formed by the gap junction protein INX-1/innexin couple the presynaptic terminals of a pair of motor neurons (AVL and DVB) to synchronize their activation in response to a pacemaker signal. Live calcium imaging reveals that inx-1/innexin mutations lead to asynchronous activation of AVL and DVB, due, in part, to loss of AVL-mediated activation of DVB by the pacemaker. In addition, loss of inx-1 leads to the ectopic activation of DVB at inappropriate times during the cycle through the activation of the L-type voltage-gated calcium channel EGL-19. We propose that electrical synapses between AVL and DVB presynaptic terminals function to ensure the precise and robust execution of a specific step in a rhythmic behavior by both synchronizing the activities of presynaptic terminals in response to pacemaker signaling and by inhibiting their activation in between cycles when pacemaker signaling is low.

Arsenite is a naturally occurring mitochondrial toxin that generates oxidative stress in vivo. Previously, we have reported that SPHK-1/sphingosine kinase plays a critical role in promoting survival following arsenite exposure in C. elegans. However, the molecular mechanism by which SPHK-1 confers resistance to arsenite-induced toxicity is not known. Using a combination of genetic, fluorescence imaging, and behavioral approaches, we addressed the cellular and molecular mechanism by which SPHK-1 signaling protects animals from arsenite-induced lethality. SPHK-1 kinase activity and localization to mitochondria are required for protection from arsenite-induced lethality. Genetic alterations leading to low SPH levels promote resistance to arsenite-mediated toxicity. SPHK-1 functions in the PMK-1/p38 MAPK pathways in the intestine to promote protection. Finally, sphk-1 mutants are sensitive to a range of oxidative stressors including juglone, paraquat and heat. SPHK-1 and PMK-1 signaling play important roles in the oxidative stress response in C. elegans.

The mitochondrial unfolded protein response (UPR(mt)) is an evolutionarily conserved adaptive response that functions to maintain mitochondrial homeostasis following mitochondrial damage. In Caenorhabditis elegans, the nervous system plays a central role in responding to mitochondrial stress by releasing endocrine signals that act upon distal tissues to activate the UPR(mt). The mechanisms by which mitochondrial stress is sensed by neurons and transmitted to distal tissues are not fully understood. Here, we identify a role for the conserved follicle-stimulating hormone G protein-coupled receptor, FSHR-1, in promoting UPR(mt) activation. Genetic deficiency of fshr-1 severely attenuates UPR(mt) activation and organism-wide survival in response to mitochondrial stress. FSHR-1 functions in a common genetic pathway with SPHK-1/sphingosine kinase to promote UPR(mt) activation, and FSHR-1 regulates the mitochondrial association of SPHK-1 in the intestine. Through tissue-specific rescue assays, we show that FSHR-1 functions in neurons to activate the UPR(mt), to promote mitochondrial association of SPHK-1 in the intestine, and to promote organism-wide survival in response to mitochondrial stress. We propose that FSHR-1 functions cell nonautonomously in neurons to activate UPR(mt) upstream of SPHK-1 signaling in the intestine.

The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved adaptive response that functions to maintain mitochondrial homeostasis following mitochondrial damage. In C. elegans, the nervous system plays a central role in responding to mitochondrial stress by releasing endocrine signals that act upon distal tissues to activate the UPRmt. The mechanisms by which mitochondrial stress is sensed by neurons and transmitted to distal tissues is not fully understood. Here, we identify a role for the conserved follicle-stimulating hormone G protein coupled receptor (GPCR), FSHR-1, in promoting UPRmt activation. Genetic deficiency of fshr-1 severely attenuates UPRmt activation and organism-wide survival in response to mitochondrial stress. FSHR-1 functions in a common genetic pathway with SPHK-1/sphingosine kinase to promote UPRmt activation, and FSHR-1 regulates the mitochondrial association of SPHK-1 in the intestine. Through tissue-specific rescue assays, we show that FSHR-1 functions in neurons to activate the UPRmt, to promote mitochondrial association of SPHK-1 in the intestine, and to promote organism-wide survival in response to mitochondrial stress. We propose that FSHR-1 functions cell non-autonomously in neurons to activate UPRmt upstream of SPHK-1 signaling in the intestine.

The transcription factor Nrf2 plays a critical role in the organism wide-regulation of the antioxidant stress response. The Nrf2 homolog SKN-1 functions in the intestine cell non-autonomously to negatively regulate neuromuscular (NMJ) function inCaenorhabditis elegans. To identify additional molecules that mediate SKN-1 signaling to the NMJ, we performed a candidate screen for suppressors of aldicarb-resistance caused by acute treatment with the SKN-1 activator, arsenite. We identified two receptor tyrosine kinases, EGL-15 (fibroblast growth factor receptor, FGFR) and DAF-2 (insulin-like peptide receptor, IR) that are required for NMJ regulation in response to stress. Through double mutant analysis, we found that EGL-15 functions downstream of SKN-1 and SPHK-1 (sphingosine kinase), and that the EGL-15 ligand EGL-17 FGF and canonical EGL-15 effectors are required for oxidative stress-mediated regulation of NMJ function. DAF-2 also functions downstream of SKN-1, independently of DAF-16/FOXO, to regulate NMJ function. Through tissue-specific rescue experiments, we found that FGFR signaling functions primarily in the hypodermis, whereas IR signaling is required in multiple tissues. Our results support the idea that the regulation of NMJ function by SKN-1 occurs via a complex organism-wide signaling network involving RTK signaling in multiple tissues.

The Nrf2 antioxidant transcription factor promotes redox homeostasis in part through reciprocal signaling between neurons and neighboring cells, but the signals involved in intertissue signaling in response to Nrf2 activation are not well defined. InCaenorhabditis elegans, activation of SKN-1/Nrf2 in the intestine negatively regulates neuropeptide secretion from motor neurons. Here, we show that sphingosine kinase (SPHK-1) functions downstream of SKN-1/Nrf2 in the intestine to regulate neuropeptide secretion from motor neurons during the oxidative stress response inC. eleganshermaphrodites. SPHK-1 localizes to mitochondria in the intestine and SPHK-1 mitochondrial localization and kinase activity are essential for its function in regulating motor neuron function. SPHK-1 is recruited to mitochondria from cytosolic pools and its mitochondrial abundance is negatively regulated by acute or chronic SKN-1 activation. Finally, the regulation of motor function by SKN-1 requires the activation of the p38 MAPK cascade in the intestine and occurs through controlling the biogenesis or maturation of dense core vesicles in motor neurons. These findings show that the inhibition of SPHK-1 in the intestine by SKN-1 negatively regulates neuropeptide secretion from motor neurons, revealing a new mechanism by which SPHK-1 signaling mediates its effects on neuronal function in response to oxidative stress.SIGNIFICANCE STATEMENTNeurons are highly susceptible to damage by oxidative stress, yet have limited capacity to activate the SKN-1/Nrf2 oxidative stress response, relying instead on astrocytes to provide redox homeostasis. InCaenorhabditis elegans, intertissue signaling from the intestine plays a key role in regulating neuronal function during the oxidative stress response. Here, through a combination of genetic, behavioral, and fluorescent imaging approaches, we found that sphingosine kinase functions in the SKN-1/Nrf2 pathway in the intestine to regulate neuropeptide biogenesis and secretion in motor neurons. These results implicate sphingolipid signaling as a new component of the oxidative stress response and suggest thatC. elegansmay be a genetically tractable model to study non-cell-autonomous oxidative stress signaling to neurons.

Activity-dependent changes in presynaptic function represent a critical mechanism by which synaptic strength is controlled. However, how changes in synaptic activity couple to presynaptic components to control synaptic vesicle release and recycling are poorly understood. Sphingosine kinase (SphK) is a sphingolipid metabolic enzyme whose activity-dependent recruitment to membrane regions within presynaptic terminals promotes neurotransmitter release. Here, we show that synaptic recruitment of SPHK-1, the SphK ortholog inCaenorhabditis elegans, is mediated by presynaptic calcium influx. Quantitative fluorescence imaging of live presynaptic terminals reveals that blocking presynaptic calcium influx reduces synaptic SPHK-1 abundance whereas increasing calcium influx increases SPHK-1 synaptic abundance. CALM-1, the calcium and integrin binding protein ortholog, colocalizes with SPHK-1 at release sites and regulates muscarinic-mediated synaptic SPHK-1 recruitment. We identify two additional sphingolipid metabolic enzymes that are concentrated at presynaptic terminals, and mutants lacking one of these, HYL-1/ceramide synthase, have defects in synaptic transmission and in synaptic vesicle cycling. Finally, we show that SPHK-1 activity is required for the recruitment of the priming protein UNC-13/Munc13 to presynaptic terminals following activation by muscarinic signaling. These findings suggest that calcium-dependent regulation of local S1P metabolism at synapses may be an important mechanism by which synaptic vesicle priming factors are recruited to release sites to promote synaptic transmission.

Aging is marked by a collapse of protein homeostasis and deterioration of adaptive stress responses that often lead to disease. During aging, the induction of stress responses decline along with protein quality control. Here, we have shown that the ability to mount an adaptive response by pretreatment with minor oxidative stress is abrogated in aged Caenorhabditis elegans. We have identified a defect in SKN-1 signaling sensitivity during aging and have also found an aging-related increase in basal proteasome expression and in vitro activity, however, adaptation of the 20S proteasome in response to stress is lost in old animals. Interestingly, increased activation of SKN-1 promotes stress resistance, but is unable to rescue declining adaptation during aging. Our data demonstrate that the aging-dependent decline in SKN-1 signaling negatively impacts adaptation of the 20S proteasome in response to acute oxidative stress.

Sphingolipids are potent lipid second messengers that regulate cell differentiation, migration, survival, and secretion, and alterations in sphingolipid signaling have been implicated in a variety of diseases. However, how sphingolipid levels are regulated, particularly in the nervous system, remains poorly understood. Here, we show that the generation of sphingosine-1-phosphate by sphingosine kinase (SphK) promotes neurotransmitter release. Electrophysiological, imaging, and behavioral analyses of Caenorhabditis elegans mutants lacking sphingosine kinase sphk-1 indicate that neuronal development is normal, but there is a significant defect in neurotransmitter release from neuromuscular junctions. SPHK-1 localizes to discrete, nonvesicular regions within presynaptic terminals, and this localization is critical for synaptic function. Muscarinic agonists cause a rapid increase in presynaptic SPHK-1 abundance, whereas reduction of endogenous acetylcholine production results in a rapid decrease in presynaptic SPHK-1 abundance. Muscarinic regulation of presynaptic SPHK-1 abundance is mediated by a conserved presynaptic signaling pathway composed of the muscarinic acetylcholine receptor GAR-3, the heterotrimeric G protein Gαq, and its effector, Trio RhoGEF. SPHK-1 activity is required for the effects of muscarinic signaling on synaptic transmission. This study shows that SPHK-1 promotes neurotransmitter release in vivo and identifies a novel muscarinic pathway that regulates SphK abundance at presynaptic terminals.

Chemical synapses are complex structures that mediate rapid intercellular signalling in the nervous system. Proteomic studies suggest that several hundred proteins will be found at synaptic specializations. Here we describe a systematic screen to identify genes required for the function or development of Caenorhabditis elegans neuromuscular junctions. A total of 185 genes were identified in an RNA interference screen for decreased acetylcholine secretion; 132 of these genes had not previously been implicated in synaptic transmission. Functional profiles for these genes were determined by comparing secretion defects observed after RNA interference under a variety of conditions. Hierarchical clustering identified groups of functionally related genes, including those involved in the synaptic vesicle cycle, neuropeptide signalling and responsiveness to phorbol esters. Twenty-four genes encoded proteins that were localized to presynaptic specializations. Loss-of-function mutations in 12 genes caused defects in presynaptic structure.

Genetic studies have suggested a functional link between cholesterol/sphingolipid metabolism and endocytic membrane traffic. Here we show that perturbing the cholesterol/sphingomyelin balance in the plasma membrane results in the massive formation of clusters of narrow endocytic tubular invaginations positive for N-BAR proteins. These tubules are intensely positive for sphingosine kinase 1 (SPHK1). SPHK1 is also targeted to physiologically occurring early endocytic intermediates, and is highly enriched in nerve terminals, which are cellular compartments specialized for exo/endocytosis. Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface. The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme. Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

Acetylcholine (ACh) is a potent neuromodulator in the brain, and its effects on cognition and memory formation are largely performed through muscarinic acetylcholine receptors (mAChRs). mAChRs are often preferentially distributed on specialized membrane regions in neurons, but the significance of mAChR localization in modulating neuronal function is not known. Here we show that the Caenorhabditis elegans homolog of the M1/M3/M5 family of mAChRs, gar-3, is expressed in cholinergic motor neurons, and GAR-3-GFP fusion proteins localize to cell bodies where they are enriched at extrasynaptic regions that are in contact with the basal lamina. The GAR-3 N-terminal extracellular domain is necessary and sufficient for this asymmetric distribution, and mutation of a predicted N-linked glycosylation site within the N-terminus disrupts GAR-3-GFP localization. In transgenic animals expressing GAR-3 variants that are no longer asymmetrically localized, synaptic transmission at neuromuscular junctions is impaired and there is a reduction in the abundance of the presynaptic protein sphingosine kinase at release sites. Finally, GAR-3 can be activated by endogenously produced ACh released from neurons that do not directly contact cholinergic motor neurons. Together, our results suggest that humoral activation of asymmetrically localized mAChRs by ACh is an evolutionarily conserved mechanism by which ACh modulates neuronal function.

The Nrf family of transcription factors mediates adaptive responses to stress and longevity, but the identities of the crucial Nrf targets, and the tissues in which they function in multicellular organisms to promote survival, are not known. Here, we use whole transcriptome RNA sequencing to identify 810 genes whose expression is controlled by the SKN-1/Nrf2 negative regulator WDR-23 in the nervous system of Caenorhabditis elegans. Among the genes identified is the synaptic cell adhesion molecule nlg-1/neuroligin. We find that the synaptic abundance of NLG-1 protein increases following pharmacological treatments that generate oxidative stress or by the genetic activation of skn-1. Increasing nlg-1 dosage correlates with increased survival in response to oxidative stress, whereas genetic inactivation of nlg-1 reduces survival and impairs skn-1-mediated stress resistance. We identify a canonical SKN-1 binding site in the nlg-1 promoter that binds to SKN-1 in vitro and is necessary for SKN-1 and toxin-mediated increases in nlg-1 expression in vivo. Together, our results suggest that SKN-1 activation in the nervous system can confer protection to organisms in response to stress by directly regulating nlg-1/neuroligin expression., Author Summary Organisms have evolved mechanisms to protect themselves at the cellular level in response to a variety of environmental stresses. Oxidative stress, caused by an imbalance in the cellular production of free radicals and endogenous antioxidant defenses, can be particularly detrimental to the nervous system. Indeed, elevated levels of oxidative stress have been linked to nearly all neurodegenerative diseases. Therefore, understanding how living creatures protect themselves against oxidative stress, from a cellular to a systemic level, is vital. Here, we have found that increased stress activates a pathway that increases the amount of certain proteins found at neuronal synapses. This presents an interesting model in which, in response to stress, neurons might attempt to enhance the strength of the synapse to prevent degeneration.

Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels., Author Summary Breathing, walking and sleeping, are examples of rhythmic behaviors that occur at regular time intervals. The time intervals are determined by pacemakers, which generate the rhythms, and the behaviors are carried out by different tissues such as neurons and muscles. How do timing signals from pacemakers get delivered to target tissues to ensure proper execution of these behaviors? To begin to address this question, we study a simple rhythmic behavior in the nematode C. elegans called the defecation motor program. In this behavior, enteric muscles contract every 50 seconds, allowing digested food to be expelled from the gut. The pacemaker is the gut itself, and here we identify a specific protein, PKA, that responds to the signal from the pacemaker by activating certain neurons that trigger enteric muscle contraction. We further demonstrate that PKA activates these neurons by controlling the entry of calcium into these neurons. We also identify two calcium channels that allow calcium to enter the neurons when PKA is activated by the signal from the pacemaker. Our results raise the possibility that PKA-mediated calcium entry might be a mechanism used in other organisms to regulate rhythmic behaviors.

Mycobacterium tuberculosis and its antigens are potent inducers of cytokine expression by mononuclear phagocytes. In this study, the ability of live M. tuberculosis to stimulate interleukin-12 (IL-12) expression by human monocytes was examined. Monocytes were purified from peripheral blood mononuclear cells by adherence and either infected with M. tuberculosis or exposed to soluble protein antigens of M. tuberculosis (purified protein derivative [PPD]). Live M. tuberculosis (10(6) to 10(7) CFU/ml) was a potent stimulus for interleukin-12 (IL-12). By using reverse transcription-PCR, p40 mRNA was detected at 3 h, peaked at 6 to 12 h, and decayed to baseline levels at 18 to 24 h following infection. Bioactive IL-12 (p70) was measured by the phytohemagglutinin blast proliferation assay and confirmed the p40 mRNA results. In contrast, soluble PPD at concentrations known to readily induce IL-1 and tumor necrosis factor alpha expression by monocytes (10 to 100 microg/ml) was a poor stimulus for IL-12 p40 mRNA expression. The different efficiencies of M. tuberculosis bacilli and PPD for IL-12 expression by monocytes was in part due to a requirement for phagocytosis. Induction of IL-12 in response to M. tuberculosis was reduced by cytochalasin D. Furthermore, phagocytosis of dead M. tuberculosis or inert 2-micron-diameter polystyrene beads by monocytes induced IL-12 p40 mRNA. In contrast, 0.5-micron-diameter beads, which can enter cells through pinocytosis, did not stimulate IL-12 expression. Functionally, IL-12 readily enhanced PPD-stimulated IFN-gamma production and CD4+ T-cell-mediated cytotoxicity by peripheral blood mononuclear cells from healthy tuberculin-positive donors but induced less enhancement when live M. tuberculosis was the antigen. These results suggest that IL-12 is upregulated as part of the early cytokine response of mononuclear phagocytes to M. tuberculosis and that the cellular events associated with phagocytosis are themselves a potent signal for IL-12 production. IL-12 released by infected macrophages in turn can further upregulate M. tuberculosis-specific CD4+ T-cell effector function.

In mammalian cells, hydrogen peroxide (H(2)O(2))-induced adaptation to oxidative stress is strongly dependent on an Nrf2 transcription factor-mediated increase in the 20S proteasome. Here, we report that both Caenorhabditis elegans nematode worms and Drosophila melanogaster fruit flies are also capable of adapting to oxidative stress with H(2)O(2) pre-treatment. As in mammalian cells, this adaptive response in worms and flies involves an increase in proteolytic activity and increased expression of the 20S proteasome, but not of the 26S proteasome. We also found that the increase in 20S proteasome expression in both worms and flies, as in mammalian cells, is important for the adaptive response, and that it is mediated by the SKN-1 and CNC-C orthologs of the mammalian Nrf2 transcription factor, respectively. These studies demonstrate that stress mechanisms operative in cell culture also apply in disparate intact organisms across a wide biological diversity.

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV–mediated secretion., Author Summary Neuropeptides produce prolonged changes in circuit activity that are associated with changes in behavioral states (e.g. mood or appetite); consequently, there is great interest in identifying molecules that are required for neuropeptide secretion. Here we show that a novel neuronal protein RIC-7 promotes neuropeptide secretion in C. elegans but has only subtle effects on neurotransmitter secretion. RIC-7 is conserved in several other nematodes; however, homologous proteins are not found in other sequenced genomes. These results suggest that the machinery responsible for neuropeptide secretion evolved more recently than factors that are required for both neurotransmitter and neuropeptide secretion.

Protein misfolding is a central mechanism for the development of neurodegenerative diseases and type 2 diabetes mellitus. The accumulation of misfolded alpha-synuclein protein inclusions in the Lewy bodies of Parkinson's disease is thought to play a key role in pathogenesis and disease progression. Similarly, the misfolding of the beta-cell hormone human islet amyloid polypeptide (h-IAPP) into toxic oligomers plays a central role in the induction of beta-cell apoptosis in the context of type 2 diabetes. In this study, we show that annexin A5 plays a role in interacting with and reducing the toxicity of the amyloidogenic proteins, h-IAPP and alpha-synuclein. We find that annexin A5 is coexpressed in human beta-cells and that exogenous annexin A5 reduces the level of h-IAPP-induced apoptosis in human islets by approximately 50% and in rodent beta-cells by approximately 90%. Experiments with transgenic expression of alpha-synuclein in Caenorhabditis elegans show that annexin A5 reduces alpha-synuclein inclusions in vivo. Using thioflavin T fluorescence, electron microscopy, and electron paramagnetic resonance, we provide evidence that substoichiometric amounts of annexin A5 inhibit h-IAPP and alpha-synuclein misfolding and fibril formation. We conclude that annexin A5 might act as a molecular safeguard against the formation of toxic amyloid aggregates.

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling., Author Summary Cells are divided into multiple subcellular compartments that perform diverse functions. In neurons, synapses mediate transmission of information between cells and they comprise hundreds of proteins dedicated for this purpose. Changes in the protein composition of synapses are thought to produce changes in synaptic transmission, such as those that occur during development, learning, and memory. Here, we describe a systematic genetic strategy for analyzing the protein composition of synapses. Using this strategy, we identified sets of genes that alter synapses in similar ways, and identified novel regulatory relationships between particular synaptic proteins. One set of genes regulated secretion of insulin-like hormones from neurons and had corresponding effects on lifespan, which is controlled by insulin signaling. These results illustrate how changes in synaptic composition can be utilized as a probe to explain changes in physiology. Our approach can be expanded to include a larger set of synaptic proteins or to analyze other subcellular compartments.

The secretion of neurotransmitters and neuropeptides is mediated by distinct organelles-synaptic vesicles (SVs) and dense-core vesicles (DCVs), respectively. Relatively little is known about the factors that differentially regulate SV and DCV secretion. Here we show that protein kinase C-1 (PKC-1), which is most similar to the vertebrate PKC eta and epsilon isoforms, regulates exocytosis of DCVs in Caenorhabditis elegans motor neurons. Mutants lacking PCK-1 activity had delayed paralysis induced by the acetylcholinesterase inhibitor aldicarb, whereas mutants with increased PKC-1 activity had more rapid aldicarb-induced paralysis. Imaging and electrophysiological assays indicated that SV release occurred normally in pkc-1 mutants. By contrast, genetic analysis of aldicarb responses and imaging of fluorescently tagged neuropeptides indicated that mutants lacking PKC-1 had reduced neuropeptide secretion. Similar neuropeptide secretion defects were found in mutants lacking unc-31 (encoding the protein CAPS) or unc-13 (encoding Munc13). These results suggest that PKC-1 selectively regulates DCV release from neurons.

We describe evidence that a regulatory B subunit of protein phosphatase 2A (PP2A) positively regulates an RTK–Ras–MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although reduction of sur-6 PP2A-B function causes few vulval induction defects in an otherwise wild-type background, sur-6 PP2A-B mutations suppress the Multivulva phenotype of an activated ras mutation and enhance the Vulvaless phenotype of mutations in lin-45 raf, sur-8, or mpk-1. Double mutant analysis suggests that sur-6 PP2A-B acts downstream or in parallel to ras, but likely upstream of raf, and functions with ksr-1 in a common pathway to positively regulate Ras signaling.

RAS GENES ENCODE SMALL GTPase proteins, which when mutated, have been shown to result in breast cancer as well as cancers in a variety of other tissues. The study of Caenorhabditis elegans and other model systems has demonstrated that Ras is part of a conserved Ras/MAPK signaling pathway involved in many aspects of development and cell regulation. The C. elegans vulva is induced by an EGF like signal that activates the Ras/MAPK pathway. Constitutively active alleles of ras lead to hyperactivity of the signal transduction pathway and result in a multivulva (Muv) phenotype where numerous pseudovulvae are formed from vulval precursor cells (VPCs). By initiating suppressor screens of activated let-60 ras, many previously unknown components of this pathway have been identified. One gene, sur-9, has been defined by a semi-dominant allele isolated in a screen for temperature sensitive mutations that suppress the let-60(n1046) allele. While homozygous, sur-9(ku258) can suppress let-60(n1046) from 80% Muv to

The Nrf family of transcription factors plays a critical role in mediating adaptive responses to cellular stress and defends against neurodegeneration, aging, and cancer. Here, we report a novel role for the Caenorhabditis elegans Nrf homolog SKN-1 in regulating synaptic transmission at neuromuscular junctions (NMJs). Activation of SKN-1, either by acute pharmacological treatment with the mitochondrial toxin sodium arsenite or by mutations that cause constitutive SKN-1 activation, results in defects in neuromuscular function. Additionally, elimination of the conserved WD40 repeat protein WDR-23, a principal negative regulator of SKN-1, results in impaired locomotion and synaptic vesicle and neuropeptide release from cholinergic motor axons. Mutations that abolish skn-1 activity restore normal neuromuscular function to wdr-23 mutants and animals treated with toxin. We show that negative regulation of SKN-1 by WDR-23 in the intestine, but not at neuromuscular junctions, is necessary and sufficient for proper neuromuscular function. WDR-23 isoforms differentially localize to the outer membranes of mitochondria and to nuclei, and the effects of WDR-23 on neuromuscular function are dependent on its interaction with cullin E3 ubiquitin ligase. Finally, whole-transcriptome RNA sequencing of wdr-23 mutants reveals an increase in the expression of known SKN-1/Nrf2-regulated stress-response genes, as well as neurotransmission genes not previously implicated in SKN-1/Nrf2 responses. Together, our results indicate that SKN-1/Nrf2 activation may be a mechanism through which cellular stress, detected in one tissue, affects cellular function of a distal tissue through endocrine signaling. These results provide insight into how SKN-1/Nrf2 might protect the nervous system from damage in response to oxidative stress., Author Summary Transcriptional programs control cellular responses in the face of environmental stress, such as dietary restriction, hypoxia, or oxidative stress. Furthermore, in order to promote survival of the organism in response to insult, communication between tissues must be established. Using the model system C. elegans, we investigate functional changes in the nervous system mediated by the transcription factor SKN-1. We establish that activation of SKN-1, either genetically or through exposure to the mitochondrial toxin arsenite, results in locomotion changes that take place at the neuromuscular junction. Furthermore, these changes in the nervous system are brought about through signaling from the intestine. Lastly, we use whole-transcriptome RNA sequencing to identify new transcriptional targets of SKN-1 that might be affecting locomotory behavior. Our results indicate that neuronal function can be regulated at the level of the synapse in response to environmental stress.

Interleukin (IL)-12 was cloned on the basis of its ability to activate natural killer (NK) cells and promote the development of cytolytic T cells. With further understanding of its activities, IL-12 has emerged as an important cytokine, affecting both immune and hematologic functions. It has been shown to be necessary for the T cell independent induction of interferon (IFN)-gamma, critical for the initial suppression of bacterial and parasitic infection; for the development of a Th1 response, critical for effective host defense against intracellular pathogens; and for the activation of differentiated T lymphocytes of both CD4+ and CD8+ phenotype. IL-12 thus functions to activate and to link the innate and acquired immune responses. The therapeutic potential of these activities is suggested by studies in tumor and microbial models. IL-12 has suppressed tumor growth in all murine models examined. Antimicrobial activity has been demonstrated in bacterial, yeast, parasitic, and viral models of infection. In many of these models, activity has been linked to production of IFN-gamma and, in the parasite model, to development of a Th1 response. In addition to the therapeutic potential associated with IL-12 activity in these disease models, the understanding of its role in immune development and interaction with other cytokines, particularly antagonists, such as IL-4 and IL-10, has clarified and extended our understanding of immune regulation and should lead to significant developments in understanding the progression of AIDS and the development of vaccine adjuvants able to direct the immune response.

Resistance to Leishmania major in mice is associated with the appearance of distinct T helper type 1 (Th1) and Th2 subsets. T cells from lymph nodes draining cutaneous lesions of resistant mice are primarily interferon gamma (IFN-gamma)-producing Th1 cells. In contrast, T cells from susceptible mice are principally Th2 cells that generate interleukin 4 (IL-4). Although existing evidence is supportive of a role for IFN-gamma in the generation of Th1 cells, additional factors may be required for a protective response to be maintained. A potential candidate is IL-12, a heterodimeric cytokine produced by monocytes and B cells that has multiple effects on T and natural killer cell function, including inducing IFN-gamma production. Using an experimental leishmanial model we have observed that daily intraperitoneal administration at the time of parasite challenge of either 0.33 micrograms IL-12 (a consecutive 5 d/wk for 5 wk) or 1.0 micrograms IL-12 per mouse (only a consecutive 5 d) caused a > 75% reduction in parasite burden at the site of infection, in highly susceptible BALB/c mice. Delay of treatment by 1 wk had less of a protective effect. Concomitant with these protective effects was an increase in IFN-gamma and a decrease in IL-4 production, as measured by enzyme-linked immunosorbent assay of supernatants generated from popliteal lymph node cells stimulated with leishmanial antigen in vitro. The reduction in parasite numbers induced by IL-12 therapy was still apparent at 10 wk postinfection. In addition, we observed that the administration of a rabbit anti-recombinant murine IL-12 polyclonal antibody (200 micrograms i.p. every other day for 25 d) at the time of infection to resistant C57Bl/6 mice exacerbated disease. These effects were accompanied by a shift in IFN-gamma production in vitro by antigen-stimulated lymph node cells indicative of a Th2-like response. These findings suggest that IL-12 has an important role in initiating a Th1 response and protective immunity.

We describe the identification and characterization of a novel gene, sur-8, that positively regulates Ras-mediated signal transduction during C. elegans vulval development. Reduction of sur-8 function suppresses an activated ras mutation and dramatically enhances phenotypes of mpk-1 MAP kinase and ksr-1 mutations, while increase of sur-8 dosage enhances an activated ras mutation. sur-8 appears to act downstream of or in parallel to ras but upstream of raf. sur-8 encodes a conserved protein that is composed predominantly of leucine-rich repeats. The SUR-8 protein interacts directly with Ras but not with the Ras(P34G) mutant protein, suggesting that SUR-8 may mediate its effects through Ras binding. A structural and functional SUR-8 homolog in humans specifically binds K-Ras and N-Ras but not H-Ras in vitro.