Pirri, Jennifer K et al. (2013) International Worm Meeting "Synaptic engineering: an ionic switch of behavior."

Alkema, Mark J, Rayes, Diego, Pirri, Jennifer K

[International Worm Meeting, 2013]

The unraveling of the human brain connectivity map is considered by many as an essential step in the understanding how the brain controls behavior and how brain malfunction underlies behavioral disorders. Although the behavioral output of neural networks depends on a delicate balance between excitatory and inhibitory synaptic connections, the neural connectivity map does not carry information about the sign of synaptic connections.. Is it possible to reverse the behavioral output of a neural circuit by changing the sign of a synapse? Does the sign of a synapse provide constraints to the development, the specification, and behavioral output of a connectome? Here, we address these questions using the neuronal circuit that mediates the C. elegans escape response. In the escape response a single pair of tyraminergic neurons coordinate the suppression of head movements with backward locomotion through the activation of an inhibitory postsynaptic receptor, the tryamine-gated chloride channel LGC-55. LGC-55 is a member of the Cys-loop ligand gated ion channel (LGIC) family, whose ion selectivity is determined by the M2 domain. Amino acid substitution of the M2 domain LGC-55 allowed us to change the selectivity from Cl- to Na+ and K+. We generated transgenic animals that express the excitatory LGC-55 cation channel under control of its endogenous promoter in an LGC-55 anion mutant background. A fluorescently tagged LGC-55 cation channel localizes opposite to tyramine release sites indistinguishable from the wild-type LGC-55 anion channel. Exogenous tyramine induces neck muscle relaxation and backward locomotion in the wild-type LGC-55 anion animals, but induces neck muscle contraction and forward locomotion in transgenic animals that express the LGC-55 cation. Similarly, touch or optogentically induced release of endogenous tyramine triggers opposite behavioral responses in animals that express the LGC-55 cation vs LGC-55 anion channel. Our data show that changing the nature of a synapse within a neural circuit can reverse its behavioral output and indicate that the C. elegans connectome is established independent of the nature of synaptic activity or behavioral output.

Hernando, Guillermina S. et al. (2011) International Worm Meeting "Contribution of LEV-8 Subunit to the Kinetics of Activation and Desensitization of C. elegans Muscle Levamisole-Sensitive Nicotinic Receptors."

Hernando, Guillermina S., Bouzat, Cecilia B., Rayes, Diego H.

[International Worm Meeting, 2011]

Caenorhabditis elegans is sensitive to the majority of anthelmintic drugs that are used against parasitic worm infections of humans and livestock. The muscle levamisole-sensitive AChR (L-AChR) is a target of several anthelmintic drugs, such as levamisole, pyrantel and morantel. L-AChRs from C. elegans muscle appear to be composed of three essential (UNC-63, UNC-38, and UNC-29) and two accessory subunits (LEV-1 and LEV-8). We here explored at the single-channel and macroscopic current levels the contribution of the alpha-type LEV-8 subunit to the kinetics of activation and desensitization of L-AChRs from C. elegans muscle. To this end, we used a primary culture method that allows differentiation of C. elegans embryonic cells into larva 1 muscle cells. Single-channel activity of L-AChRs can be readily detected from muscle cells derived from LEV-8 null mutant strain (lev-8(x-15)), thus confirming that LEV-8 is not an essential subunit. Channel conductance is similar to that of wild-type L-AChRs (~36 pS). In contrast, the duration of the slowest open component differs from that of wild-type L-AChRs, being about 3-fold more prolonged (0.28 plus or minus 0.06 and 0.91 plus or minus 0.3 ms for wild-type and mutant, respectively). A dramatic difference between recordings activated by ACh from LEV-8 null mutant and wild-type muscle cells is a time-dependent reduction in the frequency of opening events. Thus, whereas channel activity remains constant during the course of the recording in wild-type cells, it disappears within the first three minutes in the null mutant. Such a reduction is compatible with enhanced desensitization. To investigate this, we recorded macroscopic currents elicited by rapid application of ACh or levamisole in the whole-cell configuration. Our results show that the mean amplitude of currents from the null mutant is similar to that from wild-type strain. However, the decay time constant is ~4-fold reduced in the mutant, indicating faster desensitization. In addition, the steady state current, which represents receptors that remain active during the agonist-pulse, is smaller in the null mutant (41 plus or minus 10% and 19 plus or minus 10% of the peak current for wild-type and mutant, respectively). Taken together, our results reveal that L-AChRs lacking LEV-8 show increased open channel lifetime and enhanced desensitization. Thus, the properties of L-AChRs-mediated responses in muscle cells can be substantially changed whether or not the accessory LEV-8 subunit is incorporated into the pentameric receptor.

Andersen, Natalia et al. (2021) International Worm Meeting "Protective roles of imidazolium salts in C. elegans models of stress and neurodegeneration"

Andersen, Natalia, Silbestri, Gustavo, Veuthey, Tania, De Rosa, Maria Jose, Rayes, Diego

[International Worm Meeting, 2021]

In this study, we aim to evaluate the role of imidazolium salts as antioxidant and anti-aging agents. We synthesized imidazolium salts and use the nematode C. elegans to perform a screening and analyze their ability to improve oxidative stress resistance. We identified a derivate, 1-Mesithyl-3-(3-sulfonatopropyl)imidazolium (MSI), that enhances animal resistance to oxidative stress. As a first approach to delineate its mechanism of action, we evaluated MSI ability to activate transcription factors involved in cytoprotective stress responses, such as the DAF-16/FOXO and SKN-1/Nrf2 pathways. We found that MSI stress protection was not dependent on DAF-16. Nevertheless, we discovered that GST-4 detoxifying enzyme, a downstream effector of SKN-1 transcription factor, is involved in MSI-mediated oxidative stress resistance. Oxidative stress has been largely related with aging and neurodegeneration. To gain further insight into MSI role in proteostasis, we evaluated mobility as an indicator of healthspan in Huntington's, Parkinson's and Alzheimer's disease models. We found that MSI ameliorates mobility rate decline in these proteotoxic models of neurodegenerative diseases. Surprisingly, our results show that MSI did not improve mean lifespan neither in wild-type worms nor in Alzheimer's disease animal models. Overall, our results show a scenario where healthspan seems to be uncoupled to lifespan. Additional research is needed to underpin the mechanism responsible for MSI's protective role.

Veuthey, Tania et al. (2021) International Worm Meeting "Tyramine modulates the systemic stress response by stimulating the release of intestinal insulin like-peptides (ILPs)"

Rayes, Diego, Alkema, Mark, Giunti, Sebastian, Veuthey, Tania, De Rosa, Maria Jose, Florman, Jeremy

[International Worm Meeting, 2021]

Multicellular organisms trigger a complex and coordinated response against systemic stress. We have recently shown that in C. elegans, the neural stress-hormone tyramine supplies a state-dependent neural switch between acute flight- and long-term environmental-stress responses (De Rosa et al, 2019). Tyramine release during the flight response, stimulates the DAF-2/Insulin/IGF-1 signaling (IIS) pathway and precludes the nuclear translocation of the DAF-16/FOXO transcription factor through the activation of an adrenergic-like receptor TYRA-3 in the intestine. We hypothesize that tyramine stimulates the release of agonist ILPs from the intestine which acts as an autocrine and/or paracrine signal to systemically activate the DAF-2/IIS pathway. To test this hypothesis we are screening ILPs mutants for their resistance to environmental stressors (oxidative and thermal stress). The C. elegans genome encodes 40 ILPs, 28 of which expressed in the intestine. We performed a screening of intestinal peptides described as strong DAF-2 agonist, by silencing individual intestinal ILPs and testing worm resistance to environmental stressors (oxidative and thermal stress). Thus, so far we found that ins-3 and ins-7 mutants are resistant to environmental stress, like tyramine-deficient and tyra-3 mutants. We are further testing whether tyramine directly stimulates the release of these ILPs from the intestine through a Gq-protein mediated signaling pathway. These studies will provide insight into how a neurohormone coordinates systemic cellular stress responses.

Christopher J Cronin et al. (2004) West Coast Worm Meeting "The Combined UC San Diego/Caltech Quantitative Behavior Analysis System"

William Schafer, Christopher J Cronin, Zhaoyang Feng, Paul W Sternberg

[West Coast Worm Meeting, 2004]

California Institute of Technology and University of California, San Diego have each developed practical, automated movement analysis systems to quantify aspects of worm behavior and morphology (see previous meeting abstracts). To better serve the Worm Research Community, we have combined our development efforts and merged our independent system designs to create a single, unified behavior analysis system. Our combined system provides the researcher with all of the quantification tools and database functionality of the individual systems, plus provides the framework for continued future development to satisfy the needs of the community. A list of the necessary hardware is available upon request.

Kamat, Shaunak et al. (2013) International Worm Meeting "Modulation of mec-10(d)-induced necrosis by ER chaperone NRA-2."

Driscoll, Monica, Yeola, Shrutika, Bianchi, Laura, Kamat, Shaunak

[International Worm Meeting, 2013]

ER chaperones play a major role in cellular homeostasis by regulating the assembly of polypeptides, intracellular Ca2+ signaling, and degradation of misfolded proteins. Here we report on an ER - resident chaperone NRA-2 that modulates hyper-activated ion channel-induced necrosis by regulating surface expression of a death-inducing DEG/ENaC channel family member subunit MEC-10(d). nra-2 was previously identified in a screen for nicotinic acetylcholine receptor (nAChR) - interacting proteins and was found to regulate the subunit composition of the AChR receptor in C. elegans muscle1. We found that loss of function of nra-2 led to a significant increase in mec-10(d) - induced necrosis in C. elegans touch receptor neurons (TRNs), and this enhancement was rescued by TRN-specific transgenic expression of nra-2. We observed that loss of nra-2 led to a significant increase in surface localization of MEC-10(d)::GFP and a significant decrease in ER localization. Electrophysiological experiments in Xenopus oocytes revealed that NRA-2 suppresses amiloride-sensitive currents induced by hyperactivated MEC channels. Our study suggests a role for NRA-2 as an ER quality control protein that inhibits surface expression of mutant MEC-10(d) channels and is thus neuroprotective against hyperactivated ion channel-induced necrosis. 1. Ruta B. Almedom, Jana F. Liewald, Guillermina Hernando, Christian Schultheis, Diego Rayes, Jie Pan, Thorsten Schedletzky, Harald Hutter, Cecilia Bouzat, and Alexander Gottschalk. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse. The EMBO Journal, 28(17):2636-2649, July 2009.

Huang, Yung-Chi et al. (2013) International Worm Meeting "Worm migraines: Characterization of a Gain-of-Function Mutation in the Voltage-Gated Calcium Channel, UNC-2/CaV2."

Huang, Yung-Chi, Pirri, Jennifer K., Bargmann, Cornelia I., Rayes, Diego, Saheki, Yasunori, Francis, Michael M., Alkema, Mark J.

[International Worm Meeting, 2013]

The fusion of synaptic vesicles with the cell membrane is initiated by the activation of presynaptic voltage-gated calcium channels (CaV2). C. elegans has a single predicted presynaptic CaV2a subunit, encoded by the unc-2 gene. We have identified an unc-2 gain-of-function (gf) mutant, which displays an increased reversal frequency, as well as hyperactive locomotion and egg-laying. In contrast, unc-2 loss-of-function mutants are uncoordinated and sluggish. Transgenic animals that express the unc-2(gf) in the backward locomotion interneurons exhibit an increased reversal frequency, while expression in the hermaphrodite specific neurons (HSN) produces hyperactive egg-laying. This suggests that the unc-2(gf) transgene can be used to chronically hyper-activate selective neurons within a neural circuit. unc-2(gf) mutants are hypersensitive to the acetylcholine esterase inhibitor, aldicarb and show an increased frequency of endogenous synaptic release events, indicating elevated levels of neurotransmitter release. Whole cell recordings from HEK cells expressing the human CaV2.1a with the corresponding unc-2(gf) mutation revealed activation at a lower membrane potential and a higher current density than the wild type. Our findings are similar to those reported for mutations in the human CaV2.1a, CACNA1A, that cause Familial Hemiplegic Migraine (FHM1). Interestingly, transgenic animals that express unc-2 carrying corresponding FHM1 mutations display a hyperactive phenotype similar to that of the unc-2(gf) mutants. Therefore, the unc-2(gf) mutants provide an invertebrate model to study mechanisms underlying FHM1. To understand mechanisms of CaV2 function in vivo, we performed a screen for mutants that suppress the hyperactive phenotype of unc-2(gf) mutants. Genes identified from this suppressor screen may ultimately provide novel targets for the treatments of calcium channelopathies.

Christopher J Cronin et al. (2005) International Worm Meeting "The Combined UC San Diego/Caltech Quantitative Behavior Analysis System"

William R Schafer, Zhaoyang Feng, Kuang-man Huang, Paul W Sternberg, Christopher J Cronin

[International Worm Meeting, 2005]

California Institute of Technology and University of California, San Diego have each developed practical, automated movement analysis systems to quantify aspects of worm behavior and morphology (see An imaging system for standardized quantitative analysis of C. elegans behavior, Zhaoyang Feng, et al, BMC Bioinformatics 2004, 5:115, An automated system for measuring parameters of nematode sinusoidal movement, Christopher J Cronin, et al, BMC Genetics 2005, 6:5, and previous meeting abstracts). We have consolidated our development efforts and merged our independent system designs to create a single, unified behavior analysis system. The combined system provides researchers with all of the quantification tools and database functionality of the individual UCSD and Caltech systems, but also establishes a common, open software and hardware foundation for continued development by Caltech, UCSD and the community. Current efforts include expanding system capabilities for the automated analysis of mating behavior with the challenge of tracking multiple moving individuals, both in contact with and separated from each other.

RV Aroian et al. (1999) International C. elegans Meeting "Using C. elegans to Teach Embryonic Development to Undergraduates"

G Wienhausen, RV Aroian, D Johnson

[International C. elegans Meeting, 1999]

We have begun to use C. elegans as a model system in our undergraduate embryology laboratory class at the University of California, San Diego. In the past, this 10 week class has used Sea Urchin, Xenopus, Chick, and other non-genetic systems to demonstrate to students embryonic development. In this poster we will outline the 6 new weeks of C. elegans modules. These modules allow the students to use advanced techniques to explore widely applicable principals of embryonic development. These techniques include wild-type lineage analysis, mutant lineage analysis, use of specific inhibitors to alter embryonic development, immunofuorescence analysis of wild type and mutants using antibodies to various cell biology and developmental markers, genome searching, and knocking out genes using RNA-mediated interference. Our goal is to one day publish an article on our results in the Worm Breeder's Gazette. The embryonic development of more advanced model organisms such as Xenopus and chick are later used to compare and contrast to C. elegans. This work is greatly enhanced by the cooperativity of the C. elegans community in the sharing of antibodies, mutants, and other reagents such as GFP strains. Last year we used protocols, stains, and antibodies from to Geraldine Seydoux, Craig Mello, Pete Okkema, Michel Labouesse, Bruce Bowerman and the CGC. Thanks to them and others that have generously shared their reagents.

Smejkal Gary B et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Pressure mediated scheme for isolating native proteins from Caenorhabditis elegans under mild nondenaturing conditions"

Anderson Jobriah, Smejkal Gary B, Morris Krystalynne, Thomas W Kelley, Kuo Winston P, Strader Thomas E

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

The tough exterior cuticle of Caenorhabditis elegans makes this nematode very resistant to lysis and impedes proteomic and glycoproteomic analyses. So resilient is this organism, that specimens sent into space were the only survivors of the disastrous crash of the space shuttle Columbia [1]. Proteomic analysis is particularly challenging when preservation of the native conformation and biological activity of proteins is required, thus prohibiting the use of denaturing chaotropes and detergents to enhance lysis. The dilemma is that in mild physiological buffers, nematodes can withstand hydrostatic pressure hydrostatic pressure up to 20,000 psi with a 2.3% survival rate [2]. While bead beating and sonication are commonly used to disrupt nematodes, heat generated by these methods causes denaturation of proteins, and subsequently, their loss through aggregation and precipitation. By comparison, pressure cycling technology (PCT) generates minimal heat by rapidly oscillating between high and atmospheric pressure. The ProteoSOLVE CE Native Kit was developed specifically to maximize protein yields from live nematodes under mild nondenaturing conditions. In the optimized protocol, live nematodes collected from culture are concentrated on a filter and the resulting paste is mixed with the CE SiC abrasives and transferred to a PULSE Tube. The amalgam is frozen solid by placing the tube on dry ice for ten minutes, then ground with the serrated Shredder ram insert until completely extruded through perforations of the lysis disk. The Native Reagent is added, followed by 40 pressure cycles at 35,000 psi. Protein yields were six times greater with the Shredder ram than with high pressure alone, and an order of magnitude higher when the Shredder ram was used in combination with freezing and abrasives. [1] Szewczyk, N.J. et al. (2005). Astrobiology, 5, 690-705. [2] Smejkal, G.B. et al. (2008). Plant and Animal Genome XVII Conference, San Diego, CA.