8 Papers found (0.009 seconds)

Harrington, Sean et al. (2017) International Worm Meeting "The discovery of neuroactive small molecule tools using a high-throughput egg laying screening pipeline."

Yip, Chris, Harrington, Sean, Roy, Peter, Giuliani, Maximiliano, Au, Aaron

[

International Worm Meeting,

2017]

The neuromuscular circuit that governs C. elegans egg-laying is well understood and the majority of regulatory elements within it are highly conserved. The egg-laying system has been exploited previously to probe the polypharmacology of established neuromodulators such as clozapine (Karmacharya et al. (2011), Brain Res., 1393(1), 91-99) and imipramine (Weinshenker et al. (1999), J of Neurosci., 19(22), 9831-9840). Findings from these studies translated to a mouse model demonstrating the conserved nature of components of the egg-laying system. Here, we aim to identify novel small-molecule modulators of neuromuscular function by screening for those that modulate egg-laying behaviour in the worm. To do so, we have developed a high-throughput screening pipeline that takes advantage of the amenability of the egg-laying phenotype to computational counting. Using this screening pipeline, we have screened through 4642 molecules and identified 83 molecules that either stimulate or suppress egg laying behaviour at a concentration of 60 M or less. Many of these molecules have no reported relevant bioactivities in the literature. Several of our hits are bioactive in a vertebrate model (Danio rerio) suggesting the possibility of a conserved mechanism of action (Burns et al. (2015), Nature Comm., 6(1), doi:10.1038/ncomms8485). We are currently characterizing the mechanism of action of these molecules using four parallel approaches: 1) testing their interaction with a panel of human neuronal targets (NIH Psychoactive Drug Screening Project - Besnard et al. (2012), Nature, 492(7428), 215-220); 2) thermal proteome profiling using quantitative mass spectrometry to identify candidate targets in worms (Franken et al. (2015), Nature Protoc., 10(10), 1567-1593); 3) forward genetics screens to identify candidate targets (Kwok et al. (2006), Nature, 441(7089), 91-95); & 4) understanding their interaction with a panel of C. elegans egg laying mutants. We hope that our hits will become useful tools for future biological investigations, and that a subset might have therapeutic potential to modulate neuromuscular activity in more complex systems.

Sabrina G C Shore et al. (2002) West Coast Worm Meeting "The temperature sensitive lethal mutation or566 may define a new gene that functions in early morphogenesis"

Andrew D Chisholm, Sarah L Moseley, Sabrina G C Shore

[

West Coast Worm Meeting,

2002]

Mutations in the EPH receptor tyrosine kinase VAB1 and in its ephrin ligands cause incompletely penetrant defects in gastrulation cleft closure and epidermal enclosure. Fully penetrant defects in cleft closure are seen when multiple pathways are blocked (e.g. vab-1 and ptp-3 (Harrington et al., 2002, Development 129: 2141). To find genes that may have essential functions in gastrulation cleft closure we have begun to examine conditional lethal mutants for possible defects in this process. Bruce Bowermans lab identified several new temperature-sensitive (ts)-lethal mutations affecting morphogenesis in their large scale ts-lethal screens, and very kindly provided some of them to us.

Prasad BC et al. (1997) International C. elegans Meeting "THE C. ELEGANS unc-3 GENE ENCODES A HOMOLOGUE OF THE O/E FAMILY OF MAMMALIAN TRANSCRIPTION FACTORS"

Prasad BC, Reed RR, Seydoux GC

[

International C. elegans Meeting,

1997]

The expression of specialized signal transduction components in mammalian olfactory neurons is thought to be regulated by the O/E (Olf-1/EBF) family of transcription factors. The O/E proteins are expressed in cells of the olfactory neuronal lineage throughout development, and are also expressed transiently in neurons in the developing nervous system during embryogenesis. We have identified a C. elegans homologue of the mammalian O/E proteins, which displays greater than 80% similarity over 350 amino acids. The CeO/E cDNA maps to cosmid F42D1, previously shown to encode unc-3(1). We demonstrate that CeO/E is the product of the unc-3 gene, mutations in which cause defects in the axonal outgrowth of motor neurons as well as defects in dauer formation, a process requiring chemosensory input. Like its mammalian homologues, CeO/E is expressed in certain chemosensory neurons (ASI amphid neurons) throughout development, and is also expressed transiently in developing motor neurons when these cells undergo axonal outgrowth. Currently, we are defining the DNA sequence binding specificity of the CeO/E protein. These observations suggest that the O/E family of transcription factors play a central and evolutionarily conserved role in the expression of proteins essential for axonal pathfinding and neuronal differentiation. (1) Sean Eddy, WBG 12(5):86 (February 1, 1993)

SE George et al. (1999) International C. elegans Meeting "Still Searching for components of the VAB-1 pathways"

AD Chisholm, SE George

[

International C. elegans Meeting,

1999]

We are identifying the mechanisms by which the C. elegans Eph receptor tyrosine kinase, VAB-1, signals in neural and epithelial morphogenesis. VAB-1 has both kinase-dependent and kinase-independent functions during C. elegans development (George et al, Cell 92:633). The kinase-dependent (forward signaling) function of VAB-1 is mediated by its cytoplasmic kinase domain, possibly through tyrosine phosphorylation of the juxtamembrane domain. Proteins that interact with the cytoplasmic domain of Eph receptors have been identified in vertebrates using the yeast two-hybrid system. Candidate molecules include a low molecular weight protein tyrosine phosphatase and adaptor proteins such as Nck, Grb2, and Grb10. We are using a modified yeast two-hybrid screen to identify genes that function in VAB-1 forward signaling. Initial progress and methodology of the screen will be presented. The nature of the VAB-1 kinase-independent function is unknown but may involve the extracellular domain. Our genetic and biochemical studies indicate that VAB-2 (a C. elegans ephrin) is a ligand for VAB-1 and that it appears to mediate the kinase-independent function of VAB-1 (see Chin-Sang et al abstract). vab-2 mutations display synthetic lethality with vab-1 kinase mutations. We also observe synthetic lethality between vab-1 kinase mutations and a mutation in the receptor protein tyrosine phosphatase, PTP-1, suggesting that PTP-1 may be acting in a parallel pathway to the VAB-1 kinase activity (see Harrington et al abstract). To identify genes that are acting in the VAB-1 kinase-independent pathway or in parallel pathways we are performing screens to identify mutations that are synthetic lethal with vab-1 . Progress of the screen will be presented.

Griffin, Edward F et al. (2013) International Worm Meeting "Functional analysis of VPS41-mediated protection from b-Amyloid cytotoxicity."

Griffin, Edward F, Caldwell, Guy A, Gilmartin, Christopher, Caldwell, Kim A

[

International Worm Meeting,

2013]

According to the Alzheimer's Association 2012 report, 13% of Americans over 65 years of age suffer from Alzheimer's Disease (AD), resulting in an estimated cost of 200 billion dollars for related health care. Additionally, AD is the most prevalent dementia, and the sixth leading cause of death in the United States. Thus, investigating mechanisms of pathophysiology and identifying potential therapeutic targets for AD is significant. AD is characterized by the formation of plaques, composed primarily of b-amyloid 1-42 (Ab) in the brain, resulting in neurodegeneration. Our lab has observed that over-expression of VPS41 in C. elegans provides neuroprotection from Ab toxicity, and that deficiency of VPS41 in worms expressing Ab increases the toxicity of Ab (unpublished data). In yeast, VPS41 has been demonstrated to function in the tethering of vesicles, late endosomes, and AP-3-coated vesicles from the late Golgi, to the lysosome for degradation. Previously, our lab has shown that over-expression of human VPS41 is neuroprotective in a transgenic worm model of Parkinson's Disease, wherein dopaminergic neurodegeneration is induced by a-synuclein overexpression (Harrington et al., 2012, J. Neurosci.). VPS41-mediated neuroprotection from a-synuclein was dependent on the phosphorylation of VPS41 as well as its interactions with AP-3d, RAB7, VPS core proteins, and VPS39. Through this study, and others, we can conclude that VPS41 has a role in lysosomal trafficking. Yet, how this specifically relates to the processing and ameliorates the cellular impact of neurotoxic proteinaceous aggregates is undetermined. Here we report the results of a systematic RNAi screen whereby we knocked down the core components involved in lysosomal trafficking and categorized their requirement for Ab protein toxicity. Preliminary results indicate that Ab is trafficked to through the endosomes rather than through AP-3-coated vesicles from the late Golgi. In this regard, further analysis of functional effectors of Ab protein processing via the lysosomal pathway, along with subsequent evaluation in our worm neuronal model of AD (Treusch et al, 2011, Science), will assist in the elucidation of the underlying mechanism involving VPS-41.

Chin-Sang ID et al. (2000) West Coast Worm Meeting "Suppressor Analysis of Eph/Ephrin Defective Signaling in C. elegans"

Chisholm AD, Chin-Sang ID, McCleery J

[

West Coast Worm Meeting,

2000]

Mutations in the genes vab-1 and vab-2 cause similar defects in neural and epidermal morphogenesis. The phenotypes range from embryonic lethality, due to epidermal enclosure defects, and a ventral notched head in larvae and adults. vab-1 encodes an Eph receptor tyrosine kinase that is expressed in the developing nervous system, and is required in neurons for proper epidermal morphogenesis (George et al., Cell 92:633). vab-2/efn-1 encodes a GPI anchored ephrin that specifically interacts with VAB-1 in neurons to regulate neuronal and epidermal morphogenesis (Chin-Sang et al., Cell 99:781). What are the molecular consequences of the VAB-1/VAB-2 interaction? We have taken a genetic approach to identify genes that might act in the VAB-1/2 Eph/Ephrin signaling pathway or in parallel pathways. The incomplete penetrance and variability of phenotypes displayed by vab-1 and vab-2 mutants, made it difficult in the past to carry out genetic modifier screens. Therefore we sought to create strains that would make vab-1 and vab-2 mutants more suitable for genetic modifier screens. A mutation in a LAR like receptor tyrosine phosphatase, ptp-1, results in a synthetic lethality with both vab-1and vab-2, suggesting that PTP-1 may function redundantly with Eph signaling (see abstract by Harrington et al.). The ptp-1(op147);vab-2(ju1) double mutant is temperature sensitive- viable at 15 0C and 20 0C, however, at 25 0C almost completely inviable. We screened for suppressors of the ptp-1(op147);vab-2(ju1) lethality at 25 0C and isolated at least 19 suppressors from a screen of over 67,000 F1 animals. The suppressors are of two classes: those that reduce the lethality and those that suppress both the lethality and the notched head phenotype of vab-2. Five suppressors of the second class map to LGIV and are dominant suppressors of vab-2. Intriguingly, these suppressors display a low penetrance notched head phenotype (about 2-5% of the animals), however, unlike vab-1 and vab-2 the notch is always on the dorsal side. We are currently determining the specificity of the suppression effect. We will present results from the characterization of these suppressors.

Roy, Peter et al. (2021) International Worm Meeting "A C. elegans Motor-Centric Screening Pipeline Yields Novel and Selective Nematicidal Scaffolds"

[

International Worm Meeting,

2021]

Nematode parasites of humans, livestock and crops pose a significant burden to human health and welfare. Alarmingly, our arsenal of effective nematocidal compounds is being depleted. Parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides used for crop protection have been restricted or banned because of poor phylum-selectivity. Here, we present our C. elegans-based discovery pipeline focused on lethal molecules that also induce motor defects. This pipeline yielded multiple new and selective nematicidal small molecule scaffolds (i.e., structurally-related families of molecules). We show that one of these scaffolds, tentatively called the APPs, stimulates neurotransmitter release and immobilizes larvae of multiple nematode parasites of plants and mammals in vitro. At similar concentrations, APP-1 does little to model flies, fish, plants or human cells. Forward genetic screens of 100,000s of mutant genomes failed to yield resistant animals that resist the lethal effects of APP-1, suggesting that resistance to the APPs in the field may not be easily generated. Hence, the APPs represent a novel, selective and potentially useful addition to our nematocidal armament.

Martin L Hudson et al. (2003) International Worm Meeting "Functions of ephrins in embryonic and post-embryonic development: exploring connections with Kallman syndrome protein and cadherins"

Andrew D Chisholm, Martin L Hudson

[

International Worm Meeting,

2003]

During C. elegans gastrulation, embryonic cells rearrange to form tissue layers. Endodermal and mesodermal cells move into the interior of the embryo, leaving a transient ventral cleft that is sealed up by the short-range movements of ventral neuroblasts. These ventral neuroblasts and their progeny provide the substrate for the subsequent enclosure movements of the epidermis. Mutants with abnormal ventral neuroblast movements display enlarged ventral clefts that later result in epidermal enclosure defects. Previous work in our lab showed that signaling via the C. elegans Eph receptor VAB-1 and its ephrin ligand EFN-1/VAB-2 regulate neuroblast movements at the end of gastrulation (George et al. 1998; Chin-Sang et al. 1999). The divergent ephrin EFN-4 is also required for neuroblast movements but appears to function independently of the VAB-1 receptor (Chin-Sang et al. 2002). A third pathway is defined by the LAR-like receptor phosphotyrosine phosphatase PTP-3 (Harrington et al. 2002). These pathways appear to play related and partly redundant roles in ventral neuroblast movements.We have examined whether other mutants with epidermal morphogenesis phenotypes affect neuroblast movements and have used double mutant analysis to determine if these genes might define new pathways or can be placed in the VAB-1/EFN-4/PTP-3 pathways. KAL-1 is the C. elegans homolog of human Kallman syndrome protein, anosmin-1 (Rugarli et al. 2002). A kal-1 mutation displays low penetrance defects in epidermal morphogenesis. Using 4-D analysis, we find that kal-1 mutants display delayed closure of the ventral cleft. We also find that a kal-1 mutation enhances the morphogenetic phenotypes of all mutants tested (vab-1(null), efn null mutations and ptp-3), suggesting that KAL-1 may define a separate signaling pathway. We are also studying the post-embryonic functions of ephrins. EFN-4 is expressed in the primary cells of the developing vulva (Hudson and Chisholm, 2002 WCWM, abstract 144). efn-4 mutants display very low penetrance defects in vulval morphogenesis and induction. The cadherin CDH-3 is also expressed in vulval cells (Pettit et al. 1996). A cdh-3 mutation causes epidermal morphogenesis defects (6) reminiscent of those in weak efn-4 mutants. We find that the cdh-3 mutation does not enhance efn-4(null) mutant phenotypes but significantly enhances efn-1(null) mutant phenotypes, suggestive of a role for cdh-3 in efn-4 signaling. Further analysis of the phenotypes will be presented.