Jones AK et al. (1995) International C. elegans Meeting "CAN BLI-4 COMPLEMENT S.CEREVISIAE KEX2 MUTANTS?"

Thacker CM, Boone C, Jones AK, Rose AM

[International C. elegans Meeting, 1995]

The kex2/subtilisin proprotein convertase family of enzymes convert biologically inactive precursors into active secreted protein molecules. In C.elegans, the characterization of the bli-4 gene introduces a new member to the kex2/subtilisin family of endoproteases. bli-4 encodes at least four isoforms, of which the D isoform is structurally most similar to the KEX2 product in yeast. The trafficking signals of these gene products share significant sequence identity, providing an opportunity to learn about conservation of trafficking and localization signals between species. We are testing a cDNA clone of the blisterase D isoform for the ability to rescue an S.cerevisiae strain mutant in the homologous KEX2 gene. The cDNA clone has been constructed and inserted into a yeast expression vector. The resultant construct was transfected into a KEX2-deficient yeast strain. Transformants, produced on selective galactose media, were assayed for expression of blisterase D. However, preliminary experiments failed to indicate expression of the blisterase D clone. This may be a result of lack of expression of the bli-4 gene, or the blisterase product may not be appropriately folded or transported. Chimeric constructs may help to investigate the critical domain for complementation of the KEX2-deficient yeast. Further experimental approaches will include replacing the KEX2 protease domain with Bli-4, as well as the carboxy terminus, and investigating whether the KEX2 function can be complemented. If successful, this approach can also be used to generate more mutations in bli-4, in particular temperature sensitive mutants, since none have thus far been generated by direct C.elegans mutagenesis. Supported by the MRC of Canada.

AK Jones et al. (2009) European Worm Neurobiology Meeting "A mutation in the Caenorhabditis elegans nicotinic acetylcholine receptor a subunit, unc-63, provides a model for fast channel congenital myasthenia"

C Bouzat, AK Jones, A Al-Diwani, TPR Maynard, DB Sattelle, G Hernando, D Rayes, SD Buckingham, R Jones

[European Worm Neurobiology Meeting, 2009]

Authors acknowledge support from MRC and OXION. Abstract: The nematode Caenorhabditis elegans is an established model organism for studying neurobiology since many synaptic molecular components are conserved both in the worm and humans. The unc-63 a subunit of the C. elegans nicotinic acetylcholine receptor (nAChR) plays an important role in fast cholinergic synaptic transmission at the nematode neuromuscular junction (1). Here we show that worms with the unc-63(x26) allele, with its .C151Y mutation that disrupts the Cys-loop, have deficient muscle function as displayed by impaired thrashing locomotion compared to wild-type worms (2). Single-channel recordings from muscle cells of the mutant strain show reduced number of active patches, a 100-fold reduced frequency of opening events, and abnormally reduced duration of channel openings similar to that observed in patients with fast-channel congenital myasthenia syndromes (FCCMS). Interestingly, a mutation in the equivalent position of the e subunit of the human muscle nAChR (.C128S) is associated with a FCCMS due to nAChR deficiency at the endplate (3). Therefore, disruption of the Cys-loop results in qualitatively similar actions on both nematode and human receptors. Therapy for FCCMS is limited in variety and success with 3,4-diaminopyridine (3,4-DAP) and anticholinesterase drugs such as pyridostigmine bromide (PB). We show that PB and 3,4-DAP can partially rescue the motility defect seen in unc-63(x26). Conclusions: We describe the first candidate C. elegans model for screening for drugs aimed at ameliorating the consequences of mutations in nAChRs associated with FCCMS. References: (1) Culetto E et al. J Biol Chem. 2004. 279:42476-83. (2) Jones AK et al. Hum Mol Genet. Submitted 2009. (3) Milone M et al. Ann N Y Acad Sci. 1998. 841:184-8.

Lee M-H et al. (1997) International C. elegans Meeting "TOWARD THE IDENTIFICATION OF IN VIVO RNA TARGETS OF GLD-1."

Lee M-H, Schedl TB

[International C. elegans Meeting, 1997]

gld-1 is a female germ cell specific tumor suppressor gene that is essential for normal oocyte differentiation and meiotic prophase progression (Francis et al., 1995a,b). GLD-1 is a member of a small family of proteins, including the mouse/human Sam68 and Quaking proteins, that are highly related over an ~ 200 amino acid region which contains a 70 to 100 amino acid RNA binding domain called the KH motif (Jones and Schedl, 1995). Extensive mutational analysis of gld-1 has demonstrated the importance of conserved sequences for its in vivo function. GLD-1 is a cytoplasmic protein and therefore is likely to control mRNA translation or RNA stability in the cytoplasm (Jones et al., 1996). Currently, no obvious RNA targets have been identified from genetic analysis. We have employed a biochemical approach to identify in vivo RNA targets of GLD-1. The strategy is based on the ability of anti GLD-1 antibodies to immunoprecipitate (IP) GLD-1 from a worm lysate and the strong likelihood that GLD-1 present in the lysate is functional and bound to RNAs. GLD-1 was IPed with affinity purified polyclonal antibodies from a cytosol extract of adult hermaphrodites or with rabbit IgG as a control. RNAs co-IP with GLD-1, as well as with rabbit IgG, were converted into cDNAs. Non-specifically trapped RNAs from the GLD-1 IP were eliminated by subtracting cDNAs from the GLD-1 IP with cDNAs from the control IP. The difference product after 4 rounds of subtraction (DP4) was used to screen a Lambda ZAP II cDNA library constructed with the cDNAs from the GLD-1 IP. Duplicate filters were also screened with a probe made from control IP cDNA. Clones that are positive only with the DP4 probe are currently being characterized. Francis et al., 1995a Genetics 139, 579-606; Francis et al., 1995b Genetics 139, 607-630; Jones and Schedl, 1995 Genes Dev. 9, 1491-1504; Jones et al., 1996 Dev. Biol. 180, 165-183. This work was supported by NIH Grant HD25614.

James F Cregg et al. (2004) West Coast Worm Meeting "A RNAi Screen for Genes Involved in Tolerance to Nicotine"

Kathleen B Quast, Kevin J Ford, William R Schafer, James F Cregg, Caroline R Craig, Suchitra Jagannathan

[West Coast Worm Meeting, 2004]

Tobacco use is the second major cause of death worldwide. The acquisition of tolerance to nicotine is a key step in the development of nicotine addiction. Twenty seven nicotinic acetylcholine receptor subunits have been identified in C. elegans (reviewed in Jones and Sattelle, 2003). However, few of the molecules which modify nAChR function, abundance, or subcellular localization in response to nicotine exposure are known. We are performing a RNAi-based screen of the first chromosome using the Ahringer lab RNAi feeding library in a rrf-3 background. In preliminary experiments, worms fed RNAi against genes already known to be involved in nicotine responces (including unc-63 and unc-50) have shown resistance in our nicotine-induced paralysis assay. Results of this screen should provide insight into the mechanisms of nicotine tolerance.

James F Cregg et al. (2005) International Worm Meeting "A RNAi screen for genes involved in tolerance to nicotine."

Marina Ezcurra, Caroline R Craig, William R Schafer, Kathleen B Quast, James F Cregg, Kevin J Ford

[International Worm Meeting, 2005]

Despite more than fifty years of knowledge of the link between smoking and lung cancer, tobacco use remains the leading preventable cause of disease and premature death in the United States. The acquisition of tolerance to nicotine is a key step in the development of nicotine addiction. Twenty seven nicotinic acetylcholine receptor (nAChR) sub-units have been identified in C. elegans (reviewed in Jones and Sattelle, 2003). However, few of the molecules which modify nAChR function, abundance, or sub-cellular localization in response to nicotine exposure are known. We have performed a RNAi-based screen of the first chromosome, as well as a candidate gene screen of predicted neuropeptides and G-protein coupled receptors, using the Ahringer lab RNAi feeding library in a rrf-3 background. We have identified 47 candidate genes which might affect cholinergic signaling. These fall into multiple classes, including molecules involved in protein trafficking and turnover, cell structural components, kinases and phosphatases, as well as transcriptional regulators. Results of this screen should provide insight into the mechanisms of nicotine tolerance.

Clifford R et al. (1996) Midwest Worm Meeting "Searching for Proteins that Associate with GLD-1"

Clifford R, Giorgini F, Schedl TB

[Midwest Worm Meeting, 1996]

The gld-1 gene is involved in multiple aspects of C. elegans germline development. The primary, essential function of gld-1 is to direct oogenesis. In the absence of gld-1 activity, germ cells that would otherwise develop into oocytes enter meiosis normally but exit early pachytene to re-enter a mitotic cell cycle and form a tumor. The gene also plays nonessential roles in inhibiting premeiotic germ cell proliferation and promoting germ cells to adopt the male sexual fate [1,2]. gld-1 encodes a 463 amino acid protein that contains an evolutionarily conserved domain of ~200 amino acids that is also found in the mammalian Quaking and Sam68 proteins. A KH RNA binding motif lies within this domain [3]. Since GLD-1 appears to be localized exclusively to the cytoplasm, the protein is likely to regulate mRNA translation or stability [4]. To better understand how gld-1 acts in the control of germline development, we are using the yeast two-hybrid system [5] to isolate proteins that physically associate with the gene product. Through this approach we hope to identify regulators and cofactors of GLD-1. By screening an oligo(dT)-primed cDNA library (gift of R. Barstead) with a fragment of GLD-1 that includes the amino terminal half of the conserved domain, we identified the F28E10.1 gene product as a protein that specifically binds GLD-1. The F28E10.1 locus, which was identified by the C. elegans genome sequencing consortium, is predicted to encode a novel, hydrophilic protein of 118 kD. This gene maps to the region of chromosome IV that is deleted by mDf4 and may correspond to one of the lethal or sterile loci uncovered by the deficiency [6,7]. We are currently testing the biological relevance of the interaction between GLD-1 and F28E10.1 through in vivo assays. Progress will be reported. [1] Francis et al. 1995a. Genetics 139: 579-606; [2] Francis et al. 1995b. Genetics 139: 607-630; [3] Jones and Schedl. 1995. Genes Dev 9: 1491-1504; [4] Jones et al., in preparation; [5] Chien et al. 1991. PNAS 88: 9578-9582; [6] Rogalski and Riddle. 1988. Genetics 118: 61-74; [7] Cassada et al. 1981. Dev Biol 84: 193-205.

Topper, Stephen M. et al. (2013) International Worm Meeting "Ethanol induces state-dependent behavioral transition."

Vidal-Gadea, Andres, Pierce-Shimomura, Jonathan, Topper, Stephen M., Aguilar, Sara, Young, Layla

[International Worm Meeting, 2013]

Alcohol, the most widely abused drug in the world, has a wide range of effect on individuals and there are many factors that predispose one to addiction. Chief among them are stress, depression, and anxiety, all of which include altered monoamine signaling. It remains unclear how behavioral states differentially influence responses to ethanol. C. elegans displays distinct behavioral states associated with locomotion (crawling and swimming) that are mediated by dopamine and serotonin respectively. We found that ethanol-induced responses in C. elegans depended on crawl or swim behavioral state. Whereas alcohol non-specifically impaired locomotion and feeding in crawling worms on land, alcohol induced a specific transition from swim to crawl state in worms submerged in ethanol. This response was dependent on dopamine. Loss of the D1-like dopamine receptor DOP-4 impaired the EtOH-induced transition to crawl, while optogenetic inhibition of DOP-4 receptor-expressing neurons via the Arch chloride pump recovered the swim state. These results suggest that alterations of dopamine-dependent behavioral states can drastically influence EtOH-induced behaviors with implications for prevention of addiction. Acknowledgements: CGC and the Knockout Consortium for strains, Bruce/Jones Award for Alcohol and Addiction Research, Karl Deisseroff for reagents.

Natascia Ventura et al. (2008) European Worm Meeting "p53 RHEOSTATICALLY CONTROLS C. elegans LONGEVITY DEPENDING ON THE LEVEL OF MITOCHODNRIAL STRESS"

Roberto Testi, Natascia Ventura, Thomas E Johnson, Shane L Rea, Alfonso Schiavi

[European Worm Meeting, 2008]

In humans, genetic mitochondrial dysfunctions result in a number of. life-shortening diseases. At the root of most of these diseases lays. ineffectual energy production with increased free radical production. In. the nematode Caenorhabditis elegans, severe suppression of genes coding for. mitochondrial components lead to pathological phenotypes including lifespan. reduction but milder suppression results in lifespan extension (Rea, 2007).. The molecular mechanisms underlying these effects are still unknown.. The Mit mutants display multiple phenotypic alterations including. slowed development, reduced size and fertility, which are all intimately. linked with energy metabolism, DNA replication and cell cycle regulation.. The tumor suppressor p53 regulates cell cycle in response to alteration in. energy metabolism and mitochondrial dysfunction (Jones, 2005; Mandal,. 2005). In C. elegans there is one p53 homologue, CEP-1, that is required. for germ line proliferation in response to genotoxic stress (Derry, 2007).. Loss of cep-1 in C. elegans increases life span (Arum, 2007).. We found that cep-1 is required for lifespan extension of different. Mit mutants, including animals deficient in frataxin, the mitochondrial. protein defective in Friedreich''s Ataxia. Importantly, consistent with a. rheostatic role of p53 in response to different levels of stress, CEP-1. displayed opposite effects in regulating C. eleagns lifespan depending on. the level of mitochondrial disruption.. For correspondence: natascia.ventura@med.uniroma2.it

A Jones et al. (2004) European Worm Meeting "THE NICOTINIC ACETYLCHOLINE RECEPTOR FAMILY OF CAENORHABDITIS ELEGANS"

A Jones, B Esmaeili, D Sattelle

[European Worm Meeting, 2004]

Nicotinic acetylcholine receptors (nAChRs) are prototypical members of the ligand-gated ion channel superfamily. They are pentameric membrane proteins which mediate the fast action of the neurotransmitter acetylcholine (ACh) at the neuro-muscular junction and in the nervous system. The nAChR gene family of C. elegans is the largest known, consisting of potentially 50 subunits*. Why nematodes have such a diverse nAChR family, especially when considering that mammals possess 16 subunits, has yet to be fully resolved. The C. elegans nAChR subunits have been subdivided into five major groups (DEG-3 like, ACR-16 like, ACR-8 like, UNC-38 like and UNC-29 like) as well as an "orphan" group. To gain further understanding of this diverse nAChR family, a systematic strategy has been employed whereby GFP constructs will be used to determine expression patterns and gene knockdown by RNAi will be used to gain insight into receptor function. Studies of several nAChR subunits have already shown striking differences in expression patterns. For instance, acr-10 is expressed in body wall muscle whereas acr-14 is exclusive to the ventral nerve cord. Other subunits are expressed in head neurons (acr-6), chemosensory neurons (acr-20) and the pharynx (acr-7). Preliminary results using Snap-back RNAi indicate that both acr-14 and acr-10 are required for coordinated movement. *Jones and Sattelle (2004). BioEssays 26:39-49

Sandrine Fraboulet et al. (2009) European Worm Neurobiology Meeting "Chemical rescue of paralysis in a transgenic model of Caenorhabditis elegans overexpressing human Beta-amyloid"

David I C Scopes, Andrew K Jones, David B Sattelle, Aileen Moloney, Christopher D Link, Steven D Buckingham, Mark J Treherne, Hozefa Amijee, Sandrine Fraboulet

[European Worm Neurobiology Meeting, 2009]

Authors acknowledge support from MRC. Numerous studies support a role for aggregated Ab as a mediator of the toxicity that underlies Alzheimer.s disease (AD) and other diseases such as Inclusion Body myositis (IBM), an acquired muscle disease affecting people over 50 years old. In this pathology, muscle weakness and degeneration is accompanied by chronic muscle inflammation. Remarkably, despite the inflammation component of the disease, IBM patients are only poorly responsive to anti-inflammatory drugs suggesting that inflammation per se may not be the primary cause of the pathology. We have used a C. elegans transgenic line over-expressing human amyloid 1-42 peptide in the muscles as a model with which to test the efficiency of new compounds on in vivo amyloid toxicity. This C. elegans line becomes paralyzed shortly after the induction of amyloid expression in the muscles, which makes it an easily-recordable phenotype useful for exploring candidate treatments to alleviate the paralysis. We report on the actions of two novel chemicals, which inhibit amyloid aggregation and partially rescue the amyloid-induced phenotype. References: Wu Y, W.Z., Butko P, Christen Y, Lambert MP, Klein WL, Link CD, Luo Y. (2006) J. Neurosci., 26, 13102-13. Link CD, T.A., Kapulkin V, Duke K, Kim S, Fei Q, Wood DE, Sahagan BG. (2003) Neurobiol Aging., 24, 397-413. Jones, A.K., Buckingham, S.D. and Sattelle, D.B. (2005). Nat Rev Drug Discov, 4, 321-30.