Zhou, Y et al. (2017) International Worm Meeting "Genetic and chemical modulation of C. elegans intestinal antiviral innate immunity."

Zhou, Y, Tao, Y, Bedian, L, Zhong, W

[International Worm Meeting, 2017]

The RNA virus Orsay is the only known natural viral pathogen infecting the intestine cells of the nematode C. elegans. The C. elegans intestine cells resemble human intestinal epithelial cells not only in morphological features such as microvilli, but also in molecular mechanisms such as antibacterial innate immune response. As Orsay was discovered only six years ago, the C. elegans antiviral innate immune system remains largely unknown. Only two pathways, RNAi and ubiquitin-mediated protein degradation, have been discovered to be involved in the C. elegans antiviral innate immunity. To explore the genetic landscape of the C. elegans antiviral innate immunity, we conducted a genome-wide RNAi screen for genes whose inactivation sensitizes worms to Orsay infection. 110 genes were identified to be required for C. elegans antiviral innate immunity. 72 of these genes have human orthologs. Tissue-specific RNAi experiments showed that the majority of these genes function in the intestine cells to modulate antiviral innate immunity. In addition to RNAi and ubiquitin-mediated protein degradation, these genes encompass pathways in autophagy, mitochondrial unfolded protein responses, collagens, cytoskeletal organization, RNA processing, and transcription. To identify the gene products that are druggable, we screened 2000 chemicals for drugs that can alleviate Orsay infection symptoms, and discovered four innate immunity enhancing drugs, resorcinol monoacetate (RM), berberine (BBR), bismuth subsalicylate, 3,3'- diindolymethane (DIM). BBR and bismuth subsalicylate are known antidiarrhea drugs, with possible antiviral functions against human gastrointestinal viruses. Chemical-genetic experiments revealed that these drugs have different mechanism of actions: RM strengthens the collagen barrier for viral entry; BBR and DIM enhances the ubiquitin-mediated protein degradation pathway. Together, these data revealed a multifaceted antiviral innate immune system in the C. elegans intestine. Aspects of genetic and chemical modulation of this system may be conserved in human innate immunity against gastrointestinal viruses.

Jason M McEwen et al. (2005) International Worm Meeting "UNC-18/nSec1 is required for proper trafficking of UNC-64/Syntaxin-1 in C. elegans neurons"

Josh Kaplan, Jason M McEwen

[International Worm Meeting, 2005]

UNC-18/nSec1 appears to have many roles in regulating synaptic vesicle release. Studies in Drosophila and cell culture have shown that UNC-18 may act as an inhibitor of synaptic transmission 2. In C. elegans, unc-18 mutant animals show a decrease in neurotransmitter secretion at the neuromuscular junction and in both worms and mice there is a decrease in the number of docked vesicles 1,4,5. One contributing factor in how UNC-18 positively regulates synaptic release may be the availability of UNC-64/Syntaxin-1 for forming SNARE complexes. If UNC-18/nSec1 is functioning to maintain levels of UNC-64/Syntaxin at the plasma membrane, this may explain some of the synaptic transmission defects seen in unc-18 mutant worms. Studies using non-neuronal vertebrate cell types have shown that UNC-18/nSec1 is required for UNC-64/Syntaxin-1 to be properly trafficked to the plasma membrane 3.To address the role of UNC-18 in regulating anterograde transport in neurons, we looked to see if UNC-64 is properly localized in unc-18 mutant worms. Using both antibodies against UNC-64 and an UNC-64a::GFP fusion protein we showed that a larger fraction of UNC-64 is retained in the cell bodies of neurons in unc-18 animals when compared to wild type. Trafficking of other synaptic proteins such as SNB-1/Synaptobrevin, RIC-4/SNAP-25, UNC-10/RIM-1 and SYD-2 is unaffected in unc-18 mutants, suggesting that this is not a general defect in intracellular transport. Current experiments are testing whether UNC-18 regulates ER or Golgi exit of UNC-64.1. Gengyo-Ando K et al. Neuron 19932. Schulze KL et al. Neuron 19943. Rowe J, et al. J Cell Sci 20014. Voets T, et al. Neuron 20015. Weimer RM, et al. Nature Neuroscience 2003

Khare S et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Interaction of a Protein Repair System with Insulin Signaling to Extend Lifespan in Caenorhabditis elegans"

Khare S, Clarke S G, Linster C L

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The protein L-isoaspartyl-O-methyltransferase (PCM-1) functions to repair isomerized aspartyl and asparaginyl residues that spontaneously accumulate with age in a variety of organisms, including Drosophila, C. elegans, and mice. Under standard conditions, pcm-1 deletion mutant C. elegans display a normal lifespan and phenotype (Kagan et al., 1997; Banfield et al., 2008). However, a reduction in lifespan is observed in this mutant in the dauer stage. Moreover, significant defects in development, adult egg-laying, dauer formation, autophagy, and survival have been observed in pcm-1 mutant nematodes under stress conditions (Banfield et al., 2008; Gomez et al., 2008; Khare et al., 2009).An additional mutation in the insulin-like receptor protein DAF-2 prevents the oxidative stress-dependent egg-laying defect and the dauer formation defect observed in pcm-1 mutant nematodes, indicating that PCM-1 may either be working upstream or in parallel to the insulin signaling pathway to promote resistance to stress and regulate dauer formation (Khare et al., 2009). These exciting results have led us to further investigate the relationship between PCM-1 and the insulin signaling pathway in the context of aging. Interestingly, overexpression of this repair enzyme in both Drosophila and C. elegans extends adult lifespan (O' Conner et al., 1988; Banfield et al., 2008). Our preliminary results have led us to hypothesize that PCM-1 may be acting either directly or indirectly on the DAF-16 transcription factor to inhibit insulin signaling and extend lifespan. We have found that reducing DAF-16 levels by RNA interference (RNAi) reduces the lifespan extension displayed by the PCM-1 overexpressor strain. Our results from quantitative real-time PCR (qPCR) analysis reveal an up-regulation of daf-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1mutant nematodes under normal and acute thermal stress conditions. Additionally, similar to other long-lived C.elegans mutant strains, including daf-2and age-1 mutants, PCM-1 overexpressor adult animals display increased resistance to severe thermal stress. Our results support a role for protein repair in lifespan extension dependent on the insulin signaling pathway.

Michelle Po et al. (2007) International Worm Meeting "An unc-7 suppressor screen to identify novel active zone regulators."

Taizo Kawano, Michelle Po, Mei ZHEN, Magali BOUHOURS

[International Worm Meeting, 2007]

Synapses are asymmetric structures that facilitate the transmission of signals between neurons and their targets. The mature synapse consists of a presynaptic terminal in direct apposition to a postsynaptic terminal. Neurotransmission occurs when neurotransmitter-filled presynaptic vesicles dock and fuse with the plasma membrane. The neurotransmitter contents are released into the synaptic cleft and subsequently bind to receptors at the postsynapse. The active zone is an electron-dense region of the synapse that is required for the docking and fusion events of synaptic transmission. Our lab is interested in identifying genes that regulate the formation of active zones. In our study we are using the GABAergic nervous system of C. elegans as our model system. The active zones in this nervous system are visualized using a unique active zone specific marker SYD-2::GFP that was developed in our laboratory (Yeh et al., 2006). A forward genetic screen isolated an unc-7 loss-of-function mutant that shows a significant decrease in number of SYD-2::GFP puncta. We have determined that the innexin UNC-7 localizes to perisynaptic regions in addition to gap junctions. Subsequently, we showed that loss-of-function unc-9 mutants, which encodes an innexin protein that is 56% identical to UNC-7, shows the same synaptic defects as unc-7 mutants (Yeh and Ng, unpublished), suggesting a novel regulatory role for innexins at chemical synapses. We further investigated the role of innexins at the synapse by performing an unc-7 suppressor screen. We identified 10 dominant alleles of unc-1, a gene that encodes a stomatin homologue, that suppress the kinker behavior as well as the synaptic phenotype of unc-7 loss-of-function mutants. We found that UNC-7 and UNC-1 co-localize at peri-synaptic regions, indicating that the two proteins may functionally interact. We are investigating the physical properties of the potential UNC-7, UNC-9, and UNC-1 channel complexes. Current progress will be presented at the meeting. Yeh E, Kawano T, Weimer RM, Bessereau JL, Zhen M. 2005. Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans. J Neurosci. 25(15):3833-41.

Kelley L Banfield et al. (2002) West Coast Worm Meeting "Protein Repair Deficient Worms Reproduce and Age Normally"

Pamela L Larsen, Kelley L Banfield, Steven G Clarke

[West Coast Worm Meeting, 2002]

Proteins are susceptible to several types of spontaneous damage, including the isomerization of normal asparagine and aspartic acid residues to L-isoaspartyl residues. This reaction causes a kink in the protein chain by rerouting the backbone through the side-chain of the asparagine or aspartic acid residue. This damage can be corrected by the protein L-isoaspartyl-O-methyltransferase that is found in nearly all organisms examined to date. The gene coding for this protein in C. elegans is pcm-1. A recent RNAi screen by Maeda et al. (Curr Biol. 2001, 11, 171-6) found an embryonic lethal phenotype in 14% of the eggs of pcm-1 RNAi treated worms. This gene has previously been knocked out and analyzed in a him-8 mutant background. The brood size of the him-8; pcm-1 worms was not less than that of the him-8 worms, suggesting no embryonic lethality (Kagan et. al., Arch Biochem Biophys. 1997, 348, 320-8). However, the brood size of both these strains was about half that of wild type. We were concerned that phenotypes of the methyltransferase knockout worms were masked by the him-8 mutation. We backcrossed pcm-1(qa201) five times to N2. The analysis of pcm-1 in a wild-type background demonstrated that brood size and embryonic lethality are indistinguishable from wild type. We do not find the 14% embryonic lethality that was noted in the RNAi experiment. We also analyzed the adult life span at 25oC of the pcm-1 delete worms and found that it was nearly identical to that of wild-type worms. We then hypothesized that if this mutation were in a long-lived background such as daf-2(m41), then there may be a shortening of life span due to the accumulation of damaged proteins over a longer period. This however was not the case. The daf-2; pcm-1 double mutant worms lived as long as daf-2 single mutants. We are currently analyzing the two phenotypes that were noted in the him-8; pcm-1 worms, decreased dauer life span, and a failure to thrive in a mixed population of wild-type and pcm-1 worms. We are also planning to investigate several conditions that may affect the methyltransferase knockouts more severely than the wildtype worms, such as oxidative stress, pH, and temperature.

Edward Yeh et al. (2003) International Worm Meeting "A screen to identify factors involved in active zone formation."

Mei ZHEN, Edward YEH

[International Worm Meeting, 2003]

The active zone is a specialized presynaptic structure where vesicles containing neurotransmitters fuse with the plasma membrane, releasing the neurotransmitters to the post-synaptic membrane. At the EM level, electron dense active zones are surrounded by clusters of vesicles. A few components of the active zone have been identified such as UNC-10 (Koushika et al., 2001), UNC-13 (Richmond et al., 1999; Aravamudan et al., 1999) and SYD-2 (Zhen and Jin, 1999) but even fewer have been implicated in active zone establishment, differentiation or maintainance.syd-2 encodes a homolog of liprin- and has been shown to be a component of the active zone. Mutations in syd-2 give rise to larger active zones. At a previous meeting, we reported a SYD-2::GFP marker that could be used to visualize active zones in live animals. The SYD-2::GFP protein marker was placed under the promoter of unc-25 which expresses in GABAergic neurons. We have performed a SYD-2::GFP screen to identify a dditional factors involved in the formation of the active zone. An EMS mutagenesis screen was performed looking for mutations affecting the formation of the active zone. Approximately 2500 haploid genomes were screened and we are currently characterizing 5 isolated mutant alleles. hp77 and hp121 represent two loci on chromosome X, hp102 and hp198 represent two loci on chromosome IV and hp129 is on chromosome V. hp121 and hp102 have behavioural unc phenotypes, hp198 are slightly dumpy while hp77 and hp129 do not appear to have any behaviour or physical phenotypes. Initial characterization and phenotypic analysis will be presented on these mutants. It is our hope that the molecular characterization of these mutants will provide information on factors involved in the establishment, differentiation, and maintenance of active zones. Ultimately, this information will provide insight into the genetic pathways used to establish neural synapses in general.References: Aravamudan B, Fergestad T, Davis WS, Rodesch CK, and Broadie K. (1999) Nature Neuroscience. 2:965; Koushika SP, Richmond JE, Hadwiger G, Weimer RM, Jorgensen EM and Nonet M. (2001) Nature Neuroscience. 4:997-1005; Richmond JE, Davis WS and Jorgensen EM. (1999) Nature Neuroscience. 2:959-64; Zhen M and Jin Y. (1999) Nature. 401:371-5

Rebecca M Fox et al. (2005) International Worm Meeting "MAPCeL: Microarray Profiling C. elegans Cells"

Thomas Brodigan, Rebecca M Fox, Michael Krause, Kellen Olszewski, Susan J Barlow, Joan McDermott, W Clay Spencer, David M Miller, Stephen E Von Stetina

[International Worm Meeting, 2005]

Individual C. elegans cells can be easily visualized in vivo with GFP reporters but, owing to their small size, they are generally inaccessible for molecular analysis. We have now developed new methods for the isolation and gene expression profiling of GFP marked C. elegans cells (MAPCeL Microarray Profiling C. elegans Cells). In this strategy, GFP cells are isolated by FACS from in vitro cultures and RNA extracted for application to the C. elegans Affymetrix array. To demonstrate the utility of our approach, we have profiled cells in the embryonic motor circuit; we have identified genes that are differentially expressed in body muscle cells and in each of the three classes of embryonic ventral cord motor neurons (DA, DB, DD). Microarray data in each case were validated by strong correlation with genes known to be expressed in these cells as well as with GFP reporters that we constructed (~80%). For example, our MAPCeL data from inhibitory DD motor neurons confirms enrichment of transcripts required for the GABA phenotype (i.e. unc-25, unc-30, unc-47, snf-11). Conversely, the excitatory DA and DB motor neurons express cholinergic genes (unc-17, cha-1) as well as specific transcription factors that regulate cholinergic signaling (unc-3, unc-4). These data can now be used to identify novel genes with key roles in the locomotory circuit. For example, we have shown that DA motor neurons express a surprisingly diverse array of G-protein coupled receptors with the potential to modulate excitatory activity in response to neuropeptides as well as classical neurotransmitters. MAPCeL data from body muscle cells lead to the discovery that ACR-16 is a component of the levamisole-insensitive nicotinic ACh receptor (See Touroutine et al., this meeting). Finally, by identifying cohorts of genes with common expression patterns we can now search for cis-regulatory elements necessary for cell-specific expression. For example, we have identified enhancer element candidate motifs in the body wall muscle and DA motor neuron datasets (see Olszewski et al, this meeting). We conclude that MAPCeL can be used to generate reliable profiles of cell specific gene expression in the motor circuit and that these data will be valuable resources for correlating gene expression with function.1.Fox, RM, Von Stetina, SE, Barlow, SJ, Shaffer, C, Olszewski, KL, Moore, JH, Dupuy, D, Vidal, M, Miller, DM III. A gene expression fingerprint of C. elegans Embryonic motor neurons. BMC Genomics 2005, 6:42.

Klosterman S M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Regulation of tomosyn (TOM-1) trafficking in C. elegans"

Klosterman S M, Richmond J E

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

Regulated exocytosis of secretory vesicles is critically dependent on the assembly of SNARE complexes between the plasma membrane SNAREs, synatxin and SNAP-25 and vesicle-associated synaptobrevin, which render vesicles fusion competent. Tomosyn, a syntaxin-binding protein, forms a complex with syntaxin and SNAP-25 in competition with synaptobrevin predicted to limit fusogenic SNARE complex formation (Fujita et al, 1998). Consistent with these data, the highly conserved C. elegans tomosyn homolog, TOM-1, has been shown to negatively regulate both synaptic and dense-core vesicle release (Gracheva et al., 2006; Gracheva et al.,2007). TOM-1 appears to be associated with both synaptic vesicles (Takamori et al., 2006) and dense core vesicles (Gracheva et al., 2007) via an unknown mechanism. Consequently, the synaptic localization of TOM-1 is dependent on the anterograde vesicle kinesin motor (UNC-104) (McEwen et al., 2006). Given the importance of TOM-1 in the regulation of synaptic strength, we are interested in identifying the mechanism responsible for the synaptic localization of this protein. Neither syntaxin nor SNAP-25, the two established tomosyn-interacting proteins appear to be involved in TOM-1 transport to synapses (McEwen et al., 2006). In order to screen for TOM-1 trafficking defective mutants, we have generated an integrated transgene expressing TOM-1::GFP. We have verified that this protein is expressed at synapses and localizes in an UNC-104-dependent manner. We are currently screening known vesicle-associated candidate proteins by RNAi or by crossing the TOM-1::GFP transgene into reference alleles to examine their potential role in the regulation of tomosyn trafficking. Fujita Y, Shirataki H, Sakisaka T, Asakura T, Ohya T, Kotani H, YokoyamaS, Nishioka H, Matsuura Y, Mizoguchi A, Scheller RH, Takai Y (1998) Tomosyn: asyntaxin-1-binding protein that forms a novel complex in the neurotransmitterrelease process. Neuron 20:905-915. Gracheva EO, Burdina AO, Touroutine D, Berthelot-Grosjean M, Parekh H,Richmond JE (2007) Tomosyn negatively regulates CAPS-dependent peptide releaseat Caenorhabditis elegans synapses. J Neurosci 27:10176-10184. Gracheva EO, Burdina AO, Holgado AM, Berthelot-Grosjean M, Ackley BD,Hadwiger G, Nonet ML, Weimer RM, Richmond JE (2006) Tomosyn inhibits synapticvesicle priming in Caenorhabditis elegans. PLoS Biol 4:e261.McEwen JM, Madison JM, Dybbs M, Kaplan JM (2006) Antagonistic regulationof synaptic vesicle priming by Tomosyn and UNC-13. Neuron 51:303-315. Takamori S et al. (2006) Molecular anatomy of a trafficking organelle.Cell 127:831-846.