ayyadevara, srinivas et al. (2019) International Worm Meeting "A novel microtubule-binding drug attenuates and reverses protein aggregation in animal models of Alzheimer's disease."

Kakraba, Samuel, Alla, Ramani, Penthala, Narsimha Reddy, Griffin, Sue, Ganne, Akshatha, Barger, Steven, Ayyadevara, Srinivas, Liu , Ling, Shmookler Reis, Robert, Balasubramaniam, Meenakshisundaram, Bommagani, Shoban Babu, Crooks, Peter

[International Worm Meeting, 2019]

Age-progressive neurodegenerative pathologies, including Alzheimer's disease, are distinguished and diagnosed by disease-specific components of intra- or extra-cellular aggregates. Increasing evidence suggests that neuroinflammation promotes protein aggregation, and is involved in the etiology of neurological diseases. We synthesized and tested analogues of the naturally occurring tubulin-binding compound, combretastatin A-4. One such analogue, PNR502, markedly reduced the quantity of Alzheimer-associated amyloid aggregates in the BRI-A?1-42 mouse model of Alzheimer's disease, while blunting the ability of the pro-inflammatory cytokine IL-1? to raise levels of amyloid plaque and its protein precursors in a neuronal cell-culture model. In transgenic C. elegans strains that express human A?1-42 in muscle or neurons, PNR502 rescued A?-induced disruption of motility (3.8-fold, p<0.0001) or chemotaxis (1.8-fold, p<0.05), respectively. Moreover, in C. elegans with neuronal expression of A?1-42, a single day of PNR502 exposure reverses the chemotaxis deficit by 54% (p<0.01), actually exceeding the protection from longer exposure. Moreover, continuous PNR502 treatment also extends nematode lifespan 23% (p?0.001). Given that PNR502 can slow, prevent, or reverse Alzheimer-like protein aggregation in human-cell-culture and animal models, and that its principal predicted and observed binding targets are proteins previously implicated in Alzheimer's, we propose that PNR502 has therapeutic potential to inhibit cerebral A?1-42 aggregation and prevent or reverse neurodegeneration.

Sowa, J. et al. (2019) International Worm Meeting "C. elegans RIG-I homolog drh-1 mediates a transcriptional response to Orsay virus infection."

Wang, D., Somasundaram, L., Troemel, E., Sowa, J., Jiang, H.

[International Worm Meeting, 2019]

The field of C. elegans host/pathogen interactions has historically focused on infections by extracellular pathogens. However, more recently there have been several intracellular pathogens found infecting C. elegans in the wild. These include the Orsay virus and several species of microsporidia, a phylum of eukaryotic intracellular pathogens. Previously, our lab identified a common set of genes that are highly upregulated by both microsporidia and the Orsay virus that we have termed the Intracellular Pathogen Response (IPR) (Bakowski et al, 2014; Reddy et al., 2017). These genes are distinct from genes induced by extracellular pathogens and many other classical stressors. Importantly, mutants with constitutive expression of IPR genes have increased resistance to microsporidia and Orsay virus, indicating the IPR provides defense (Reddy et al., 2019). Previous work demonstrated that the anti-viral RNAi response against Orsay virus in C. elegans involves the RIG-I homolog drh-1, and we now report that drh-1 is also required for IPR induction upon Orsay virus infection. Interestingly, drh-1 is not required for IPR induction by microsporidia or other known IPR triggers. In fact, drh-1 mutants had stronger IPR induction in response to some other triggers. We further found that IPR activation in response to virus does not require known RNAi factors, suggesting that the signaling role of drh-1 in IPR activation is distinct from its previously known role in antiviral RNAi. Previous analysis of transgenic worms expressing the Orsay virus RNA1, which encodes an RNA-dependent RNA polymerase, showed that RNA1 alone was sufficient to activate an IPR reporter, and that this activation required the RDRP activity of RNA1 (Jiang et al, 2017). Here we show this activation is dependent on drh-1, and demonstrate that several IPR genes are induced by this trigger. Together these results indicate that drh-1 triggers the IPR in response to viral dsRNA replication intermediates, and highlight a similarity between worms and mammals, with both detecting viral dsRNAs via a RIG-I -like receptor to activate a protective transcriptional response.

Chen, Barbara et al. (2021) International Worm Meeting "Evaluating the effects of individual Orsay virus proteins on the C. elegans Intracellular Pathogen Response"

Chen, Barbara, Cooper, Emily, Sowa, Jessica

[International Worm Meeting, 2021]

To successfully withstand a pathogen attack, organisms must first recognize the presence of an invader and then mount an immune response. C. elegans lack adaptive immunity and have no known professional immune cells, meaning that they rely exclusively on epithelial innate immunity for pathogen defense. The goal of this project is to better understand the interactions between viruses and host innate immune signaling. The Orsay virus, a natural pathogen of C. elegans, is a positive single-stranded RNA virus with an unusually small genome containing only four proteins (Felix et al., 2011). Orsay viral infection induces an innate immune response in C. elegans known as the Intracellular Pathogen Response (IPR) (Bakowski et al., 2014; Reddy et al., 2017, 2019). The IPR is a transcriptional innate immune response that provides defense against stress and intracellular pathogens (Reddy et al., 2017). The activity of the RNA-dependent RNA polymerase encoded by the Orsay virus RNA1 genome segment is known to trigger induction of the IPR through a mechanism dependent on the DRH-1 receptor, and expression of the RNA-dependent RNA polymerase alone is sufficient to turn on the IPR in the absence of infection (Sowa et al., 2020). However, the individual effects of the other three Orsay viral proteins on IPR induction have not yet been investigated. In this project, we will assess the effects of the Orsay viral capsid, delta, and capsid-delta fusion proteins on induction of the IPR. Thus far, molecular cloning has been used to construct plasmids for heat-shock-inducible overexpression of the capsid, delta, and capsid-delta fusion proteins. These constructs will be used to create transgenic lines overexpressing each of the three viral proteins. To visualize IPR activation levels in these strains, we will use the pals-5p::GFP IPR transcriptional reporter strain, in which the promoter for pals-5 (a gene which is part of the IPR) drives the expression of GFP when the IPR is triggered (Bakowski et al., 2014). By assessing the degree of IPR activation in the viral protein overexpression strains vs non-transgenic siblings, under different stress conditions we will determine whether the Orsay viral capsid, delta, or capsid-delta fusion proteins affect IPR signaling.

Katherine M Walstrom et al. (2002) Mid-west Worm Meeting "Yet Another Germline Helicase"

Byram Ozer, Jens-Christian Paul, Katherine M Walstrom

[Mid-west Worm Meeting, 2002]

RNA helicase A (RHA) is conserved in many organisms and has numerous cellular functions. Human RHA is required for RNA export from the nucleus (Tang et al. (1999) MCB 19, 3540) and the Drosophila homolog, MLE, is required for dosage compensation in male flies (Kuroda et al. (1991) Cell 66, 935). We are characterizing tm329, a strain containing a deletion in rha-1, the C. elegans homolog of RHA. These worms develop normally between 17-20C, but above 23C hermaphrodites accumulate oocytes in the uterus or have atrophied gonads (small, few germ cells). In males, sperm development occurs at lower temperatures but appears to be defective at 25C. Double-mutant worms, gld-1 (q266); rha-1 (tm329), were constructed since human RHA and the human homologue of GLD-1 (Sam68) interact (Reddy et al. (2000) Oncogene 19, 3570), but no epistasis or enhancement was observed in hermaphrodites between 17-25 C. We are continuing experiments to identify the relationship between RHA-1 and other known germline and spermatogenesis proteins. We are grateful to the C. elegans Gene Knockout Consortium for the tm329 strain and to T. Schedl for the rha-1 (q266) strain and for helpful advice.

Villeneuve AM et al. (2000) West Coast Worm Meeting "RNAi Screen for Components of the C. elegans Meiotic Machinery"

Colaiacovo MP, Villeneuve AM, Stanfield GM, Reddy K

[West Coast Worm Meeting, 2000]

Meiosis is the specialized cell division process by which diploid organisms generate haploid gametes. In preparation for the meiosis I division, homologous chromosomes condense, recognize each other, synapse, and undergo recombination. During this process a proteinaceous structure known as the synaptonemal complex (SC) forms between homologs maintaining them in a side-by-side alignment. Crossing over occurs in the context of the SC, leading to formation of chiasmata that connect the homologs and allow them to orient toward opposite poles of the meiosis I spindle. We are using a functional genomics strategy to identify genes required for these key meiotic prophase events, taking advantage of a genome-wide survey of germline gene expression conducted by V. Reinke and collaborators in the lab of S. Kim. We defined a subset of 206 germline-enriched genes whose expression profiles most closely match those of known meiosis genes, and designed a screen to identify those genes for which RNAi elicits defects in meiotic prophase. Defects in pairing, synapsis or recombination that lead to chromosome non-disjunction are initially identified by the production of inviable embryos and an increased frequency of male progeny. Defects that lead to arrest in meiotic progression are initially identified by a sterile phenotype. Candidates fitting either of these profiles are then subjected to cytological analysis to investigate possible meiotic defects. The efficacy of our screening procedure has been validated using genes encoding known meiotic recombination and synapsis components as positive controls. Our initial screening has already identified several distinct phenotypic classes with defects in chromosome behavior and/or morphology. These define genes with putative roles in meiotic chromosome synapsis (see accompanying poster by Reddy et al.), as well as genes that may have both mitotic and meiotic roles. A summary of the results of this ongoing screen will be presented.

Guruprasada R. Sure et al. (2006) East Asia C. elegans Meeting "Identifying the players involved in the transport of mitochondria in Caenorhabditis elegans neurons"

Swetha Mohan, Guruprasada R. Sure, Shaili N Johri, Sandhya P Koushika

[East Asia C. elegans Meeting, 2006]

The growth, maintenance, and survival of neurons rely critically on organelle transport between the cell body and the synapse. Mitochondria are involved in the ATP production, calcium homeostasis and synaptic vesicles recycling. Mitochondria, unlike other organelles are transported bi-directionally and remain stationary 75% of the time. In neurodegenerative diseases such as Alzheimer""s and Parkinson""s, abnormal mitochondrial distribution has been observed. We are trying to identify the molecular players involved in the transport of mitochondria using classical genetic approaches. To address this question we take a classical genetic approach using a transgenic strain jsIs609 in which GFP is expressed in the mechanosensory neurons and targeted to the mitochondrial matrix. We have performed EMS mutagenesis and screened around 3000 genomes and isolated 17 mutants. These fall into different classes, such as 100% increase in number of mitochondria in axons, 50-75% fewer mitochondria in the axons, abnormal accumulation of mitochondria in minor neurites and large spherical mitochondria in the cell bodies of the neurons. We are currently performing complementation analyses of these mutants. We have also analyzed the distribution of mitochondria in mutants with known roles for the transport of organelles and in the dynamics of mitochondria, such as anc-1, unc-16, unc-116, rab-7 and unc-73. In unc-116 Kinesin-I mutants fewer mitochondria are observed in the axons whereas unc-16 ortholog of Sunday driver have more number of mitochondria in the axons. To analyze the mitochondrial behavior/dynamics with respect to metabolism we have performed starvation analyses. We find that the cell bodies of the neurons are fragmented during starvation but the axons are spared and are normal. We are currently imaging mitochondrial motility and dynamics across the developmental stages. # Guruprasada Reddy Sure and Swetha Mohan contributed equally to this work.

Martin, Natalia et al. (2015) International Worm Meeting "Natural genetic variation in Caenorhabditis elegans response to bacterial pathogens."

Aballay, Alejandro, Martin, Natalia

[International Worm Meeting, 2015]

Caenorhabditis elegans responds to pathogenic bacteria by inducing specific innate immune pathways and pathogen avoidance behaviors. These mechanisms share the requirement of microbe and/or damage associate molecular patterns recognition. As a result, these two distinct physiological responses could have common molecular components. This is the case of the neuronal G-protein coupled receptor (GPCR) related to the mammalian neuropeptide Y receptor, NPR-1. Previous studies from our laboratory, aimed to identify the molecular mechanisms involved in neural-immune communications, showed that the GPCR NPR-1 is required to mount an immune response to different bacterial pathogens (1). However, NPR-1 was also shown to be essential to promote an avoidance behavior to the pathogen Pseudomonas aeruginosa (1, 2), which highlights the importance of NPR-1 in the different physiological responses to pathogenic bacteria.In this study, we aimed to identify key components of the NPR-1 neuronal circuit that could be beneficial for C. elegans survival in nature. We compared the response to pathogens of animals carrying an npr-1 mutation in the wild-type (N2) background with that of different C. elegans wild isolates that bear the same mutation in the npr-1 gene. We identified several isolates that showed a differential response to P. aeruginosa, due to differences in their genetic backgrounds. In order to map and characterize the mechanisms that compensate the effect of the npr-1 mutation in these wild isolates, we used a combination of single nucleotide polymorphism and whole genome sequencing mapping strategy (3). Using this approach we map the compensatory phenotype to a region in chromosome V of one particular environmental isolates and we are currently in the process of identifying the responsible gene among the potential candidates.References: 1. Styer et al. Science 322, 460 (Oct 17, 2008); 2. Reddy et al. Science 323, 382 (Jan 16, 2009); 3. Doitsidou et al. PLoS One 5, e15435, doi:10.1371/journal.pone.0015435 (2010).

Sfarcic, I. et al. (2019) International Worm Meeting "Nanoluciferase-based method for detecting gene expression in C. elegans."

Bui, T., Troemel, E., Sfarcic, I.

[International Worm Meeting, 2019]

Gene expression in C. elegans is commonly monitored with the use of promoter::GFP fusions. However, GFP has poor signal-to-noise properties, particularly in tissues with high autofluorescence, like the C. elegans intestine. We developed a very sensitive, plate-based assay to quantitatively monitor gene expression with the use of nanoluciferase (NanoLuc, Promega), which exceeds the brightness of other glow-type luciferases like Firefly and Renilla by ~150-fold. First, we used a strain that constitutively expresses a single-copy NanoLuc gene under the intestinal promoter vha-6p. We combined worm lysate from vha-6p::nanoluc animals with the substrate furimazine (Promega) in 96-well plates and measured luminescent signal on a conventional plate reader. We optimized grinding temperature, plate type, substrate incubation time and explored the limitations of sample storage. With optimized conditions, we detected a robust signal over background (wild type N2) in all life stages of single worms from L1s (~15-fold) to adults (~450-fold). We even saw a 5-fold signal over background in 1:100 dilutions of single worms. In contrast, we were unable to detect a signal over background of a single-copy of GFP driven by the vha-6 promoter in less than 1000 adults using a plate reader. Further, we measured a ~280-fold luminescent signal over background when we 'hid' a single vha-6p::nanoluc animal in a pool of 5000 N2s. Our results demonstrate that NanoLuc is suitable for plate-reader based reporter assays and has virtually no background signal in wild type worms. Additionally, we tested NanoLuc as an inducible genetic reporter under pals-5 promoter control. The pals-5 gene is used as a read-out for the Intracellular Pathogen Response (IPR), which is a defense program induced by diverse, natural intracellular pathogens (Reddy et al, 2019). The IPR is also activated by proteotoxic stress. With NanoLuc, we measured a ~50-fold increase in luminescent signal after infection with the intracellular pathogen N. parisii, a ~300-fold increase upon treatment with the proteasome inhibitor bortezomib and a ~4-fold increase upon prolonged heat stress. Overall, we present NanoLuc as a powerful constitutive and inducible genetic reporter in C. elegans.

Byrnes, Catherine et al. (2021) International Worm Meeting "Nematode hunters: a citizen science approach to identifying new systems for the study of host-virus interactions"

Sowa, Jessica, Byrnes, Catherine

[International Worm Meeting, 2021]

C. elegans is a popular model organism that has proved very useful for studying the cell biology of intracellular infections. However, its use as a model for the study of host-virus interactions has been limited by the fact that only one natural viral pathogen of C. elegans has been identified to date (Felix and Wang, 2019; Franz et al., 2014). The goal of this project is to identify novel natural nematode viruses capable of infecting C. elegans by mobilizing ordinary citizens to collect wild nematodes. Studying the interactions of different types of viruses with their host's cells can provide new insights into cell biology and host-pathogen interactions. To date, only four viruses naturally infecting Caenorhabditis nematodes have been identified, and of those only one (Orsay virus) infects C. elegans (Felix et al., 2011; Frezal et al., 2019). In the past, identification of intracellular pathogens in wild-caught nematodes has relied on detection by microscopy of morphological changes caused by the infection (Felix et al., 2011; Troemel et al., 2008). This approach is relatively low throughput and requires an expert screener. Our approach instead uses a fluorescent reporter-based method, taking advantage of a set of genes which are expressed at low levels in basal conditions but highly upregulated during infection by intracellular pathogens (Bakowski et al., 2014; Reddy et al., 2017, 2019). Co-culturing infected nematodes together with C. elegans expressing these intracellular infection reporters produces fluorescence which is easily detected on a fluorescence dissecting microscope. By using this method on a large sampling of wild-caught nematodes, we hope to identify novel nematode viruses which can be transmitted to C. elegans. In the pilot phase of this project, we established protocols for wild nematode collection which require minimal supplies and can be performed at home by people with no particular science background after viewing a series of short training videos. We have successfully cultured wild nematodes from these samples in the lab, and have established systems for sample intake, expansion and frozen stocking of the strains, performing co-culture experiments, and sharing experimental results with the original collectors. In the fall of 2021, we hope to expand this project by partnering with educators at a variety of levels on a larger scale who would be interested in incorporating nematode hunting into their science curriculum.

William R Goodyer et al. (2005) International Worm Meeting "Meiotic Recombination Initiation Requires HTP-3, a Chromosome Core Component Essential for Homolog Alignment and Synapsis"

William R Goodyer, Monique Zetka

[International Worm Meeting, 2005]

A distinctive feature of meiosis is the assembly of a proteinaceous core, composed of cohesins and meiosis-specific components, between replicated sister chromatids. The nematode non-cohesin chromosome core component HIM-3 is required for critical meiotic events including homolog alignment, but not for the initiation of recombination. While recombination is known to depend on the presence of double-strand DNA breaks (DSBs) mediated by the conserved topoisomerase SPO-11, the regulation of DSB formation during meiosis remains poorly understood in any system. We are investigating the localization and function of HTP-3 (HIM Three Paralog), which we have identified as a HIM-3 interacting protein in yeast two-hybrid screens. Consistent with this interaction, immunostainings with anti-HTP-3 antibodies localize the protein to meiotic chromosome cores and HIM-3 fails to localize in htp-3(RNAi) germ lines. Furthermore, in the absence of HTP-3, sister chromatid cohesion remains intact, but meiotic defects consistent with a failure to recruit HIM-3 to chromosome cores are observed: the abrogation of homolog alignment, synapsis, and accompanying nuclear reorganization. Unlike him-3 mutants, however, we found that these defects were accompanied by an inability to initiate meiotic DSBs. First, RAD-51 a DSB repair enzyme required for recombination is detectable in htp-3(RNAi) germ lines at levels not different from those of spo-11(ok79) null mutants in which DSBs fail to form. Our inability to detect RAD-51 foci in htp-3(RNAi) germ lines is not attributable to a defect in the localization of RAD-51 since the protein readily localizes to artificially provided DSBs induced by gamma-irradiation. Furthermore, the DSB-dependent decondensation phenotype of diakinesis chromosomes in rad-51 null mutants is rescued in a rad-51(lg8701); htp-3(RNAi) background, indicating a requirement for HTP-3 in DSB formation, rather than RAD-51 function. Recently, HIM-17 mediated changes in histone modification have been suggested to be required for meiotic DSBs (Reddy and Villeneuve 2004), however, we show that HTP-3 acts independently of HIM-17. Our results indicate that HTP-3 represents a novel regulator of DSB formation and provides the first evidence for the requirement of a chromosome core component in mediating the initiation of recombination in a multicellular organism. Furthermore, HTP-3 also localizes to chromatin of mitotic germ line nuclei, in a cohesin-dependent manner, prior to entry into meiosis thereby providing a link between mitotic chromosome organization, meiotic chromosome axis morphogenesis and critical early meiotic events. Supported by NSERC & CIHR