Sowa, Jessica et al. (2015) International Worm Meeting "Olfactory Regulation of C. elegans Reproductive Aging."

Wang, Meng, Mutlu, Ayse Sena, Sowa, Jessica

[International Worm Meeting, 2015]

As women age, they experience both a decline in fertility and an increased risk of miscarriage and birth defects. In addition to purely genetic contributions, reproductive aging is thought to be regulated by the complex interactions of genetic signaling pathways and environmental conditions. However, the molecular mechanisms integrating environmental and genetic signals to regulate reproductive aging remain unknown. Caenorhabditis elegans experience their diet of bacteria as a source of both metabolic and sensory input. We took advantage of this relationship as a convenient method of introducing environmental variation by raising C. elegans on different bacterial diets to investigate the effect of environment on reproductive aging. We found that C. elegans exposed to different bacterial environments show significant differences in the duration of their reproductive span. To further dissect the molecular mechanisms underlying these reproductive span differences we chose to focus on two strains of Escherichia coli (OP50 and HB101) that gave drastically different effects on C. elegans reproduction. We found that worms raised on OP50 reproduce longer and maintain their fertility later than worms raised on HB101. This effect is mediated by a pair of olfactory neurons which perceive a volatile odorant signal from the HB101 E.coli. The presence or absence of this environmental cue affects germline proliferation and maintenance in C. elegans, ultimately contributing to the timing of reproductive senescence. We have identified a pair of olfactory neurons, the AWB neurons,that are specifically required for this olfaction-mediated reproductive adaptation as well as for chemotaxis of C. elegans to the smell of the HB101 bacterial diet. Optogenetic activation of the AWB neurons confirmed that AWB activation was sufficient to induce reproductive span shortening in the absence of HB101. Finally, we identified neuropeptide release as the mechanism of AWB neurotransmission in response to the HB101 odorant. Together, our results reveal a novel pathway for the regulation of reproductive aging in C. elegans, and suggest the relevance of environment-sensitive signaling mechanisms in regulating the onset and progression of reproductive aging.

Sowa, Jessica et al. (2017) International Worm Meeting "The role of the decapping enzyme EOL-1 in intracellular infection response."

Troemel, Emily, Sowa, Jessica

[International Worm Meeting, 2017]

Microsporidia comprise a phylum of obligate intracellular parasites most closely related to fungi. Although they infect a wide range of animal species and can cause lethal infections in immunocompromised patients, relatively little is known about the mechanisms of host defense against microsporidia. Nematocida parisii is a species of microsporidia that is a natural pathogen of C. elegans. N. parisii infects the C. elegans intestine, causing fusion of intestinal cells and ultimately lethality (Troemel et al, PLoS Biology 2008; Balla et al, Nature Microbiology 2016). Our lab has profiled the C. elegans transcriptional response to N. parisii (Bakowski et al, PLoS Pathogens 2014), with the goal of identifying features of the host response to microsporidia infection. One gene found to be induced by N. parisii infection was eol-1, a de-capping and exonuclease enzyme that has been reported by Yun Zhang's lab to be involved in regulating pathogen-induced aversive olfactory learning (Shen et al, J. Neuroscience 2014). Our RNAseq data shows that eol-1 is strongly up-regulated during N. parisii infection, beginning early at 8 hours post infection. In uninfected worms, we find that EOL-1 is expressed primarily in the spermatheca and URX neurons based on analysis of EOL-1 transcriptional and translational fusions*. Interestingly we find that when worms are exposed to N. parisii, eol-1 expression is induced in the intestine, which is the tissue targeted by N. parisii. Our preliminary results from analysis of eol-1 mutants suggest that eol-1 plays a role in host response to N. parisii infection. *Thanks to Yun Zhang's lab for sending eol-1 strains.

Sowa, Jessica et al. (2013) International Worm Meeting "Regulation of C. elegans Reproductive Aging by a Novel Gene-Environment Signaling Mechanism."

Wang, Meng, Sowa, Jessica

[International Worm Meeting, 2013]

Reproductive senescence is a hallmark of aging, the onset of which can be modulated by both genetic and environmental factors. However, the molecular mechanisms that integrate environmental and genetic signals to regulate the onset and progression of reproductive aging remain largely unknown. Here we report the first known instance of a gene-environment signaling mechanism functioning to regulate reproductive senescence in Caenorhabditis elegans. We found that C. elegans fed the standard lab diet of OP50 E. coli reproduce significantly longer than C. elegans fed the alternate diet of HB101 E. coli. This effect is mediated by the AWB olfactory neurons, which perceive a volatile odorant signal produced by HB101 E. coli. The presence or absence of this sensory cue specifically affects germline proliferation and maintenance, ultimately contributing to the timing of reproductive senescence. The effect of the HB101 odorant on reproductive span was found to be independent of all previously identified pathways for the regulation of reproductive aging in C. elegans, indicating that it acts through a novel regulatory mechanism. Furthermore, we have found that serotonin signaling plays a role as a downstream effector of this response. Together, these studies describe a previously unknown regulatory mechanism for reproductive senescence, and suggest the significance of gene-environment interactions in the regulation of reproductive aging.

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.

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.

Sullivan-Brown, Jessica et al. (2021) International Worm Meeting "Characterization of RNAi phenotypes in C. elegans from understudied genes in a Cell and Molecular Biology course"

Sowa, Jessica, Sullivan-Brown, Jessica

[International Worm Meeting, 2021]

The characterization of relatively understudied genes in C. elegans using RNA interference is an excellent approach for designing undergraduate research-based labs. Here we describe a successful lab course in which Cell and Molecular Biology students at West Chester University of Pennsylvania (WCU) make novel discoveries associated with knock down of dcaf-13, a likely oncogene in humans. Each class represents the flow of a typical study of gene function, beginning with a journal club reading the primary literature. Then students use bioinformatics to identify the human protein sequence of DCAF13, perform a BLAST search to find C. elegans homologs, and use WormBase to identify the C. elegans gene sequence. An RNAi knock down experiment is then performed with dcaf-13. Throughout the lab, students characterize the resulting phenotypes, using IMAGEJ/FIJI for quantification of worm length and fluorescence microscopy for GFP analysis of an intestinal cell fate marker. Students then isolate RNAs using Trizol RNA extraction and perform cDNA synthesis. Students then design primers to test if dcaf-13 knock down affects the expression of different cell cycle genes by performing RT-PCR. At the end of the course, students present their work orally in a poster presentation and in written communication in a primary research style paper. We have recently added a component in which students design a future experiment in the format of a small grant proposal and after peer review, two projects are chosen to perform in lab. The lab serves two primary functions: to provide students opportunities to advance their experimental techniques so they are competitive in the job market and create an experience that mimics a typical research project by making real contributions to science. Our goals are to publish the work the students generated. Importantly, this framework can be applied to other understudied genes in C. elegans in which the RNAi phenotypes are not well documented, benefiting both students and the C. elegans community.

Neve, Isaiah et al. (2015) International Worm Meeting "Modified OP50 E. coli expressing dsRNA elicite a robust RNA interference response in C. elegans."

Wang, Meng, Sowa, Jessica, Neve, Isaiah

[International Worm Meeting, 2015]

In the majority of research laboratories using C. elegans as a model, they sustain their animals on the B strain E. coli OP50. This strain is easy to manipulate in the laboratory, and has provided us with a key platform for defining many genetic pathways. Simultaneously, the series of revolutionary realizations which culminated in the 2006 Nobel Prize, quickly allowed the technique of dsRNA feeding to incite RNA interference (RNAi) to become a key technique in many laboratories. Two major resources are available to the RNAi wielding researcher, the Ahringer and Vidal libraries respectively. These libraries house thousands of E. coli clones with unique plasmids expressing dsRNA corresponding to individual C. elegans genes. Countless experimental insights have been facilitated using these resources. However, both libraries are housed in the HT115 E. coli K12 strain background. Several recent studies (Brooks et al., 2009; Soukas et al., 2009; Pang & Curran 2014) have revealed the importance of bacterial environment in eliciting certain genetically induced phenotypes. With the realization that gene-environment interactions may play a key role in the elucidation of genetic pathways, we have generated an OP50 strain capable of dependably housing and robustly expressing dsRNA plasmid vectors. Furthermore, when C. elegans are fed this OP50 strain, they activate a substantial RNAi response against the target gene of interest. We anticipate that this resource will allow the further characterization of gene-environment specific relationships. .

Maaike C. W. van den Berg et al. (2006) European Worm Meeting "Sex-Dependent Resistance to the Fungal Pathogen Cryptococcus neoformans"

Maaike C. W. van den Berg, Jessica Z. Woerlee, Robin C. May, Hansong Ma

[European Worm Meeting, 2006]

Maaike C. W. van den Berg1,2, Jessica Z. Woerlee2, Hansong Ma1,2 & Robin C. May1. Cryptococcus neoformans is the causative agent of cryptococcosis, a fatal fungal disease of immunocompromised patients. Our group and others have made use of C. elegans as an alternative host for Cryptococcus, in order to investigate the molecular basis of host-pathogen interactions. Using this system, we now report that male C. elegans show greater resistance to killing by Cryptococcus than hermaphrodite animals and that this resistance can be induced in hermaphrodite animals by inappropriate activation of the male sex-determination pathway. Resistance is molecularly determined, rather than resulting from behavioural changes or reproductive differences, and requires the activity of the stress-response transcription factor DAF-16. Finally, we demonstrate that resistance to Cryptococcus neoformans correlates broadly with longevity within the Caenorhabditis genus. Our results suggest that many of the molecular determinants of longevity and immunity are the same and that differential regulation of these determinants may underlie much sex-dependent and species-dependent variation.

Hodul, M. et al. (2019) International Worm Meeting "WD40-repeat proteins promote the stability of the deubiquitinating enzyme USP-46 and surface levels of the glutamate receptor GLR-1."

Ganji, R., Raman, M., Dahlberg, C., Hodul, M., Juo, P.

[International Worm Meeting, 2019]

Addition and removal of ubiquitin regulates the abundance of glutamate receptors at synapses. We previously showed that the conserved deubiquitinating enzyme (DUB) Ubiquitin Specific Protease-46 (USP-46) regulates the abundance of the glutamate receptor GLR-1 in C. elegans (1). usp-46 loss-of-function mutants have decreased levels of GLR-1 in the ventral nerve cord (VNC) and corresponding defects in glutamatergic behavior. We showed that USP-46 deubiquitinates GLR-1 and protects it from lysosomal degradation. While an increasing number of neuronal DUB substrates are beginning to be defined, the mechanisms that regulate DUBs are poorly understood. USP-46 function appears to be tightly controlled in vivo because overexpression of usp-46 by itself does not increase GLR-1 abundance. We, and others, showed that two WD40-repeat proteins, WDR-48 and WDR-20, can interact with USP-46 and stimulate its catalytic activity (2-4). Furthermore, we found that overexpression of WDR-48, WDR-20 and USP-46 in neurons results in increased levels of GLR-1 and glutamatergic behavior (4). Here we show that USP-46, WDR-48, and WDR-20 act together and are required to regulate surface levels of GLR-1 in the VNC based on analysis of GLR-1 tagged with pH-sensitive superecliptic pHluorin (5). We found that WDR-48 promotes USP-46 protein levels in HEK293 cells and in GLR-1-expressing interneurons. We show that USP-46 is ubiquitinated and degraded in the proteasome and that WDR-48 promotes USP-46 stability by preventing USP-46 ubiquitination. This effect is mediated by direct binding of WDR-48 to USP-46, because point mutations that disrupt this interaction abrogate WDR-48 regulation of USP-46 and GLR-1 surface abundance. Interestingly, overexpression of wdr-20, but not wdr-48, promotes GLR-1 surface abundance in an usp-46-dependent manner suggesting that regulation of wdr-20 expression may provide a novel mechanism to control synaptic GLR-1. Together, these data suggest a model where WDR-48 increases the stability of USP-46 and, together with WDR-20, promotes USP-46 activity, surface levels of GLR-1, and glutamatergic behavior. (1) Kowalski et al. (2011) J Neurosci 31:1341-1354. (2) Kee et al. (2010) J Biol Chem 285:11252-11257. (3) Sowa et al. (2009) Cell 138:389-403. (4) Dahlberg and Juo. (2014). J Biol Chem 289:3444-3456. (5) Hoerndli et al. (2013). Neuron 80:1421-1437.

Hodul, Molly et al. (2017) International Worm Meeting "Regulation of the deubiquitinating enzyme USP-46 and the glutamate receptor GLR-1 by WD40 repeat proteins."

Juo, Peter, Hodul, Molly, Dahlberg, Caroline L.

[International Worm Meeting, 2017]

Ubiquitin-mediated endocytosis and degradation of glutamate receptors controls their synaptic abundance and is implicated in modulating synaptic strength, learning, and memory. Ubiquitination of GLR-1, a C. elegans AMPA-type glutamate receptor, promotes receptor internalization and degradation in the lysosome (1). Ubiquitination can be reversed by deubiquitinating enzymes (DUBs). We previously found that the DUB Ubiquitin-Specific Protease 46 (USP-46) regulates GLR-1 levels (2). USP-46 removes ubiquitin from GLR-1 and likely acts at endosomes to protect GLR-1 from lysosomal degradation. Consistently, usp-46 null mutants have decreased GLR-1 at synapses in the ventral nerve cord (VNC) and corresponding defects in GLR-1-dependent behaviors (2). In contrast, overexpression of usp-46 does not have the converse effect, suggesting that USP-46 activity is tightly controlled in neurons. Very little is known about how DUBs are regulated in vivo. Two WD-40 repeat proteins, WDR-48 and WDR-20, were previously shown to interact with USP-46 in mammalian cells (3-5). We showed that the C. elegans homologs of these WDR proteins bind USP-46 and stimulate DUB catalytic activity in vitro (6). Overexpression of wdr-48 and wdr-20 in neurons in vivo results in increased synaptic levels of GLR-1, and this effect is further enhanced by co-expression of usp-46 (6). Here we investigate several mechanisms by which the WDR proteins regulate USP-46 and GLR-1. First, we found that overexpression of wdr-48 and wdr-20 increase the abundance of USP-46 in HEK cells and in neurons in vivo. We will take advantage of point mutations known to disrupt the binding between each WDR and USP-46 to test if their direct interaction is required for the increased levels of USP-46. Second, we have preliminary evidence suggesting that WDR-48 recruits USP-46 to endosomes where it is presumed to act on ubiquitinated GLR-1. Finally, we recently found that wdr-20 but not wdr-48 transcription is regulated by chronic synaptic activity-blockade, which has previously been shown to increase synaptic GLR-1 (7). Thus, WDR-48 and WDR-20 are emerging as key regulators that appear to use multiple mechanisms to control USP-46 and its effects on GLR-1. 1. Burbea et al. (2002) Neuron 2. Kowalski et al. (2011) J Neurosci 3. Sowa et al. (2009) Cell 4. Cohn et al. (2009) JBC 5. Kee et al. (2010) JBC 6. Dahlberg & Juo (2014) JBC 7. Grunwald et al. (2004) PNAS