Hedtke SM et al. (2019) Front Genet "Genomic Epidemiology in Filarial Nematodes: Transforming the Basis for Elimination Program Decisions."

Lau CL, Hedtke SM, Crawford KE, Graves PM, Boussinesq M, Kuesel AC, Boakye DA, Grant WN

[Front Genet, 2019]

Onchocerciasis and lymphatic filariasis are targeted for elimination, primarily using mass drug administration at the country and community levels. Elimination of transmission is the onchocerciasis target and global elimination as a public health problem is the end point for lymphatic filariasis. Where program duration, treatment coverage, and compliance are sufficiently high, elimination is achievable for both parasites within defined geographic areas. However, transmission has re-emerged after apparent elimination in some areas, and in others has continued despite years of mass drug treatment. A critical question is whether this re-emergence and/or persistence of transmission is due to persistence of local parasites-i.e., the result of insufficient duration or drug coverage, poor parasite response to the drugs, or inadequate methods of assessment and/or criteria for determining when to stop treatment-or due to re-introduction of parasites <i>via</i> human or vector movement from another endemic area. We review recent genetics-based research exploring these questions in <i>Onchocerca volvulus</i>, the filarial nematode that causes onchocerciasis, and <i>Wuchereria bancrofti</i>, the major pathogen for lymphatic filariasis. We focus in particular on the combination of genomic epidemiology and genome-wide associations to delineate transmission zones and distinguish between local and introduced parasites as the source of resurgence or continuing transmission, and to identify genetic markers associated with parasite response to chemotherapy. Our ultimate goal is to assist elimination efforts by developing easy-to-use tools that incorporate genetic information about transmission and drug response for more effective mass drug distribution, surveillance strategies, and decisions on when to stop interventions to improve sustainability of elimination.

Jeong J et al. (2019) Nanotoxicology "Developing adverse outcome pathways on silver nanoparticle-induced reproductive toxicity via oxidative stress in the nematode Caenorhabditis elegans using a Bayesian network model."

Choi I, Song T, Choi J, Chatterjee N, Jeong J, Cha YK

[Nanotoxicology, 2019]

An adverse outcome pathway (AOP) is a framework that organizes the mechanistic or predictive relationships between molecular initiating events (MIEs), key events (KEs), and adverse outcomes (AOs). Previously, we intensively investigated the molecular mechanism that underlies toxicity caused by AgNPs in the nematode Caenorhabditis elegans. Using transcriptomics, functional genetics, and various molecular/biochemical tools, we identified oxidative stress as the major mechanism underlying toxicity and reproduction failure as the outcome. With this information, here we conducted a case study of building an AOP to link oxidative stress with reproductive toxicity. To validate this AOP, we filled the gaps by conducting further experiments on its elements, such as NADPH oxidase, ROS formation, PMK-1 P38 MAPK activation, HIF-1 activation, mitochondrial damage, DNA damage, and apoptosis. The establishment of a causal link between the MIE and AO is critical for the construction of an AOP. Therefore, causal relationships between each KE and AO were verified by using functional genetic mutants of each KE. By combining these experimental data with our previously published results, we established causal relationships between the MIE, KEs, and AO using a Bayesian network (BN) model, culminating in an AOP entitled 'NADPH oxidase and P38 MAPK activation leading to reproductive failure in C. elegans ( https://aopwiki.org/aops/207)' . Overall, our approach shows that an AOP can be developed using existing data and further experiments can be conducted to fill the gaps between the MIE, KEs, and the AO. This study also shows that BN modeling has the potential to identify causal relationships in an AOP.

Hipkiss AR (2007) Mech Ageing Dev "On why decreasing protein synthesis can increase lifespan."

Hipkiss AR

[Mech Ageing Dev, 2007]

An explanation is offered for the increased lifespan of Caenorhabditis elegans when mRNA translation is inhibited due to loss of the initiation factor IFE-2 [Hansen, M., Taubert, T., Crawford, D., Libina, N., Lee, S.-J., Kenyon, C., 2007. Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Ageing Cell 6, 95-110; Pan, K.Z., Palter, J.E., Rogers, A.N., Olsen, A., Chen, D., Lithgow, G.J., Kapahi, P., 2007. Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans. Ageing Cell 6, 111-119; Syntichaki, P., Troulinaki, K., Tavernarakis, N., 2007. eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans. Nature 445, 922-926]. It is suggested that the general reduction of protein synthesis, due to the decreased frequency of mRNA translation, also lowers the cellular load of erroneously synthesized polypeptides which the constitutive protein homeostatic apparatus (proteases and chaperones proteins) normally eliminates. This situation results in "spare" proteolytic and chaperone function which can then deal with those proteins modified post-synthetically, e.g. by oxidation and/or glycation, which are thought to contribute to the senescent phenotype. This increased availability of proteolytic and chaperone functions may thereby contribute to the observed increase in organism stress resistance and lifespan.

Gross, E. et al. (2013) International Worm Meeting "The neuroglobin GLB-5 regulates C. elegans responses to hypoxic exposure."

Soltesz, Z., Gross, E., Zelmanovich, V., de-Bono, M.

[International Worm Meeting, 2013]

Fine-tuned O2 metabolism is essential for most animals, requiring animals to monitor and adapt to changes in O2 levels. We previously identified a polymorphic neuroglobin, GLB-5, that acts in O2-sensing neurons and enables C. elegans to respond to small changes in O2 concentration (Persson et al, 2009). Here we show that GLB-5 is essential for fast behavioural recovery after exposure to hypoxia. Whereas glb-5(Haw) animals recovered from hypoxia within minutes, glb-5(Bri) recovered slowly, over four hours. By combining genetics, biochemistry, and Calcium imaging we provide evidence that glb-5 enables fast recovery of O2-sensing neurons after prolonged hypoxic exposure. We designed mutagenesis screens to explore how GLB-5 regulates recovery from hypoxia. One mutant we identified disrupts a conserved chaperone that regulates the activity of the O2 sensing neurons. The chaperone regulates the spatial organization of soluble guanylate cyclases in these neurons, altering the way they interact with the neuroglobin to control the O2 response properties of these neurons. Persson A, Gross E, Laurent P, Busch KE, Bretes H, de Bono M (2009) Natural variation in a neural globin tunes oxygen sensing in wild Caenorhabditis elegans. Nature 458: 1030-1033.

Preston S et al. (2017) FASEB J "Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain."

Sternberg PW, Ansell BRE, Andrews KT, Nowell C, Chang BCH, Hofmann A, Crawford S, Korhonen PK, Baell J, Gijs MAM, Fisher GM, Young ND, Preston S, Mouchiroud L, Gasser RB, Jabbar A, Auwerx J, Davis RA, McGee SL, Cornaglia M

[FASEB J, 2017]

As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mito-ribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

Lapierre L et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "The TOR-regulated process of autophagy is required for lifespan extension in germline-less C. elegans"

Lapierre L, Van Gilst M, Nunez L, Gelino S, Hansen M, Melendez A

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

Autophagy is a key biological process by which cellular components are degraded and recycled, and this process plays important roles during organismal development and aging. For instance, we and others have recently shown that autophagy is upregulated in several C. elegans mutants with extended longevity (1). Such TOR mutants require autophagy genes to live long (1, 2), suggesting a critical role for autophagy in the lifespan extension induced by low TOR signaling. Removal of germline stem cells in C. elegans also extends lifspan, potentially in a conserved fashion as signals from the reproductive system also extend the lifespan of flies and mice (3). The cellular mechanisms by which germline stem cell ablation extends lifespan are presently unclear, but are likely to be affected by the nutritional status of the animal (4). We therefore asked if TOR and the TOR-regulated process of autophagy are critically involved in the longevity response induced by the lack of germline stem cells in C. elegans. We find that long-lived, germline-less animals, mimicked by a mutation in the glp-1/Notch receptor, had decreased levels of TOR, and autophagy was induced in these animals. Moreover, RNAi of several different autophagy genes significantly shortened the lifespan of adult glp-1 mutants while having no effect on wild-type animals, indicating a specific role for autophagy in the extended longevity of germline-less worms. Taken together, these observations suggest the existence of links between TOR, autophagy, and aging in C. elegans lacking a germline. We are currently investigating these novel links in more molecular detail. (1) Hansen et al., PLoS Genetics, 2008 (2) Toth et al., Autophagy, 2008(3) Kenyon, Nature, 2010 (4) Crawford et al., Aging Cell, 2007

Hart, Michael et al. (2015) International Worm Meeting "Age-related deterioration of neurons in male C. elegans."

Hart, Michael, Hobert, Oliver

[International Worm Meeting, 2015]

Aging neurons undergo changes in structural and functional integrity that result in neuronal dysfunction associated with both normal aging and neurodegenerative diseases. C. elegans provide a powerful model for studying the development of the nervous system, and have been instrumental in the study of pathways and processes involved in aging. Neurons in hermaphrodite C. elegans show mild morphological changes with increasing age, including neurite sprouting and branching, axon beading and defasciculation, and synapse deterioration (1, 2, 3). The nervous system of male C. elegans shares 294 neurons in common with hermaphrodites, but also contains 89 additional sex-specific neurons and a significantly more complex connectivity, resulting in the male-specific mating behavior (4). With these differences in mind, we analyzed aging male C. elegans for neuronal age-related changes. Using fluorescent neuronal reporters, we found that the posterior male nervous system can undergo dramatic age-related changes prior to death, including progressive axon denervation and degeneration, which is not seen in hermaphrodites. This male-specific neuronal degeneration occurs in different neuron types at different frequencies. We have identified neuron specification and signaling mutants that can rescue the age-related neuronal degeneration phenotype in males, independent of general tissue deterioration. We are currently testing the hypothesis that inherent excitability of the male-mating circuit may contribute to these male-specific age-related neuronal changes (5).1) Tank EM, Rodgers KE and Kenyon C. 2011, J. Neurosci 31(25):9279-9288. 2) Toth ML et al. 2012, J. Neurosci 32(26): 8778-8790. 3) Pan C-L et al. 2011, PNAS 108: 22 2010. 4) Jarrell TA et al. 2012, Science 337, 437. 5) Guo X et al. 2012, Neurobiol Aging 33(7):1483.

Aronoff R et al. (1996) West Coast Worm Meeting "PLASMID RESCUE FINALLY. IS SMG-4 A PROTEIN WITH AN RRM AND A KE RICH DOMAIN?"

Hodgkin JA, Baran R, Aronoff R

[West Coast Worm Meeting, 1996]

The seven smg genes mediate mRNA surveillance, a system that detects and destroys aberrant mRNA, in the nematode C. elegans. Previously cloned smg genes encode an RNA helicase, a protein kinase and several novel products, some of which contain motifs implicated in protein:protein interactions and can be shown to co-immunoprecipitate in vitro (Anderson lab, unpub'd results). Rescue of smg-4 mutants, assayed in an unc-54(r293);smg-4 (ma116) strain, was previously obtained with yeast artificial chromosomes, Y1D1 and Y42F5. Phage were isolated from a library generated from Y42F5 genomic DNA, and one clone, gmRB162, was found to rescue smg-4 mutant animals. Plasmid subclones were tested in further rescue assays, and the predicted gene sequence analyzed. The smallest rescuing subclone of 3.6Kbp, pgm185, could encode two potential open reading frames, which may be part of an operon. Subclones containing either ORF will not rescue. However, when only one or the other ORF is disrupted by site directed mutagenesis, only mutation of the downstream ORF will abrogate rescuing activity. So it seems the rescuing activity requires upstream sequences for its expression, and the downstream ORF mediates smg-4+ function. A 1.5Kb RNA is detected in both total and pA+ RNA, which could encode either predicted gene product. Restriction fragment length polymorphisms have been found associated with mutant smg-4 alleles by southern blotting. For instance, 2.5Kbp is deleted from this region in smg-4 (r1169), an allele generated with DEO by S. Kuchma. This 2.5Kb includes a domain which contains a good match to the octamer consensus sequence of the RNA Recognition Motif (RRM), an 80 a.a. RNA binding motif. In addition, the ORF has a lysine and glutamic acid (KE) rich domain which shares sequence identity with a few genes, of particular note, the yeast gene SPB. Analysis of cDNAs, sequencing of mutant alleles, and expression and more rescue studies are currently in progress.

Cheng X et al. (2022) Autophagy "Proteotoxic stress disrupts epithelial integrity by inducing MTOR sequestration and autophagy overactivation."

Zhang H, Zheng H, Zhang P, Cheng X, Zheng K, Zhao H

[Autophagy, 2022]

Macroautophagy/autophagy, an evolutionarily conserved degradation system, serves to clear intracellular components through the lysosomal pathway. Mounting evidence has revealed cytoprotective roles of autophagy; however, the intracellular causes of overactivated autophagy, which has cytotoxic effects, remain elusive. Here we show that sustained proteotoxic stress induced by loss of the <u>RI</u>NG and <u>Ke</u>lch repeat-containing protein C53A5.6/RIKE-1 induces sequestration of LET-363/MTOR complex and overactivation of autophagy, and consequently impairs epithelial integrity in <i>C. elegans</i>. In C53A5.6/RIKE-1-deficient animals, blocking autophagosome formation effectively prevents excessive endosomal degradation, mitigates mislocalization of intestinal membrane components and restores intestinal lumen morphology. However, autophagy inhibition does not affect LET-363/MTOR aggregation in animals with compromised C53A5.6/RIKE-1 function. Improving proteostasis capacity by reducing DAF-2 insulin/IGF1 signaling markedly relieves the aggregation of LET-363/MTOR and alleviates autophagy overactivation, which in turn reverses derailed endosomal trafficking and rescues epithelial morphogenesis defects in C53A5.6/RIKE-1-deficient animals. Hence, our studies reveal that C53A5.6/RIKE-1-mediated proteostasis is critical for maintaining the basal level of autophagy and epithelial integrity.<b>Abbreviations:</b> ACT-5: actin 5; ACTB: actin beta; ALs: autolysosomes; APs: autophagosomes; AJM-1: apical junction molecule; ATG: autophagy related; C. elegans: Caenorhabditis elegans; CPL-1: cathepsin L family; DAF: abnormal dauer formation; DLG-1: Drosophila discs large homolog; ERM-1: ezrin/radixin/moesin; EPG: ectopic P granule; GFP: freen fluorescent protein; HLH-30: helix loop helix; HSP: heat shock protein; LAAT-1: lysosome associated amino acid transporter; LET: lethal; LGG-1: LC3, GABARAP and GATE-16 family; LMP-1: LAMP (lysosome-associated membrane protein) homolog; MTOR: mechanistic target of rapamycin kinase; NUC-1: abnormal nuclease; PEPT-1/OPT-2: Peptide transporter family; PGP-1: P-glycoprotein related; RAB: RAB family; RIKE-1: RING and Kelch repeat-containing protein; SLCF-1: solute carrier family; SQST-1: sequestosome related; SPTL-1: serine palmitoyl transferase family.

Andrew Bretscher et al. (2009) European Worm Neurobiology Meeting "Sensory neurons regulating C. elegans carbon dioxide avoidance behaviour"

Eiji Kodama, Andrew Bretscher, Mario De Bono

[European Worm Neurobiology Meeting, 2009]

CO2 comprises only ~0.038% of earth.s atmosphere but local CO2 concentrations can rise much higher in the presence of aerobically respiring organisms. Previous data have shown that well-fed C. elegans avoid CO2 levels greater than 0.5% 1,2. This worm can alter behaviour in response to both changes in CO2 levels, d[C]/dt, and to absolute CO2 concentrations [C]. These responses are highly regulated. For example starvation strongly suppresses CO2 avoidance as does exposure to hypoxia. We are investigating how the C. elegans nervous system coordinates responses to CO2. Recent laser ablation studies show that the anteriorly located BAG neurons are required for acute CO2 avoidance 1. BAG neurons also respond to changes in O2 3 and we have shown that O2 levels can modulate CO2 avoidance 2. These data raise two alternatives: 1) BAG responses to O2 modulate neural circuits mediating CO2 responses; 2) BAG neurons respond to both O2 and CO2. To test whether the BAG neurons are CO2 sensors, we applied CO2 to N2 animals expressing the high-affinity genetically encoded calcium sensor YC3.60 in this neuron. The neurons responded with a large change in YFP/CFP fluorescence, indicative of a rise in calcium. Calcium transients were observed as long as CO2 levels remained high and peak activity coincided with CO2 increases. Concentrations as low as 0.25% elicited robust CO2 responses. unc-13 and unc-31 mutants expressing pBAG::YC3.60 retained N2-like responses to CO2. This suggests that pre-synaptic input is not required for CO2 responses and that BAG neurons are primary CO2 sensors. CO2-induced activity in BAG requires the cGMP-gated ion channel subunits TAX-2 and TAX-4. At a behavioural level TAX-2 and TAX-4 promote CO2 avoidance, but other pathways are also important. Employing in vivo calcium imaging to test hypotheses based on earlier genetic work we are currently seeking other sensory neurons that display pronounced CO2 sensitivity. References 1. Hallem, E. A. & Sternberg, P. W. Acute carbon dioxide avoidance in Caenorhabditis elegans. Proc Natl Acad Sci U S A 105, 8038-8043 (2008). 2. Bretscher AJ, Busch KE, de Bono M. Carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans. Proc Natl Acad Sci U S A 105, 8044-9 (2008). 3. Zimmer M, Gray JM, Pokala N, Chang AJ, Karow DS, Marletta MA, Hudson ML, Morton DB, Chronis N, Bargmann CI. Neurons detect increases and decreases in oxygen levels using distinct guanylate cyclases. Neuron 61, 865-79 (2009).