Ren, Karen et al. (2015) International Worm Meeting "Molecular and cellular characterization of SKN-1 and the multidrug resistance protein MRP-7 that inhibits toxicant-associated dopaminergic neurodegeneration."

Wong, Garry, Rudgalvyte, Martina, Ren, Karen, Nass, Richard, Shi, Jing, Trinidad, Jonathan

[International Worm Meeting, 2015]

Background: Methylmercury (MeHg) is a ubiquitous environmental toxicant that primarily targets the central nervous system and has been associated with the development of the dopamine (DA) neurodegenerative disorder Parkinson's disease (PD). We have previously developed a Caenorhabditis elegans (C. elegans) model of MeHg toxicity that shows that low, chronic exposure confers DA neuron degeneration that is largely dependent on the transcription factor SKN-1/Nrf2 and the multidrug resistance protein MRP-7. Aims: In this study we asked how SKN-1 and MRP-7 modulates whole animal and DA neuron vulnerability to MeHg. Methods: We utilize a reverse genetic screen, immunofluorescence, transgenic C. elegans, RT-PCR, Western analysis, and neuronal morphology to characterize the role that SKN-1, MRP-7, and post-translational modifications play in MeHg-associated toxicity. Results and Conclusion: Over 17,000 genes were screened for whole animal sensitivity to MeHg, and 92 genes were identified that affect whole animal and/or DA neuron pathology. These genes are strongly biased towards molecular mechanisms that affect the mitochondria and the ubiquitin-proteosome system (UPS). We also show that genetic knockdown of MRP-7 results in a 2-fold increase in Hg levels and a loss of DA neuron integrity. Chronic exposure to low concentrations of MeHg induces MRP-7 gene expression athat affects transporter localization, organelle vulnerability, and specific post-translational modifications that modulate cellular toxicity. Furthermore, we demonstrate that SKN-1 regulates DA neuron synaptic neurotransmission-associate gene expression that modulates the DA neuron vulnerability. These studies show that a DA neuron-associated multidrug resistance protein and post-translational modifications play a critical role in neuronal vulnerability. Support: NIH, BRG, Alzheimer's Disease Foundation, and FNDR Fund to RN, IUCRG to RN and JT.

Coleman JJ et al. (2009) Cell Host Microbe "The tangled web of signaling in innate immunity."

Coleman JJ, Mylonakis E

[Cell Host Microbe, 2009]

Similarities in innate immune signaling exist between mammals and the nematode Caenorhabditis elegans. Now, Ziegler et al. (2009) and Ren et al. (2009) demonstrate that a protein kinase C delta homolog in C. elegans is involved in innate immunity, providing evidence that the conservation of immune signaling networks extends further than previously thought.

Nass, Richard et al. (2015) International Worm Meeting "The identification and characterization of SKN-1-associated acetylation pathways involved in dopamine neurodegeneration in Parkinson's disease models."

Shi, Jing, Trinidad, Jonathan, Ren, Karen, Nass, Richard

[International Worm Meeting, 2015]

Background: Idiopathic Parkinson's disease (PD) is an oxidative stress-related disorder that result in abnormal dopamine (DA) signaling and cell death. Although the origin of the pathogenesis in PD remains unclear, corollary evidence suggests both genetic and environmental contributions. The overlapping molecular determinants involved in the neuropathology in both genetic- and toxicant-associated PD models are largely ill defined. Aims: In this study we asked what are the common genes, molecular pathways and mechanisms involved in PD-associated DA neuron vulnerability. Methods: We utilized reverse genetics, biochemical assays, immunofluorescence, transgenic C. elegans, RT-PCR, Western analysis, LC/MS, and neuronal morphology analysis to characterize expression and localization of the PD-associated transcription factor SKN-1 and a gene involved in protein acetylation play in several genetic- and toxicant-associated C. elegans PD models. Results: In this study we demonstrate that a gene involved in protein acetylation renders the DA neurons up to 15-fold more resistant to PD-associated genetic mutations or neurotoxicants, and that overexpression results in a 2-fold increase in DA neurodegeneration. We have also generated a C. elegans mutant that results in highly dysfunctional protein acetylation and an age dependent complete loss of DA neurons in young adult nematodes. The generation of the first C. elegans acetylome as well as reverse genetics and biochemical assays indicates SKN-1 modulates the protein acetylation that effect PD-associated DA neuron vulnerability. Conclusions: This study identifies novel genes and molecular pathways involved in DA neuron vulnerability, and provides in vivo evidence that a common epigenetic mechanism likely plays a significant role in PD-associated neurodegeneration. Support: NIH, BRG, Alzheimer's Disease Foundation, and FNDR Fund to RN, IUCRG to RN and JT.

Roussell DL et al. (1994) Parasitol Today "Germ-line determination in Caenorhabditis and Ascaris: will a helicase begin to unravel the mystery?"

Bennett KL, Roussell DL, Gruidl ME

[Parasitol Today, 1994]

How cell lineages are established during development in higher eukaryotes is being addressed by geneticists and by developmental and molecular biologists. In Drosophila melanogaster, a gene corresponding to a germ-line-specific RNA helicase, vasa, has been shown to be a component o f the posteriorly localized germ granules o f the developing embryo. A putative RNA helicase, glh-I r which appears germ-line specific in its expression, has recently been reported from the free-living nematode Caenorhabditis elegans. Parasitologists studying the nematode Ascaris lumbricoides var. suum have found it to be a useful complement to Caenorhabditis. Deborah Roussell, Michael Gruidl and Karen Bennett predict that Ascaris will be valuable in determining the role played by germ-line helicases in development.

Fletcher, Marissa et al. (2015) International Worm Meeting "The DAF-7/TGF-beta pathway is required for lifespan extension mediated by dietary restriction in C. elegans."

Fletcher, Marissa, Kim, Dennis

[International Worm Meeting, 2015]

The neuromodulator DAF-7/TGF-beta is a key regulator of animal physiology and behavior. DAF-7 is expressed in the chemosensory neurons and expression levels have been shown to respond to levels of food availability. Signaling through DAF-7 regulates dauer formation and fat metabolism, as well as feeding, egg laying, and avoidance behaviors (Ren et al. 1996; Greer et al. 2008; Meisel et al. 2014). Additionally, daf-7 mutations have been observed to extend lifespan in a DAF-16 dependent manner(Shaw et al. 2007). Here, we show that lifespan extension by dietary restriction is dependent on DAF-7. We also observe dynamic DAF-7 expression throughout the aging process, and we are characterizing how DAF-7 signaling regulates the organismal response to changing food levels that promotes longevity under conditions of dietary restriction. Understanding how limited nutritional resources elicit a response in this neuroendocrine pathway to confer lifespan benefits may allow us to gain insight into how DR may be acting to delay the aging process in evolutionarily diverse species.

Tylon Stephney et al. (2002) East Coast Worm Meeting "Alae Mutant Defects Assessed by SEM and TEM"

Adam Hartley, Charles S Rubin, Carolyn Marks, David H Hall, Min Ren, Tylon Stephney

[East Coast Worm Meeting, 2002]

We have previously reported movement defects in adult C. elegans after RNAi (dsRNA feeding) with the gene PKC4, which encodes a novel Ser/Thr protein kinase (Ren et al., 2001). The PKC4 protein is expressed highly in the seam cells in late-stage embryos and again in late L4 stage, at the time of morphogenesis for seam-cell derived cuticular structures, the alae. It seemed likely that the movement defects, a sloppy zig-zag body motion of variable amplitude when placed on standard agar plates, were the result of absent or poorly formed alae. We have fixed adult animals for examination by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and compared these PKC4 RNAi animals to adult wild type. We will show SEM evidence for the normal extent of adult alae and several other hallmark features of the body surface, and TEM data for the corresponding seam cell cytology which underlies the alae. As predicted, the "knockout" animals often show alae defects or complete loss of alae locally, with a penetrance which agrees well with the prevalence of movement defects. The most common defect is a merger of the tripartite ala structure into a large smooth ridge of cuticle, or less often, a zone of smooth cuticle with no ridge at all. In severely affected PKC4 RNAi animals, some tissue defects can be seen in seam cells, including cell swelling and general disorder in the cytoplasm.

Carolyn Marks et al. (2002) West Coast Worm Meeting "Alae Mutant Defects Assessed by SEM and TEM"

Min Ren, Tylon Stephney, David H Hall, Carolyn Marks, Hui Feng, Charles S Rubin, Adam Hartley

[West Coast Worm Meeting, 2002]

We previously reported movement defects in adult C. elegans after RNAi (dsRNA feeding) with the gene PKC4, which encodes a novel Ser/Thr protein kinase (Ren et al., 2001). The PKC4 protein is expressed highly in the seam cells in late-stage embryos and again in late L4 stage, at the time of morphogenesis for seam-cell derived cuticular structures, the alae. It seemed likely that the movement defects, a sloppy zig-zag body motion of variable amplitude when placed on standard agar plates, were the result of absent or poorly formed alae. We have fixed adult animals for examination by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and compared these PKC4 RNAi animals to adult wild type. We will show SEM evidence for the normal extent of adult alae and several other hallmark features of the body surface, and TEM data for the corresponding seam cell cytology which underlies the alae. As predicted, the "knockout" animals often show alae defects or complete loss of alae locally, with a penetrance which agrees well with the prevalence of movement defects. The most common defect is a merger of the tripartite ala structure into a large smooth ridge of cuticle, or less often, a zone of smooth cuticle with no ridge at all. In severely affected PKC4 RNAi animals, some tissue defects can be seen in seam cells, which can wander away from the midline, or in the local hypodermis, which can show local swelling . Misassembled plaques of secreted material are sometimes seen in the cuticle in regions corresponding to the missing alae.

Prasad SS et al. (1985) Worm Breeder's Gazette "identification and isolation of the coding regions around unc-22."

Baillie DL, Prasad SS

[Worm Breeder's Gazette, 1985]

Our laboratory has been intensively studying the region around unc- 22 gene over the last eight years using conventional genetic analysis. We have obtained cosmids from John Sulston that cover approximately 500 kb. of DNA. These cosmids were identified by means of Tcl RFLD flanking sequences (isolated by Karen Beckenbach) in the region which lies immediately to the left and right of unc-22 gene. We are currently examining the region flanked by dpy-20 on the left and unc-22 on the right. Five overlapping cosmids have been identified which cover this region, (these include D. Moerman's and R. Waterston's unc-22 clones). We are using the method suggested by T. Snutch to identify putative coding regions (Divergent regions between C. elegans and C. briggsae include mostly non-coding DNA sequences whereas the highly conserved regions are likely to code for the proteins essential for nematode growth, development and reproduction). We have restriction mapped and subcloned Hind III and Pst I fragments from these cosmids into pUC19. Nick translated individual fragments have been used to probe Bristol and C. briggsae DNA. This technique helps us to identify the conserved regions or the coding sequences in C. briggsae DNA. We are now in the process of screening B. Meyers's lambda gt10 cDNA library with the probes which show homology to the C. briggsae DNA. We intend to identify and isolate the major coding regions that lie between unc-22 and dpy-20 regions using this approach. The isolation of cDNA clones will be used to study gene expression and regulation during nematode development. The isolated cDNA clones will be sequenced in order to identify the nature of protein coded for by particular gene(s) in this region.

GM Sandoval et al. (1999) International C. elegans Meeting "Investigating A Role For Cholinergic Signaling In The Dauer Formation Pathway"

G Ruvkun, GM Sandoval

[International C. elegans Meeting, 1999]

Animals with mutations in the daf-7 TGF- b gene (Ren at al. Science 274, 1996) arrest at the dauer larval stage in the presence of favorable conditions for reproductive growth. Exogenous application of the muscarinic receptor agonist oxotremorine rescues dauers allowing them to enter the reproductive life cycle (Heidi Tissembaum, personal communication) suggesting a potential role for muscarinic signaling in the dauer/nondauer developmental switch. To address more directly the role of cholinergic signaling pathways we made double mutants between daf-c genes and animals mith mutations that have decreased cholinergic signaling. We observed a synthetic L1 lethality in the double mutant of the insulin-like receptor daf-2( e1370) and a vesicular transporter of acetylcholine unc-17 (e245) (Alfonso, A. et al. Science 261, 1993). We are conducting a screen to suppress this lethality to isolate mutations in components of the cholinergic signaling pathway which may mediate dauer formation. We are also using a reverse genetic approach to analyze components of the cholinergic signaling pathway. By sequence comparisons in the C. elegans genome, we have identified three muscarinic receptor homologues. We have analyzed the expression of a GFP promoter fusion of two of these genes. We are currently analyzing the expression of the third. We have not observed expression of our constructs in neurons identified as having a role in the dauer signaling pathway. Both constructs are expressed in a small set of neurons with one construct also exhibiting expression in intestinal cells. Deletion mutants of these genes will be generated and analyzed for dauer phenotypes to address whether there is muscarinic input into the dauer pathway.

Rasmussen, Neal R et al. (2021) MicroPubl Biol

Reiner, David J, Smith, Harold E, Rasmussen, Neal R

[MicroPubl Biol, 2021]

The C. elegans dauer is an alternative L3 stage larva that forms under harsh environmental conditions, including low food, high temperature, and high concentration of constitutively secreted dauer pheromone. Genetic screens identified genes conferring dauer-constitutive and dauer-defective phenotypes (Daf-c and Daf-d, respectively; Hu, 2007). Double mutant analysis using principles of epistasis and parallelism ordered genes controlling the dauer process into a network (Gottlieb and Ruvkun, 1994; Thomas et al., 1993). Molecular genetic cloning of genes provided identities with similarity to orthologs in Drosophila and mammals. Taken together, these approaches arrived at a model of four main signaling axes controlling entry into dauer: upstream and parallel TGF-beta (DAF-7, mutated to Daf-c) and receptor guanylyl cyclase (DAF-11, mutated to Daf-c) signals reflect parallel processing by sensory neurons, revealed by laser ablation experiments (Birnby et al., 2000; Ren et al., 1996; Schackwitz et al., 1996). Downstream, serial Insulin/IGF-like growth factor receptor (DAF-2, mutated to Daf-c; (Kimura et al., 1997) and nuclear hormone receptor (DAF-12/NHR, mutated to Daf-d; (Antebi et al., 2000) signals control and execute tissue-specific changes in the animal (Fig.1C, DAF-12 not shown). Mutants for each signaling axis also control diverse developmental and metabolic outputs in addition to the dauer decision. The four-axis model of signaling control of dauer formation neglects potential positive- and negative-feedback loops and is thus likely reductive. Still, these approaches have provided a robust framework for further investigation into the control of the dauer developmental decision by sensory and endocrine signaling modalities.