Allen, Parker et al. (2021) International Worm Meeting "Role of the conserved cholinesterase family member CEST-1.1 and its modular metabolite products in life span control"

Curtis, Brian, Kruempel, Joseph, Hu, Patrick, Wrobel, Chester, Allen, Parker, Schroeder, Frank, Higgins-Chen, Albert

[International Worm Meeting, 2021]

Although the role of the C. elegans FoxO transcription factor DAF-16 in promoting longevity is well established, how DAF-16/FoxO extends life span remains poorly understood. While the identities of thousands of DAF-16/FoxO target genes are known, mechanistic links between DAF-16/FoxO-dependent regulation of specific genes and DAF-16/FoxO-dependent life span extension are lacking. We have discovered that a conserved cholinesterase family member encoded by the DAF-16/FoxO target gene cest-1.1 is required for full life span extension in the context of reduced DAF-2 insulin-like signaling and sufficient to extend life span when expressed in a wild-type background. A functional CEST-1.1::GFP fusion protein is expressed specifically in the intestine and localizes to the apical plasma membrane. Comparative metabolomic analysis using HPLC-high resolution mass spectrometry revealed that CEST-1.1 is required for the biosynthesis of two structurally novel nucleoside-like ascarosides termed uglas#1 and uglas#11. These surprising findings support a role for an unprecedented class of metabolites in life span control and orthogonally expand the landscape of biogenic small molecules that may influence aging.

Cendrine Tourette et al. (2007) International Worm Meeting "Classification of oligonucleotide microarray data for mutant polyglutamine toxicity in cell-sorted neurons using gene networks C.elegans."

Cendrine Tourette, Christian Neri, Alex, J Parker, Anne Louise

[International Worm Meeting, 2007]

The limitations of cellular and rodent models preclude the detailed analysis of molecular and genetic factors involved in polyglutamine neuronal toxicity in vivo. Invertebrates provide convenient and robust experimental systems to study the regulators of polyglutamine neuronal toxicity in whole organisms and at the genomic level. We have previously described C.elegans models of the early phases of mutant polyglutamine (polyQ) cytotoxicity in neurons, namely the dysfunction of touch receptor neurons before cell death produced by the expression of N-ter huntingtin fused to a fluorescent protein under the control of the mec-3 promoter (Parker et al., PNAS 2001). We have illustrated the value of these animals to defining disease targets and active compounds for neuroprotection in Huntingtons disease (Parker et al., Nature Genet 2005). We analyzed the effects of mutant polyQ pathogenicity using FACS-purified neurons and oligonucleotide microarrays from Agilent. Data classification using C. elegans gene networks and the comparison of our data to microarray data from other models of mutant huntingtin neurotoxicity will be presented.

Alex Parker et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Studying the modifier genes of neuronal cell response to mutant huntingtin expression in C. elegans"

Christian Neri, Alex Parker, Maryline Mage, Emmanuel Lambert, Sabine Cloax, Cendrine Tourette, Salima Abderrahmane, Beate Gertsbrein

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Huntington"s disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in huntingtin. Although HD is inherited, its age of onset shows great variability, suggesting neuronal dysfunction and death in HD is influenced by modifier genes. To study how the neuronal cell may respond to mutant htt, we have developed C. elegans transgenics that overexpress mutant N-terminal htt fused to fluorescent proteins in touch receptor neurons. This model shows neuronal dysfunction without cell death, an effect accompanied by aggregate formation and dystrophy of neuronal processes (Parker et al., PNAS 2001). To study the pathways underlying these transgenic phenotypes, we use a combination of C. elegans mutants, RNAi screens, microarray analysis, and physiological analysis. This effort has led us to identify sirtuin activation as a neuroprotective mechanism against the effects of mutant htt expression, an effect mediated by daf-16/FOXO, (Parker et al., Nature Genetics 2005). Our findings indicated that modulators of organismal longevity like sir2 and FOXO may modify early-stage pathogenesis of neurodegenerative diseases, defining a new strategy with therapeutic potential. We will present an update on the progress made with our models in better understanding how mutant htt may be toxic to the neuronal cell and how neuroprotection may be achieved.

Vazquez-manrique, Rafael et al. (2011) International Worm Meeting "AMPK activation is protective in models of neuron dysfunction and death in Huntington's disease."

Deglon, Nicole, Neri, Christian, Offner, Nicolas, Vazquez-manrique, Rafael, Cambon, Karine, Orfila, Anne-marie, Darbois, Aurelie

[International Worm Meeting, 2011]

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the huntingtin protein (Htt). Expression of the first exon of Htt containing an expanded polyQ produces neuronal dysfunction and axonal dystrophy in mechanosensory neurons in C. elegans (Parker et al., 2001). Genetic pathway analysis using this nematode model has emphasized a role for the longevity-promoting factor daf-16/FoxO in the protection of neurons from expanded polyQ toxicity (Parker et al., 2005). Here, we investigated the role of AMP-activated protein kinase (AMPK), a well-known energy sensor involved in lifespan and health span extension, on neuronal dysfunction in expanded-polyQ nematodes and vulnerability to cell death in mutant Htt striatal cells (HdhQ111 knock-in mice). In C. elegans, activating this enzyme (aak-2/AMPK) with metformin reduces the neuronal dysfunction caused by expanded polyQ expression. In contrast, aak-2 mutants show enhanced neuronal impairment. In striatal cells from HdhQ111 knock-in mice, reducing AMPK levels by siRNA treatment enhances the susceptibility to cell death of mutant htt cells, and metformin treatment has the opposite effect. Additionally, overexpressing AMPK reduced striatal neurodegeneration in a rat lentiviral-based fragment model of HD pathogenesis. Collectively, our results suggest that AMPK activation has therapeutic potential to protect from neuron dysfunction and degeneration in HD.

Nichols, Courtney Rose et al. (2013) International Worm Meeting "A role for the insulin signaling pathway in development of neuronal aging markers in a polyglutamine protein aggregation C. elegans model."

Parker, J. Alex, Nichols, Courtney Rose, Driscoll, Monica, Neri, Christian, Vayndorf, Elena, Taylor, Barbara

[International Worm Meeting, 2013]

Neurodegenerative diseases, such as Huntington's, Alzheimer's, and Parkinson's disease, result in the progressive loss of neuronal structure and function with age. Many neurodegenerative disorders are caused by genetic mutations and characterized by toxic aggregation of proteins within neurons. We explored the mechanism of accelerated neuronal aging in a polyglutamine (polyQ) protein aggregation model through genetic manipulation. Caenorhabditis elegans expressing the first 57 amino acids of human huntingtin protein with a toxic polyglutamine chain (polyQ128) fluorescently labeled with CFP in YFP-labeled mechanosensory neurons (Parker et al, 2001) were used to monitor neuronal aging phenotypes. We found an age-associated increase in aberrant neuronal morphology, protein aggregation, and functional impairment of the mechanosensory neurons expressing toxic 128 polyQ. We also tested the hypothesis that the insulin signaling pathway is involved in morphological and functional health of aging mechanosensory neurons using neuron-specific RNA interference (RNAi). Furthermore, we measured levels of endogenous oxidative stress and lifespan of aging animals that contain toxic polyQ128 repeats following RNAi manipulation of the insulin signaling pathway. Overall, we found that DAF-16/FOXO is neuroprotective in this accelerated aging model, which corroborates previous findings on the role of DAF-16/FOXO in polyQ128 young adults (Parker et al. 2005). To further characterize overall muscle and neuronal health, we measured action potentials of pharyngeal muscle contractions and neurons that innervate the pharynx using microfluidic electropharyngeography (EPG). In total, our observations support that insulin signaling via DAF-16/FOXO is a mechanism through which accelerated aging and neurodegeneration occurs in this model.

Tauffenberger A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Glucose rescues age-dependent proteotoxicity in C.elegans"

Vaccaro A, Parker A, Tauffenberger A

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

As the population shifts to an older demographic, it has become important to find new therapeutic ways to combat neurological disease. To model neurodegeneration we use a C. elegans model for Huntington's Disease (HD). HD is a late-onset fatal neurodegenerative disease caused by an expanded CAG trinucleotide repeat that codes for glutamine (Q), and is characterized by the intracellular accumulation of mutant proteins. Our transgenic C. elegans strains expressing normal (19Q) and expanded mutant (128Q) polyQ in mechanosensory neurons show a progressive and polyQ size dependent loss-of-mechanosensation and increased-aggregation phenotypes in vivo1. The difference between mutant (128Q) and controls (19Q) is easily distinguished and the effects of genetic and pharmacological modifiers can be rapidly derived from small populations of animals2. Disrupted metabolism is one of a number of mechanisms that may contribute to neurodegeneration. Indeed, conditions that reprogram metabolism like dietary restriction are active areas of investigation in efforts to identify new therapeutic targets. We have observed that opposite to the effects on organismal lifespan, excess glucose suppresses toxicity in worm models of proteotoxicity. Furthermore, many of the genes essential for glucose-mediated suppression of proteotoxicity are in common with genes required for dietary restriction. These data demonstrate that glucose enrichment and dietary restriction utilize many of the same genetic pathways with opposite metabolic endpoints both of which are beneficial in models of neurodegeneration. A summary of our recent data will be presented. 1.Parker, J.A. et al. Expanded polyglutamines in Caenorhabditis elegans cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death. Proc Natl Acad Sci U S A 98, 13318-23 (2001). 2.Parker, J.A. et al. Resveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons. Nat Genet 37, 349-50 (2005).

Reinke, Valerie et al. (2011) International Worm Meeting "The C. elegans transcriptome."

Leng, J, Gerstein, Mark, Miller, III, David M, Reinke, Valerie, Robilotto, Rebecca, Slack, Frank J, Spencer, W Clay, Kato, Masaomi, Strasbourger, Pnina, Wang, Guilin, Green, Phil, High, Amber, MacCoss, Michael, Hillier, LaDeana, Thompson, Owen, Ratsch, Gunnar, Zeller, Georg, Ewing, Brent, Waterston, Robert H, Merrihew, Gennifer, Henz, S

[International Worm Meeting, 2011]

As part of the modENCODE consortium, we are characterizing the C. elegans transcriptome using tiling arrays, RNA-seq, RT-PCR and mass spectrometry. Our earlier studies on whole animals of various stages and conditions and on specific cells and tissues led to a much improved set of protein coding genes covering greater than 95% of all genes including more than 12,413 trans-spliced leaders, 20,515 different trans-spliced transcript start sites, 28,199 polyA sites, 111,786 confirmed splice junctions, >7,000 inferred non-coding (nc) RNAs, and over 50 new miRNAs (1-5). More recently, we have (1) analyzed biological replicates with RNA-seq for different stages and conditions, validating the observed expression levels; (2) closed gaps in RNA-seq coverage of weakly expressed genes with RT-PCR; (3) characterized the RNA content of more finely staged embryos with RNA-seq; (4) tested methods that deplete rRNA to allow direct analysis by RNA-seq of ncRNAs and smaller samples, such as specific embryonic cells and tissues; (5) analyzed polyA+ RNA from selected stages of C. briggsae, C. remanei, C. brenneri and C. japonica; (6) analyzed miRNAs under additional stresses and conditions; and (7) characterized the proteins present in 12 size fractions from 16 different stages and conditions. All of the data are available through the modENCODE Data Coordinating Center and increasingly through WormBase. Our goal is to provide the community with a comprehensive description of the transcripts of the C. elegans genome, providing information about their specific utilization where possible. References 1. Hillier et al. Genome Research PMID: 19181841 2. Gerstein et al Science PMID: 21177976 3. Lu et al. Genome Reseaarch PMID: 21177971 4. Allen et al. Genome Research PMID: 21177958 5. Spencer et al. Genome Research PMID: 21177967.

Arzu Atalay et al. (2005) International Worm Meeting "Functional analysis of CGH-1-associated proteins in C.elegans"

T Keith Blackwell, Arzu Atalay, Peter Boag, Rosa E Navarro, Jiayun Lu

[International Worm Meeting, 2005]

Two conserved aspects of oogenesis are storage of translationally repressed mRNA and a high frequency of apoptosis. In C.elegans approximately half of the developing oocytes die through a germline-specific physiological apoptosis pathway (1). Previous studies in our laboratory reported that cgh-1 (Conserved Germline Helicase-1) is required for gametogenesis and protection from physiological germline apoptosis in C.elegans (2). cgh-1 RNAi leads to markedly increased physiological germline cell death and sterility. CGH-1 is specifically expressed in the germline and early embryo. It associates with P granules, and is present in additional predicted RNA storage particles. In Drosophila and Xenopus CGH-1 orthologs are involved in translational control of oocyte mRNA. In yeast, the CGH-1 ortholog Dhh1p is required for decapping- and deadenylation-mediated RNA degradation, which occurs in discrete cytoplasmic foci called P (processing) bodies. Yeast Dhh1p is concentrated in P bodies and is critical for their decapping and subsequent degradation functions (3). Biochemical studies in yeast and two-hybrid screens in yeast and C.elegans have identified numerous other proteins including P-body associated proteins- that interact with CGH-1 either directly or indirectly. We are investigating whether RNAi knockdown of these genes influences the frequency of physiological apoptosis, or affects other phenotypes associated with CGH-1. The vast majority of these genes do not affect the frequency of germ cell death, supporting the idea that this process responds to specific cues during oogenesis. We are particularly interested in studying how CGH-1 interacts functionally with C.elegans counterparts of other proteins that are present in P bodies in yeast or other organisms. For example, we are investigating how the decapping/degradation machinery influences the localization and levels of CGH-1 RNA-protein particles in the embryo. Since there are several parallels between deadenylation-dependent mRNA degradation pathways and regulation of maternal RNA storage events, we are also investigating whether CGH-1 functions in the regulation of specific maternal mRNAs. (1)Gumienny et al. (1999) Development 126: 1011-22 (2)Navarro et al. (2001) Development 128: 3221-32 (3)Sheth and Parker (2003) Science 300: 805-8

Ali MY et al. (1996) West Coast Worm Meeting "GENETIC INTERACTION AMONG MEMBERS OF THE KINESIN FAMILY AND THE EFFECT OF DPY-20 MUTATION"

Ali MY, Hori Y, Siddiqui ZK, Siddiqui SS

[West Coast Worm Meeting, 1996]

Previously mutants in three kinesin genes have been characterized in C. elegans (osm-3, unc-116, unc-104) (Shakir et al., 1993; Patel et al., 1993; Otsuka et al., 1991). Mutants in osm-3 are known to be defective in osmotic aviodance and other chemosensory behavior such as FITC uptake in all amphid and phasmid neurons (Culotti and Russel, 1978; Parker et al., 1986; Shakir et al., 19993a, 1993b, and Tabish et al., 1995). However osm-3 mutants display normal locomotion. Mutants in unc-104 are severely uncoordinated, but display normal FITC uptake. Similarly, unc-116 mutants are defective in locomotion but have normal FITC uptake. We have constructed various double and triple mutants to reveal the genetic interaction among various kinesin mutants. Mutants in dpy-20 are normal in locomotion and show normal FITC uptake. Our results are the following: Gene FITC Locomotion Brood Size Remarks osm-3 - normal average abnormal chemosensory (p802) behavior unc-116 + slow low defective locomotion (e2310) unc-104 + paralyzed low immobile (NJ479) unc-104 + semi-paralyzed average abnormal locomotion (e1265) dpy-20 + normal average Dpy and normal locomotion (e2017) osm-3 - paralyzed low osm-3 improves the locomotion (p802) (less than of unc-104 (NJ479) unc-104 unc-104) (NJ479) osm-3 - slow average almost the same (p802) locomotory behavior unc-104 as unc-104 (e1265) (e1265) osm-3 - slow low phenotype is almost (p802) the same as unc-116 unc-116 but size is smaller (e2310) and fatter than unc-116 dpy-20 + severely low dpy-20 reduce the (e2017) paralyzed brood size of unc-104 unc-104 (NJ479) dpy-20 + slow average dpy-20 does not affect the (e2017) brood size of unc-104 unc-104 (e1265) dpy-20 + paralyzed low dpy-20 reduce the viability (e2017) of unc-116 unc-116 (e2310) dpy-20 - normal average dpy-20 does not seem (e2017) to affect any behavior osm-3 osm-3 (p802) dpy-20 - severely low lethal (e2017) paralyzed unc-104 (NJ479) osm-3 (p802) In summary osm-3 has a dual role: (1) Improving the locomotion and brood size of unc-104 (NJ479). (2) Reducind the viability of unc-104 (NJ479) very significantly in the presence of dpy-20 (e2017).

Boateng, Ruby et al. (2015) International Worm Meeting "Characterization of a novel role for ETR-1 in reproduction."

Golden, Andy, Allen, Anna, Boateng, Ruby

[International Worm Meeting, 2015]

ETR-1, an ELAV-Type RNA-binding protein, is canonically known for its involvement in muscle development, however we have identified novel roles for this protein in both the somatic and germline cells of the C. elegans gonad. RNAi depletion of ETR-1 results in animals paralyzed at the two-fold (PAT) embryonic stage, indicating a failure in normal muscle development, and in a reduced brood compared to control depleted animals, indicating a reproductive role. We hypothesized that the reduced brood size might potentially be due to decreased germ cell numbers, reduced ovulation rate and/or an increase in apoptosis. The number of germ cells and ovulation rate were found to be similar in ETR-1- and control-depleted animals, however only ETR-1-depleted animals exhibit an increased number of apoptotic cells. Interestingly, a recent RNAi based suppressor screen by Allen et. al. (2014) has shown that co-depletion of ETR-1 and WEE-1.3 (an essential regulator of the meiotic cell cycle and oocyte maturation) restores fertility to the otherwise sterile wee-1.3(RNAi) animals. We investigated this further using a somatic RNAi defective worm strain and determined that this ability of ETR-1 to suppress is dependent on ETR-1 being depleted in the soma. ETR-1 depletion only in the germline also resulted in a brood size that was significantly different from its control, uncovering a novel role for ETR-1 in the germline. Further supporting this, ETR-1 is expressed in both the somatic muscle sheath cells of hermaphrodites and the germ cells of both hermaphrodite and male gonads. We hypothesize that these roles may be controlled by different isoforms of ETR-1. To address this, we are utilizing CRISPR-Cas9 technology to generate genome-edited lines in which green fluorescent protein (GFP) endogenously tags specific isoforms of ETR-1. Ultimately, these studies will deepen our understanding of the role of ETR-1 within both the somatic and germline tissue. This increased understanding will benefit the reproductive biology field and add to our knowledge of the role of the human ETR-1 homologue, CUG-BP, in myotonic muscular dystrophy.