High-Throughput Isolation and Mapping of C. elegans Mutants Susceptible to Pathogen Infection

We present a novel strategy that uses high-throughput methods of isolating and mapping C. elegans mutants susceptible to pathogen infection. We show that C. elegans mutants that exhibit an enhanced pathogen accumulation (epa) phenotype can be rapidly identified and isolated using a sorting system that allows automation of the analysis, sorting, and dispensing of C. elegans by measuring fluorescent bacteria inside the animals. Furthermore, we validate the use of Amplifluor® as a new single nucleotide polymorphism (SNP) mapping technique in C. elegans. We show that a set of 9 SNPs allows the linkage of C. elegans mutants to a 5–8 megabase sub-chromosomal region

C. elegans Germline-Deficient Mutants Respond to Pathogen Infection Using Shared and Distinct Mechanisms

Reproduction extracts a cost in resources that organisms are then unable to utilize to deal with a multitude of environmental stressors. In the nematode C. elegans, development of the germline shortens the lifespan of the animal and increases its susceptibility to microbial pathogens. Prior studies have demonstrated germline-deficient nematodes to have increased resistance to Gram negative bacteria. We show that germline-deficient strains display increased resistance across a broad range of pathogens including Gram positive and Gram negative bacteria, and the fungal pathogen Cryptococcus neoformans. Furthermore, we show that the FOXO transcription factor DAF-16, which regulates longevity and immunity in C. elegans, appears to be crucial for maintaining longevity in both wild-type and germline-deficient backgrounds. Our studies indicate that germline-deficient mutants glp-1 and glp-4 respond to pathogen infection using common and different mechanisms that involve the activation of DAF-16

Insulin Production and Signaling in Renal Tubules of Drosophila Is under Control of Tachykinin-Related Peptide and Regulates Stress Resistance

The insulin-signaling pathway is evolutionarily conserved in animals and regulates growth, reproduction, metabolic homeostasis, stress resistance and life span. In Drosophila seven insulin-like peptides (DILP1-7) are known, some of which are produced in the brain, others in fat body or intestine. Here we show that DILP5 is expressed in principal cells of the renal tubules of Drosophila and affects survival at stress. Renal (Malpighian) tubules regulate water and ion homeostasis, but also play roles in immune responses and oxidative stress. We investigated the control of DILP5 signaling in the renal tubules by Drosophila tachykinin peptide (DTK) and its receptor DTKR during desiccative, nutritional and oxidative stress. The DILP5 levels in principal cells of the tubules are affected by stress and manipulations of DTKR expression in the same cells. Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype. Thus, stress seems to induce hormonal release of DTK that acts on the renal tubules to regulate DILP5 signaling. Manipulations of S6 kinase and superoxide dismutase (SOD2) in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation. Our findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone

Nucleolar Proteins Suppress Caenorhabditis elegans Innate Immunity by Inhibiting p53/CEP-1

The tumor suppressor p53 has been implicated in multiple functions that play key roles in health and disease, including ribosome biogenesis, control of aging, and cell cycle regulation. A genetic screen for negative regulators of innate immunity in Caenorhabditis elegans led to the identification of a mutation in NOL-6, a nucleolar RNA-associated protein (NRAP), which is involved in ribosome biogenesis and conserved across eukaryotic organisms. Mutation or silencing of NOL-6 and other nucleolar proteins results in an enhanced resistance to bacterial infections. A full-genome microarray analysis on animals with altered immune function due to mutation in nol-6 shows increased transcriptional levels of genes regulated by a p53 homologue, CEP-1. Further studies indicate that the activation of innate immunity by inhibition of nucleolar proteins requires p53/CEP-1 and its transcriptional target SYM-1. Since nucleoli and p53/CEP-1 are conserved, our results reveal an ancient immune mechanism by which the nucleolus may regulate immune responses against bacterial pathogens

Drug discovery: Insights from the invertebrate Caenorhabditis elegans

Therapeutic drug development is a long, expensive, and complex process that usually takes 12–15 years. In the early phases of drug discovery, in particular, there is a growing need for animal models that ensure the reduction in both cost and time. Caenorhabditis elegans has been traditionally used to address fundamental aspects of key biological processes, such as apoptosis, aging, and gene expression regulation. During the last decade, with the advent of large-scale platforms for screenings, this invertebrate has also emerged as an essential tool in the pharmaceutical research industry to identify novel drugs and drug targets. In this review, we discuss the reasons why C. elegans has been positioned as an outstanding cost-effective option for drug discovery, highlighting both the advantages and drawbacks of this model. Particular attention is paid to the suitability of this nematode in large-scale genetic and pharmacological screenings. High-throughput screenings in C. elegans have indeed contributed to the breakthrough of a wide variety of candidate compounds involved in extensive fields including neurodegeneration, pathogen infections and metabolic disorders. The versatility of this nematode, which enables its instrumentation as a model of human diseases, is another attribute also herein underscored. As illustrative examples, we discuss the utility of C. elegans models of both human neurodegenerative diseases and parasitic nematodes in the drug discovery industry. Summing up, this review aims to demonstrate the impact of C. elegans models on the drug discovery pipeline.Fil: Giunti, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Andersen, Natalia Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Rayes, Diego Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: de Rosa, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentin

The disruption of proteostasis in neurodegenerative diseases

Cells count on surveillance systems to monitor and protect the cellular proteome which, besides being highly heterogeneous, is constantly being challenged by intrinsic and environmental factors. In this context, the proteostasis network (PN) is essential to achieve a stable and functional proteome. Disruption of the PN is associated with aging and can lead to and/or potentiate the occurrence of many neurodegenerative diseases (ND). This not only emphasizes the importance of the PN in health span and aging but also how its modulation can be a potential target for intervention and treatment of human diseases.info:eu-repo/semantics/publishedVersio

Age structure changes and extraordinary lifespan in wild medfly populations

The main purpose of this study was to test the hypotheses that major changes in age structure occur in wild populations of the Mediterranean fruit fly (medfly) and that a substantial fraction of individuals survive to middle age and beyond (> 3-4 weeks). We thus brought reference life tables and deconvolution models to bear on medfly mortality data gathered from a 3-year study of field-captured individuals that were monitored in the laboratory. The average time-to-death of captured females differed between sampling dates by 23.9, 22.7, and 37.0 days in the 2003, 2004, and 2005 field seasons, respectively. These shifts in average times-to-death provided evidence of changes in population age structure. Estimates indicated that middle-aged medflies (> 30 days) were common in the population. A surprise in the study was the extraordinary longevity observed in field-captured medflies. For example, 19 captured females but no reference females survived in the laboratory for 140 days or more, and 6 captured but no reference males survived in the laboratory for 170 days or more. This paper advances the study of aging in the wild by introducing a new method for estimating age structure in insect populations, demonstrating that major changes in age structure occur in field populations of insects, showing that middle-aged individuals are common in the wild, and revealing the extraordinary lifespans of wild-caught individuals due to their early life experience in the field. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd/The Anatomical Society of Great Britain and Ireland

An essential role for XBP-1 in host protection against immune activation in C. elegans

The detection and compensatory response to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), termed the unfolded protein response (UPR), represents a conserved cellular homeostatic mechanism with important roles in normal development and in the pathogenesis of disease1. The IRE1–XBP1/Hac1 pathway is a major branch of the UPR that has been conserved from yeast to human2, 3, 4, 5, 6. X-box binding protein 1 (XBP1) is required for the differentiation of the highly secretory plasma cells of the mammalian adaptive immune system7, 8, but recent work also points to reciprocal interactions between the UPR and other aspects of immunity and inflammation9, 10, 11. We have been studying innate immunity in the nematode Caenorhabditis elegans, having established a principal role for a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway in mediating resistance to microbial pathogens12. Here we show that during C. elegans development, XBP-1 has an essential role in protecting the host during activation of innate immunity. Activation of the PMK-1-mediated response to infection with Pseudomonas aeruginosa induces the XBP-1-dependent UPR. Whereas a loss-of-function xbp-1 mutant develops normally in the presence of relatively non-pathogenic bacteria, infection of the xbp-1 mutant with P. aeruginosa leads to disruption of ER morphology and larval lethality. Unexpectedly, the larval lethality phenotype on pathogenic P. aeruginosa is suppressed by loss of PMK-1-mediated immunity. Furthermore, hyperactivation of PMK-1 causes larval lethality in the xbp-1 mutant even in the absence of pathogenic bacteria. Our data establish innate immunity as a physiologically relevant inducer of ER stress during C. elegans development and indicate that an ancient, conserved role for XBP-1 may be to protect the host organism from the detrimental effects of mounting an innate immune response to microbes.Howard Hughes Medical Institute (Summer Research Fellowship)National Institutes of Health (U.S.) (NIH grant R01-GM084477)Burroughs Wellcome Fund (Career Award in the Biomedical Sciences)Ellison Medical Foundation (New Scholar Award