Host-Microbe-Drug-Nutrient Screen Identifies Bacterial Effectors of Metformin Therapy.

Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT.

azyx-1 is a new gene that overlaps with zyxin and affects its translation in C. elegans, impacting muscular integrity and locomotion.

Overlapping genes are widely prevalent; however, their expression and consequences are poorly understood. Here, we describe and functionally characterize a novel zyx-1 overlapping gene, azyx-1, with distinct regulatory functions in Caenorhabditis elegans. We observed conservation of alternative open reading frames (ORFs) overlapping the 5' region of zyxin family members in several animal species, and find shared sites of azyx-1 and zyxin proteoform expression in C. elegans. In line with a standard ribosome scanning model, our results support cis regulation of zyx-1 long isoform(s) by upstream initiating azyx-1a. Moreover, we report on a rare observation of trans regulation of zyx-1 by azyx-1, with evidence of increased ZYX-1 upon azyx-1 overexpression. Our results suggest a dual role for azyx-1 in influencing zyx-1 proteoform heterogeneity and highlight its impact on C. elegans muscular integrity and locomotion.

Mass Spectrometry-Driven Discovery of Neuropeptides Mediating Nictation Behavior of Nematodes.

Neuropeptides regulate animal physiology and behavior, making them widely studied targets of functional genetics research. While the field often relies on differential -omics approaches to build hypotheses, no such method exists for neuropeptidomics. It would nonetheless be valuable for studying behaviors suspected to be regulated by neuropeptides, especially when little information is otherwise available. This includes nictation, a phoretic strategy of Caenorhabditis elegans dauers that parallels host-finding strategies of infective juveniles of many pathogenic nematodes. We here developed a targeted peptidomics method for the model organism C. elegans and show that 161 quantified neuropeptides are more abundant in its dauer stage compared with L3 juveniles. Many of these have orthologs in the commercially relevant pathogenic nematode Steinernema carpocapsae, in whose infective juveniles, we identified 126 neuropeptides in total. Through further behavioral genetics experiments, we identify flp-7 and flp-11 as novel regulators of nictation. Our work advances knowledge on the genetics of nictation behavior and adds comparative neuropeptidomics as a tool to functional genetics workflows.

Adult-restricted gene knock-down reveals candidates that affect locomotive healthspan in C. elegans.

Understanding how we can age healthily is a challenge at the heart of biogerontological interest. Whereas myriad genes are known to affect the lifespan of model organisms, effects of such interventions on healthspan-the period of life where an animal is considered healthy, rather than merely alive-are less clear. To understand relationships between life- and healthspan, in recent years several platforms were developed with the purpose of assessing both readouts simultaneously. We here relied on one such platform, the WorMotel, to study effects of adulthood-restricted knock-down of 130 Caenorhabditis elegans genes on the locomotive health of the animals along their lifespans. We found that knock-down of six genes affected healthspan while lifespan remained unchanged. For two of these, F26A3.4 and chn-1, knock-down resulted in an improvement of healthspan. In follow-up experiments we showed that knockdown of F26A3.4 indeed improves locomotive health and muscle structure at old age.

Global analysis of neuropeptides in cestodes identifies Attachin, a SIFamide homolog, as a stimulant of parasite motility and attachment.

Many anthelmintics target the neuromuscular system, in particular by interfering with signaling mediated by classical neurotransmitters. Although peptidergic signaling has been proposed as a novel target for anthelmintics, current knowledge of the neuropeptide complement of many helminth groups is still limited, especially for parasitic flatworms (cestodes, trematodes, and monogeneans). In this work, we have characterized the neuropeptide complement of the model cestode Hymenolepis microstoma. Peptidomic characterization of adults of H. microstoma validated many of the neuropeptide precursor (npp) genes previously predicted in silico, and identified novel neuropeptides that are conserved in parasitic flatworms. Most neuropeptides from parasitic flatworms lack significant similarity to those from other animals, confirming the uniqueness of their peptidergic signaling. Analysis of gene expression of ten npp genes by in situ hybridization confirmed that all of them are expressed in the nervous system and identified cryptic features, including the first evidence of dorsoventral asymmetry, as well as a new population of peripheral peptidergic cells that appears to be conserved in the trematode Schistosoma mansoni. Finally, we characterized in greater detail Attachin, an SIFamide homolog. Although its expression is largely restricted to the longitudinal nerve cords and cerebral commissure in H. microstoma, it shows widespread localization in the larval nervous system of Echinococcus multilocularis and Mesocestoides corti. Exogenous addition of a peptide corresponding to the highly conserved C-terminus of Attachin stimulated motility and attachment of M. corti larvae. Altogether, this work provides a robust experimental foothold for the characterization of peptidergic signaling in parasitic flatworms. Cover Image for this issue: https://doi.org/10.1111/jnc.15418.

CEH-60/PBX regulates vitellogenesis and cuticle permeability through intestinal interaction with UNC-62/MEIS in Caenorhabditis elegans.

The onset of sexual maturity involves dramatic changes in physiology and gene expression in many animals. These include abundant yolk protein production in egg-laying species, an energetically costly process under extensive transcriptional control. Here, we used the model organism Caenorhabditis elegans to provide evidence for the spatiotemporally defined interaction of two evolutionarily conserved transcription factors, CEH-60/PBX and UNC-62/MEIS, acting as a gateway to yolk protein production. Via proteomics, bimolecular fluorescence complementation (BiFC), and biochemical and functional readouts, we show that this interaction occurs in the intestine of animals at the onset of sexual maturity and suffices to support the reproductive program. Our electron micrographs and functional assays provide evidence that intestinal PBX/MEIS cooperation drives another process that depends on lipid mobilization: the formation of an impermeable epicuticle. Without this lipid-rich protective layer, mutant animals are hypersensitive to exogenous oxidative stress and are poor partners for mating. Dedicated communication between the hypodermis and intestine in C. elegans likely supports these physiological outcomes, and we propose a fundamental role for the conserved PBX/MEIS interaction in multicellular signaling networks that rely on lipid homeostasis.

Identification of healthspan-promoting genes in Caenorhabditis elegans based on a human GWAS study.

To find drivers of healthy ageing, a genome-wide association study (GWAS) was performed in healthy and unhealthy older individuals. Healthy individuals were defined as free from cardiovascular disease, stroke, heart failure, major adverse cardiovascular event, diabetes, dementia, cancer, chronic obstructive pulmonary disease (COPD), asthma, rheumatism, Crohn's disease, malabsorption or kidney disease. Six single nucleotide polymorphisms (SNPs) with unknown function associated with ten human genes were identified as candidate healthspan markers. Thirteen homologous or closely related genes were selected in the model organism C. elegans for evaluating healthspan after targeted RNAi-mediated knockdown using pathogen resistance, muscle integrity, chemotaxis index and the activity of known longevity and stress response pathways as healthspan reporters. In addition, lifespan was monitored in the RNAi-treated nematodes. RNAi knockdown of yap-1, wwp-1, paxt-1 and several acdh genes resulted in heterogeneous phenotypes regarding muscle integrity, pathogen resistance, chemotactic behaviour, and lifespan. Based on these observations, we hypothesize that their human homologues WWC2, CDKN2AIP and ACADS may play a role in health maintenance in the elderly.

Mass spectrometric evidence for neuropeptide-amidating enzymes in Caenorhabditis elegans.

Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules and are widely involved in the regulation of various physiological processes. The nematode Caenorhabditis elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans However, the enzymes required for the last step in the production of many bioactive peptides, the carboxyl-terminal amidation reaction, have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in WT animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase and a peptidylglycine α-amidating monooxygenase had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans All MS data are available via ProteomeXchange with the identifier PXD008942. In summary, the key steps in neuropeptide processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.

Exploring neuropeptide signalling through proteomics and peptidomics.

INTRODUCTION: Neuropeptides are neuro-endocrine signaling molecules capable of signaling as neurotransmitters, neuromodulators or neurohormones. Studying how neuropeptide signaling is integrated in endocrine pathways and how neuropeptides regulate endogenous processes is crucial to understanding how multicellular organisms respond to environmental and internal cues. Areas covered: This review will cover proteomics and peptidomics approaches used in researching peptide signaling systems and breakthroughs that were achieved in this field. Both differential mass spectrometry and reverse genetic approaches are commonly used to study neuropeptidergic signaling. The field of proteomics quickly developed in the past decades and expanded from gel-based approaches to include advanced liquid chromatography and mass spectrometry. We explore how proteomics is used to reveal neuropeptide maturation and identify downstream targets of neuropeptide signaling pathways. We show how peptidomics differs from standard proteomics approaches and how it is used to study both neuropeptide processing and signal pathway identification. Expert commentary: Thanks to recent advancements in isolation techniques and increased sensitivity of equipment, proteomics and peptidomics studies of neuropeptide signaling are contributing increasingly to elucidating functional implications of endocrine signaling. Further technical progress should allow for full peptidomic profiling of single neurons, eventually providing us with a complete comprehension of endocrine signaling.

In vitro aggregating ß-lactamase-polyQ chimeras do not induce toxic effects in an in vivo Caenorhabditis elegans model.

BACKGROUND: A series of human diseases are caused by the misfolding and aggregation of specific proteins or peptides into amyloid fibrils; nine of these diseases, referred to as polyglutamine diseases, are associated with proteins carrying an expanded polyglutamine (polyQ) region. While the presence of this latter is thought to be the determinant factor for the development of polyQ diseases, the non-polyQ regions of the host proteins are thought to play a significant modulating role. METHOD: In order to better understand the role of non-polyQ regions, the toxic effects of model proteins bearing different polyQ regions (containing up to 79 residues) embedded at two distinct locations within the ß-lactamase (BlaP) host enzyme were evaluated in Caenorhabditis elegans. This small organism can be advantageous for the validation of in vitro findings, as it provides a multicellular context yet avoids the typical complexity of common studies relying on vertebrate models. Several phenotypic assays were performed in order to screen for potential toxic effects of the different BlaP-polyQ proteins. RESULTS: Despite the significant in vitro aggregation of BlaP-polyQ proteins with long polyQ regions, none of the BlaP-polyQ chimeras aggregated in the generated transgenic in vivo models. CONCLUSION: The absence of a toxic effect of the expression of BlaP-polyQ chimeras may find its cause in biochemical mechanisms present in vivo to cope with protein aggregation (e.g. presence of chaperones) or in C. elegans' limitations such as its short lifespan. It is plausible that the aggregation propensities of the different BlaP chimeras containing embedded polyQ sequences are too low in this in vivo environment to permit their aggregation. These experiments emphasize the need for several comparative and in vivo verification studies of biologically relevant in vitro findings, which reveal both the strengths and limitations of widely used model systems.

Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2.

The antiglycemic drug metformin, widely prescribed as first-line treatment of type II diabetes mellitus, has lifespan-extending properties. Precisely how this is achieved remains unclear. Via a quantitative proteomics approach using the model organism Caenorhabditis elegans, we gained molecular understanding of the physiological changes elicited by metformin exposure, including changes in branched-chain amino acid catabolism and cuticle maintenance. We show that metformin extends lifespan through the process of mitohormesis and propose a signaling cascade in which metformin-induced production of reactive oxygen species increases overall life expectancy. We further address an important issue in aging research, wherein so far, the key molecular link that translates the reactive oxygen species signal into a prolongevity cue remained elusive. We show that this beneficial signal of the mitohormetic pathway is propagated by the peroxiredoxin PRDX-2. Because of its evolutionary conservation, peroxiredoxin signaling might underlie a general principle of prolongevity signaling.

Functional neuropeptidomics in invertebrates.

Neuropeptides are key messengers in almost all physiological processes. They originate from larger precursors and are extensively processed to become bioactive. Neuropeptidomics aims to comprehensively identify the collection of neuropeptides in an organism, organ, tissue or cell. The neuropeptidome of several invertebrates is thoroughly explored since they are important model organisms (and models for human diseases), disease vectors and pest species. The charting of the neuropeptidome is the first step towards understanding peptidergic signaling. This review will first discuss the latest developments in exploring the neuropeptidome. The physiological roles and modes of action of neuropeptides can be explored in two ways, which are largely orthogonal and therefore complementary. The first way consists of inferring the functions of neuropeptides by a forward approach where neuropeptide profiles are compared under different physiological conditions. Second is the reverse approach were neuropeptide collections are used to screen for receptor-binding. This is followed by localization studies and functional tests. This review will focus on how these different functional screening methods contributed to the field of invertebrate neuropeptidomics and expanded our knowledge of peptidergic signaling. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

Ageing with elegans: a research proposal to map healthspan pathways.

Human longevity continues to increase world-wide, often accompanied by decreasing birth rates. As a larger fraction of the population thus gets older, the number of people suffering from disease or disability increases dramatically, presenting a major societal challenge. Healthy ageing has therefore been selected by EU policy makers as an important priority ( http://www.healthyageing.eu/european-policies-and-initiatives ); it benefits not only the elderly but also their direct environment and broader society, as well as the economy. The theme of healthy ageing figures prominently in the Horizon 2020 programme ( https://ec.europa.eu/programmes/horizon2020/en/h2020-section/health-demographic-change-and-wellbeing ), which has launched several research and innovation actions (RIA), like "Understanding health, ageing and disease: determinants, risk factors and pathways" in the work programme on "Personalising healthcare" ( https://ec.europa.eu/research/participants/portal/desktop/en/opportunities/h2020/topics/693-phc-01-2014.html ). Here we present our research proposal entitled "ageing with elegans" (AwE) ( http://www.h2020awe.eu/ ), funded by this RIA, which aims for better understanding of the factors causing health and disease in ageing, and to develop evidence-based prevention, diagnostic, therapeutic and other strategies. The aim of this article, authored by the principal investigators of the 17 collaborating teams, is to describe briefly the rationale, aims, strategies and work packages of AwE for the purposes of sharing our ideas and plans with the biogerontological community in order to invite scientific feedback, suggestions, and criticism.

Identification and Targeted Quantification of Endogenous Neuropeptides in the Nematode Caenorhabditis elegans Using Mass Spectrometry.

The nematode Caenorhabditis elegans lends itself as an excellent model organism for peptidomics studies. Its ease of cultivation and quick generation time make it suitable for high-throughput studies. The nervous system, with its 302 neurons, is probably the best-known and studied endocrine tissue. Moreover, its neuropeptidergic signaling pathways display numerous similarities with those observed in other metazoans. Here, we describe two label-free approaches for neuropeptidomics in C. elegans: one for discovery purposes, and another for targeted quantification and comparisons of neuropeptide levels between different samples. Starting from a detailed peptide extraction procedure, we here outline the liquid chromatography tandem mass spectrometry (LC-MS/MS) setup and describe subsequent data analysis approaches.

Large-scale deorphanization of Nematostella vectensis neuropeptide G protein-coupled receptors supports the independent expansion of bilaterian and cnidarian peptidergic systems.

Neuropeptides are ancient signaling molecules in animals but only few peptide receptors are known outside bilaterians. Cnidarians possess a large number of G protein-coupled receptors (GPCRs) - the most common receptors of bilaterian neuropeptides - but most of these remain orphan with no known ligands. We searched for neuropeptides in the sea anemone Nematostella vectensis and created a library of 64 peptides derived from 33 precursors. In a large-scale pharmacological screen with these peptides and 161 N. vectensis GPCRs, we identified 31 receptors specifically activated by 1 to 3 of 14 peptides. Mapping GPCR and neuropeptide expression to single-cell sequencing data revealed how cnidarian tissues are extensively connected by multilayer peptidergic networks. Phylogenetic analysis identified no direct orthology to bilaterian peptidergic systems and supports the independent expansion of neuropeptide signaling in cnidarians from a few ancestral peptide-receptor pairs.

Large-scale sampling of C. elegans on E. coli HT115 RNAi-compatible bacteria.

As opposed to standard culturing, growing large numbers of C. elegans worms on Nematode Growth Medium-based RNA interference (RNAi) plates requires multiple transferring steps to prevent starvation, which increase handling time and reduce practical efficiency. We here provide an optimized method to grow four times more worms in RNAi conditions, thus saving on required resources and handling steps.

Endocytic coelomocytes are required for lifespan extension by axenic dietary restriction.

A rather peculiar but very potent means of achieving longevity is through axenic dietary restriction (ADR), where animals feed on (semi-)defined culture medium in absence of any other lifeform. The little knowledge we already have on ADR is mainly derived from studies using the model organism Caenorhabditis elegans, where ADR more than doubles organismal lifespan. What is underlying this extreme longevity so far remains enigmatic, as ADR seems distinct from other forms of DR and bypasses well-known longevity factors. We here focus first on CUP-4, a protein present in the coelomocytes, which are endocytic cells with a presumed immune function. Our results show that loss of cup-4 or of the coelomocytes affects ADR-mediated longevity to a similar extent. As the coelomocytes have been suggested to have an immune function, we then investigated different central players of innate immune signalling, but could prove no causal links with axenic lifespan extension. We propose that future research focuses further on the role of the coelomocytes in endocytosis and recycling in the context of longevity.

Dry-freezing Steinernema carpocapsae infective juveniles for robust preservation of stocks.

Cryopreservation allows strains to be stored, eliminating genetic drift and maintenance costs. Existing cryopreservation methods for the economically-important entomopathogenic nematode Steinernema carpocapsae involve multiple incubation and filtration steps to precondition the animals. The standard protocol for freezing the model organism Caenorhabditis elegans in buffer is simpler, and a recent C. elegans dry-freezing protocol allows stocks to survive multiple freeze-thaws, a possibility during a power failure. Here we report the efficacy of C. elegans cryopreservation protocols adapted for S. carpocapsae . We show that dry freezing with disaccharides, but not glycerol-based or trehalose-DMSO-based freezing buffer, allows reliable recovery of infective juveniles.

Automated scoring of nematode nictation on a textured background.

Entomopathogenic nematodes, including Steinernema spp., play an increasingly important role as biological alternatives to chemical pesticides. The infective juveniles of these worms use nictation-a behavior in which animals stand on their tails-as a host-seeking strategy. The developmentally-equivalent dauer larvae of the free-living nematode Caenorhabditis elegans also nictate, but as a means of phoresy or "hitching a ride" to a new food source. Advanced genetic and experimental tools have been developed for C. elegans, but time-consuming manual scoring of nictation slows efforts to understand this behavior, and the textured substrates required for nictation can frustrate traditional machine vision segmentation algorithms. Here we present a Mask R-CNN-based tracker capable of segmenting C. elegans dauers and S. carpocapsae infective juveniles on a textured background suitable for nictation, and a machine learning pipeline that scores nictation behavior. We use our system to show that the nictation propensity of C. elegans from high-density liquid cultures largely mirrors their development into dauers, and to quantify nictation in S. carpocapsae infective juveniles in the presence of a potential host. This system is an improvement upon existing intensity-based tracking algorithms and human scoring which can facilitate large-scale studies of nictation and potentially other nematode behaviors.

Automated scoring of nematode nictation on a textured background.

Entomopathogenic nematodes including Steinernema spp. play an increasingly important role as biological alternatives to chemical pesticides. The infective juveniles of these worms use nictation - a behavior in which animals stand on their tails - as a host-seeking strategy. The developmentally-equivalent dauer larvae of the free-living nematode Caenorhabditis elegans also nictate, but as a means of phoresy or "hitching a ride" to a new food source. Advanced genetic and experimental tools have been developed for C. elegans , but time-consuming manual scoring of nictation slows efforts to understand this behavior, and the textured substrates required for nictation can frustrate traditional machine vision segmentation algorithms. Here we present a Mask R-CNN-based tracker capable of segmenting C. elegans dauers and S. carpocapsae infective juveniles on a textured background suitable for nictation, and a machine learning pipeline that scores nictation behavior. We use our system to show that the nictation propensity of C. elegans from high-density liquid cultures largely mirrors their development into dauers, and to quantify nictation in S. carpocapsae infective juveniles in the presence of a potential host. This system is an improvement upon existing intensity-based tracking algorithms and human scoring which can facilitate large-scale studies of nictation and potentially other nematode behaviors.