The role of phagocytic cells in aging: insights from vertebrate and invertebrate models.

While the main role of phagocytic scavenger cells consists of the neutralization and elimination of pathogens, they also keep the body fluids clean by taking up and breaking down waste material. Since a build-up of waste is thought to contribute to the aging process, these cells become particularly pertinent in the research field of aging. Nevertheless, a direct link between their scavenging functions and the aging process has yet to be established. Integrative approaches involving various model organisms hold promise to elucidate this potential, but are lagging behind since the diversity and evolutionary relationship of these cells across animal species remain unclear. In this perspective, we review the current knowledge associating phagocytic scavenger cells with aging in vertebrate and invertebrate animals, as well as put forward important questions for further exploration. Additionally, we highlight future challenges and propose a constructive approach for tackling them.

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.

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.

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.

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.

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.

Yolk-deprived Caenorhabditis elegans secure brood size at the expense of competitive fitness.

Oviparous animals support reproduction via the incorporation of yolk as a nutrient source into the eggs. In Caenorhabditis elegans, however, yolk proteins seem dispensable for fecundity, despite constituting the vast majority of the embryonic protein pool and acting as carriers for nutrient-rich lipids. Here, we used yolk protein-deprived C. elegans mutants to gain insight into the traits that may yet be influenced by yolk rationing. We show that massive yolk provisioning confers a temporal advantage during embryogenesis, while also increasing early juvenile body size and promoting competitive fitness. Opposite to species that reduce egg production under yolk deprivation, our results indicate that C. elegans relies on yolk as a fail-safe to secure offspring survival, rather than to maintain offspring numbers.

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.

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.

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.

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.

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.

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.

Identification of Non-Canonical Translation Products in C. elegans Using Tandem Mass Spectrometry.

Transcriptome and ribosome sequencing have revealed the existence of many non-canonical transcripts, mainly containing splice variants, ncRNA, sORFs and altORFs. However, identification and characterization of products that may be translated out of these remains a challenge. Addressing this, we here report on 552 non-canonical proteins and splice variants in the model organism C. elegans using tandem mass spectrometry. Aided by sequencing-based prediction, we generated a custom proteome database tailored to search for non-canonical translation products of C. elegans. Using this database, we mined available mass spectrometric resources of C. elegans, from which 51 novel, non-canonical proteins could be identified. Furthermore, we utilized diverse proteomic and peptidomic strategies to detect 40 novel non-canonical proteins in C. elegans by LC-TIMS-MS/MS, of which 6 were common with our meta-analysis of existing resources. Together, this permits us to provide a resource with detailed annotation of 467 splice variants and 85 novel proteins mapped onto UTRs, non-coding regions and alternative open reading frames of the C. elegans genome.

Conditional gene expression in invertebrate animal models.

A mechanistic understanding of biology requires appreciating spatiotemporal aspects of gene expression and its functional implications. Conditional expression allows for (ir)reversible switching of genes on or off, with the potential of spatial and/or temporal control. This provides a valuable complement to the more often used constitutive gene (in)activation through mutagenesis, providing tools to answer a wider array of research questions across biological disciplines. Spatial and/or temporal control are granted primarily by (combinations of) specific promoters, temperature regimens, compound addition, or illumination. The use of such genetic tool kits is particularly widespread in invertebrate animal models because they can be applied to study biological processes in short time frames and on large scales, using organisms amenable to easy genetic manipulation. Recent years witnessed an exciting expansion and optimization of such tools, of which we provide a comprehensive overview and discussion regarding their use in invertebrates. The mechanism, applicability, benefits, and drawbacks of each of the systems, as well as further developments to be expected in the foreseeable future, are highlighted.

DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans.

Transcription factors govern many of the time- and tissue-specific gene expression events in living organisms. CEH-60, a homolog of the TALE transcription factor PBX in vertebrates, was recently characterized as a new regulator of intestinal lipid mobilization in Caenorhabditis elegans. Because CEH-60's orthologs and paralogs exhibit several other functions, notably in neuron and muscle development, and because ceh-60 expression is not limited to the C. elegans intestine, we sought to identify additional functions of CEH-60 through DNA adenine methyltransferase identification (DamID). DamID identifies protein-genome interaction sites through GATC-specific methylation. We here report 872 putative CEH-60 gene targets in young adult animals, and 587 in L2 larvae, many of which are associated with neuron development or muscle structure. In light of this, we investigate morphology and function of ceh-60 expressing AWC neurons, and contraction of pharyngeal muscles. We find no clear functional consequences of loss of ceh-60 in these assays, suggesting that in AWC neurons and pharyngeal muscle, CEH-60 function is likely more subtle or redundant with other factors.

Comparison of size distribution and (Pro249-Ser258) epitope exposure in in vitro and in vivo derived Tau fibrils.

BACKGROUND: Although several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity. METHODS: Tau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors. RESULTS: We observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients. CONCLUSIONS: This study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.

RPamide neuropeptides NLP-22 and NLP-2 act through GnRH-like receptors to promote sleep and wakefulness in C. elegans.

Sleep and wakefulness are fundamental behavioral states of which the underlying molecular principles are becoming slowly elucidated. Transitions between these states require the coordination of multiple neurochemical and modulatory systems. In Caenorhabditis elegans sleep occurs during a larval transition stage called lethargus and is induced by somnogenic neuropeptides. Here, we identify two opposing neuropeptide/receptor signaling pathways: NLP-22 promotes behavioral quiescence, whereas NLP-2 promotes movement during lethargus, by signaling through gonadotropin-releasing hormone (GnRH) related receptors. Both NLP-2 and NLP-22 belong to the RPamide neuropeptide family and share sequence similarities with neuropeptides of the bilaterian GnRH, adipokinetic hormone (AKH) and corazonin family. RPamide neuropeptides dose-dependently activate the GnRH/AKH-like receptors GNRR-3 and GNRR-6 in a cellular receptor activation assay. In addition, nlp-22-induced locomotion quiescence requires the receptor gnrr-6. By contrast, wakefulness induced by nlp-2 overexpression is diminished by deletion of either gnrr-3 or gnrr-6. nlp-2 is expressed in a pair of olfactory AWA neurons and cycles with larval periodicity, as reported for nlp-22, which is expressed in RIA. Our data suggest that the somnogenic NLP-22 neuropeptide signals through GNRR-6, and that both GNRR-3 and GNRR-6 are required for the wake-promoting action of NLP-2 neuropeptides.