Systematic mapping of organism-scale gene-regulatory networks in aging using population asynchrony.

In aging, physiologic networks decline in function at rates that differ between individuals, producing a wide distribution of lifespan. Though 70% of human lifespan variance remains unexplained by heritable factors, little is known about the intrinsic sources of physiologic heterogeneity in aging. To understand how complex physiologic networks generate lifespan variation, new methods are needed. Here, we present Asynch-seq, an approach that uses gene-expression heterogeneity within isogenic populations to study the processes generating lifespan variation. By collecting thousands of single-individual transcriptomes, we capture the Caenorhabditis elegans "pan-transcriptome"-a highly resolved atlas of non-genetic variation. We use our atlas to guide a large-scale perturbation screen that identifies the decoupling of total mRNA content between germline and soma as the largest source of physiologic heterogeneity in aging, driven by pleiotropic genes whose knockdown dramatically reduces lifespan variance. Our work demonstrates how systematic mapping of physiologic heterogeneity can be applied to reduce inter-individual disparities in aging.

High-Throughput Behavioral Aging and Lifespan Assays Using the Lifespan Machine.

Genetically identical animals kept in a constant environment display a wide distribution of lifespans, reflecting a large non-genetic, stochastic aspect to aging conserved across all organisms studied. This stochastic component means that in order to understand aging and identify successful interventions that extend the lifespan or improve health, researchers must monitor large populations of experimental animals simultaneously. Traditional manual death scoring limits the throughput and scale required for large-scale hypothesis testing, leading to the development of automated methods for high-throughput lifespan assays. The Lifespan Machine (LSM) is a high-throughput imaging platform that combines modified flatbed scanners with custom image processing and data validation software for the life-long tracking of nematodes. The platform constitutes a major technical advance by generating highly temporally resolved lifespan data from large populations of animals at an unprecedented scale and at a statistical precision and accuracy equal to manual assays performed by experienced researchers. Recently, the LSM has been further developed to quantify the behavioral and morphological changes observed during aging and relate them to lifespan. Here, we describe how to plan, run, and analyze an automated lifespan experiment using the LSM. We further highlight the critical steps required for the successful collection of behavioral data and high-quality survival curves.

An mTOR/RNA pol I axis shapes chromatin architecture in response to fasting.

Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom. Yet, how it impacts on 3D genome organization is unknown. Here, we show that fasting induces a reversible and large-scale spatial reorganization of chromatin in C. elegans . This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, a major regulator of ribosome biogenesis. Remarkably, loss of transcription by RNA Pol I, but not RNA Pol II nor Pol III, induces a similar 3D genome reorganization in fed animals, and prevents the restoration of the fed-state architecture upon restoring nutrients to fasted animals. Our work documents the first large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.

Neuronal temperature perception induces specific defenses that enable C. elegans to cope with the enhanced reactivity of hydrogen peroxide at high temperature.

Hydrogen peroxide is the most common reactive chemical that organisms face on the microbial battlefield. The rate with which hydrogen peroxide damages biomolecules required for life increases with temperature, yet little is known about how organisms cope with this temperature-dependent threat. Here, we show that Caenorhabditis elegans nematodes use temperature information perceived by sensory neurons to cope with the temperature-dependent threat of hydrogen peroxide produced by the pathogenic bacterium Enterococcus faecium. These nematodes preemptively induce the expression of specific hydrogen peroxide defenses in response to perception of high temperature by a pair of sensory neurons. These neurons communicate temperature information to target tissues expressing those defenses via an insulin/IGF1 hormone. This is the first example of a multicellular organism inducing their defenses to a chemical when they sense an inherent enhancer of the reactivity of that chemical.

The Earth's environment is full of reactive chemicals that can cause harm to organisms. One of the most common is hydrogen peroxide, which is produced by several bacteria in concentrations high enough to kill small animals, such as the roundworm Caenorhabditis elegans. Forced to live in close proximity to such perils, C. elegans have evolved defenses to ensure their survival, such as producing enzymes that can break down hydrogen peroxide. However, this battle is compounded by other factors. For instance, rising temperatures can increase the rate at which the hydrogen peroxide produced by bacteria reacts with the molecules and proteins of C. elegans. In 2020, a group of researchers found that roundworms sense these temperature changes through special cells called sensory neurons and use this information to control the generation of enzymes that break down hydrogen peroxide. This suggests that C. elegans may pre-emptively prepare their defenses against hydrogen peroxide in response to higher temperatures so they are better equipped to shield themselves from this harmful chemical. To test this theory, Servello et al. ­ including some of the authors involved in the 2020 study ­ exposed C. elegans to a species of bacteria that produces hydrogen peroxide. This revealed that the roundworms were better at dealing with the threat of hydrogen peroxide when growing in warmer temperatures. Experiments done in C. elegans lacking a class of sensory cells, the AFD neurons, showed that these neurons increased the roundworms' resistance to the chemical when temperatures increase. They do this by repressing the activity of INS-39, a hormone that stops C. elegans from switching on their defense mechanism against peroxides. This is the first example of a multicellular organism preparing its defenses to a chemical after sensing something (such as temperature) that enhances its reactivity. It is possible that other animals may also use this 'enhancer sensing' strategy to anticipate and shield themselves from hydrogen peroxide and potentially other external threats.

Omnipose: a high-precision morphology-independent solution for bacterial cell segmentation.

Advances in microscopy hold great promise for allowing quantitative and precise measurement of morphological and molecular phenomena at the single-cell level in bacteria; however, the potential of this approach is ultimately limited by the availability of methods to faithfully segment cells independent of their morphological or optical characteristics. Here, we present Omnipose, a deep neural network image-segmentation algorithm. Unique network outputs such as the gradient of the distance field allow Omnipose to accurately segment cells on which current algorithms, including its predecessor, Cellpose, produce errors. We show that Omnipose achieves unprecedented segmentation performance on mixed bacterial cultures, antibiotic-treated cells and cells of elongated or branched morphology. Furthermore, the benefits of Omnipose extend to non-bacterial subjects, varied imaging modalities and three-dimensional objects. Finally, we demonstrate the utility of Omnipose in the characterization of extreme morphological phenotypes that arise during interbacterial antagonism. Our results distinguish Omnipose as a powerful tool for characterizing diverse and arbitrarily shaped cell types from imaging data.

A hierarchical process model links behavioral aging and lifespan in C. elegans.

Aging involves a transition from youthful vigor to geriatric infirmity and death. Individuals who remain vigorous longer tend to live longer, and within isogenic populations of C. elegans the timing of age-associated vigorous movement cessation (VMC) is highly correlated with lifespan. Yet, many mutations and interventions in aging alter the proportion of lifespan spent moving vigorously, appearing to "uncouple" youthful vigor from lifespan. To clarify the relationship between vigorous movement cessation, death, and the physical declines that determine their timing, we developed a new version of the imaging platform called "The Lifespan Machine". This technology allows us to compare behavioral aging and lifespan at an unprecedented scale. We find that behavioral aging involves a time-dependent increase in the risk of VMC, reminiscent of the risk of death. Furthermore, we find that VMC times are inversely correlated with remaining lifespan across a wide range of genotypes and environmental conditions. Measuring and modelling a variety of lifespan-altering interventions including a new RNA-polymerase II auxin-inducible degron system, we find that vigorous movement and lifespan are best described as emerging from the interplay between at least two distinct physical declines whose rates co-vary between individuals. In this way, we highlight a crucial limitation of predictors of lifespan like VMC-in organisms experiencing multiple, distinct, age-associated physical declines, correlations between mid-life biomarkers and late-life outcomes can arise from the contextual influence of confounding factors rather than a reporting by the biomarker of a robustly predictive biological age.

A complex systems approach to aging biology.

Having made substantial progress understanding molecules, cells, genes and pathways, aging biology research is now moving toward integration of these parts, attempting to understand how their joint dynamics may contribute to aging. Such a shift of perspective requires the adoption of a formal complex systems framework, a transition being facilitated by large-scale data collection and new analytical tools. Here, we provide a theoretical framework to orient researchers around key concepts for this transition, notably emergence, interaction networks and resilience. Drawing on evolutionary theory, network theory and principles of homeostasis, we propose that organismal function is accomplished by the integration of regulatory mechanisms at multiple hierarchical scales, and that the disruption of this ensemble causes the phenotypic and functional manifestations of aging. We present key examples at scales ranging from sub-organismal biology to clinical geriatrics, outlining how this approach can potentially enrich our understanding of aging.

Caenorhabditis elegans processes sensory information to choose between freeloading and self-defense strategies.

Hydrogen peroxide is the preeminent chemical weapon that organisms use for combat. Individual cells rely on conserved defenses to prevent and repair peroxide-induced damage, but whether similar defenses might be coordinated across cells in animals remains poorly understood. Here, we identify a neuronal circuit in the nematode Caenorhabditis elegans that processes information perceived by two sensory neurons to control the induction of hydrogen peroxide defenses in the organism. We found that catalases produced by Escherichia coli, the nematode's food source, can deplete hydrogen peroxide from the local environment and thereby protect the nematodes. In the presence of E. coli, the nematode's neurons signal via TGFß-insulin/IGF1 relay to target tissues to repress expression of catalases and other hydrogen peroxide defenses. This adaptive strategy is the first example of a multicellular organism modulating its defenses when it expects to freeload from the protection provided by molecularly orthologous defenses from another species.

Cells of all kinds often wage chemical warfare against each other. Hydrogen peroxide is often the weapon of choice on the microscopic battlefield, where it is used to incapacitate opponents or to defend against attackers. For example, some plants produce hydrogen peroxide in response to infection to fight off disease-causing microbes. Individual cells have also evolved defenses to prevent or repair 'injuries' caused by hydrogen peroxide. These are similar across many different species. They include enzymes called catalases, which break down hydrogen peroxide, and others to repair damage. However, scientists still do not fully understand how animals and other multicellular organisms might coordinate these defenses across their cells. Caenorhabditis elegans is a microscopic species of worm that lives in rotting fruits. It often encounters the threat of cellular warfare: many types of bacteria in its environment generate hydrogen peroxide, and some can make enough to kill the worms outright. Like other organisms, C. elegans also produces catalases to defend itself against hydrogen peroxide attacks. However, it must activate its defenses at the right time; if it did so when they were not needed, this would result in a detrimental energy 'cost' to the worm. Although C. elegans is a small organism containing only a defined number of cells, exactly why and how it switches its chemical defenses on or off remains unknown. Schiffer et al. therefore set out to determine how C. elegans controls these defenses, focusing on the role of the brain in detecting and processing information from its environment. Experiments looking at the brains of genetically manipulated worms revealed a circuit of sensory nerve cells whose job is to tell the rest of the worm's tissues that they no longer need to produce defense enzymes. Crucially, the circuit became active when the worms sensed E. coli bacteria nearby. Bacteria in the same family as E. coli are normally found in in the same habitat as C. elegans and these bacteria are also known to make enzymes of their own to eliminate hydrogen peroxide around them. These results indicate that C. elegans can effectively decide, based on the activity of its circuit, when to use its own defenses and when to 'freeload' off those of neighboring bacteria. This work is an important step towards understanding how sensory circuits in the brain can control hydrogen peroxide defenses in multicellular organisms. In the future, it could help researchers work out how more complex animals, like humans, coordinate their cellular defenses, and therefore potentially yield new strategies for improving health and longevity.

Transcriptomics-Based Screening Identifies Pharmacological Inhibition of Hsp90 as a Means to Defer Aging.

Aging strongly influences human morbidity and mortality. Thus, aging-preventive compounds could greatly improve our health and lifespan. Here we screened for such compounds, known as geroprotectors, employing the power of transcriptomics to predict biological age. Using age-stratified human tissue transcriptomes and machine learning, we generated age classifiers and applied these to transcriptomic changes induced by 1,309 different compounds in human cells, ranking these compounds by their ability to induce a "youthful" transcriptional state. Testing the top candidates in C. elegans, we identified two Hsp90 inhibitors, monorden and tanespimycin, which extended the animals' lifespan and improved their health. Hsp90 inhibition induces expression of heat shock proteins known to improve protein homeostasis. Consistently, monorden treatment improved the survival of C. elegans under proteotoxic stress, and its benefits depended on the cytosolic unfolded protein response-inducing transcription factor HSF-1. Taken together, our method represents an innovative geroprotector screening approach and was able to identify a class that acts by improving protein homeostasis.

DAF-16/FOXO and HLH-30/TFEB function as combinatorial transcription factors to promote stress resistance and longevity.

The ability to perceive and respond to harmful conditions is crucial for the survival of any organism. The transcription factor DAF-16/FOXO is central to these responses, relaying distress signals into the expression of stress resistance and longevity promoting genes. However, its sufficiency in fulfilling this complex task has remained unclear. Using C. elegans, we show that DAF-16 does not function alone but as part of a transcriptional regulatory module, together with the transcription factor HLH-30/TFEB. Under harmful conditions, both transcription factors translocate into the nucleus, where they often form a complex, co-occupy target promoters, and co-regulate many target genes. Interestingly though, their synergy is stimulus-dependent: They rely on each other, functioning in the same pathway, to promote longevity or resistance to oxidative stress, but they elicit heat stress responses independently, and they even oppose each other during dauer formation. We propose that this module of DAF-16 and HLH-30 acts by combinatorial gene regulation to relay distress signals into the expression of specific target gene sets, ensuring optimal survival under each given threat.

Measuring and modeling interventions in aging.

Many dietary, pharmaceutical, and genetic interventions have been found to increase the lifespan of laboratory animals. Several are now being explored for clinical application. To understand the physiologic action and therapeutic potential of interventions in aging, researchers must build quantitative models. Do interventions delay the onset of aging? Slow it down? Merely ameliorate some of its symptoms? If interventions slow some aging mechanisms but accelerate others, can we detect or predict the systemic consequences? Statistical and analytic models provide a crucial framework in which to answer these questions and clarify the systems-level effect of molecular interventions in aging. This review provides a brief survey of approaches to modeling lifespan data and places them in the context of recent experimental work.

Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway.

BACKGROUND: Many pathogens secrete toxins that target key host processes resulting in the activation of immune pathways. The secreted Pseudomonas aeruginosa toxin Exotoxin A (ToxA) disrupts intestinal protein synthesis, which triggers the induction of a subset of P. aeruginosa-response genes in the nematode Caenorhabditis elegans. RESULTS: We show here that one ToxA-induced C. elegans gene, the Tribbles pseudokinase ortholog nipi-3, is essential for host survival following exposure to P. aeruginosa or ToxA. We find that NIPI-3 mediates the post-developmental expression of intestinal immune genes and proteins and primarily functions in parallel to known immune pathways, including p38 MAPK signaling. Through mutagenesis screening, we identify mutants of the bZIP C/EBP transcription factor cebp-1 that suppress the hypersusceptibility defects of nipi-3 mutants. CONCLUSIONS: NIPI-3 is a negative regulator of CEBP-1, which in turn negatively regulates protective immune mechanisms. This pathway represents a previously unknown innate immune signaling pathway in intestinal epithelial cells that is involved in the surveillance of cellular homeostasis. Because NIPI-3 and CEBP-1 are also essential for C. elegans development, NIPI-3 is analogous to other key innate immune signaling molecules such as the Toll receptors in Drosophila that have an independent role during development.

Extended Twilight among Isogenic C. elegans Causes a Disproportionate Scaling between Lifespan and Health.

Although many genetic factors and lifestyle interventions are known to affect the mean lifespan of animal populations, the physiological variation displayed by individuals across their lifespans remains largely uncharacterized. Here, we use a custom culture apparatus to continuously monitor five aspects of aging physiology across hundreds of isolated Caenorhabditis elegans individuals kept in a constant environment from hatching until death. Aggregating these measurements into an overall estimate of senescence, we find two chief differences between longer- and shorter-lived individuals. First, though long- and short-lived individuals are physiologically equivalent in early adulthood, longer-lived individuals experience a lower rate of physiological decline throughout life. Second, and counter-intuitively, long-lived individuals have a disproportionately extended "twilight" period of low physiological function. While longer-lived individuals experience more overall days of good health, their proportion of good to bad health, and thus their average quality of life, is systematically lower than that of shorter-lived individuals. We conclude that, within a homogeneous population reared under constant conditions, the period of early-life good health is comparatively uniform, and the most plastic period in the aging process is end-of-life senescence.

The temporal scaling of Caenorhabditis elegans ageing.

The process of ageing makes death increasingly likely, involving a random aspect that produces a wide distribution of lifespan even in homogeneous populations. The study of this stochastic behaviour may link molecular mechanisms to the ageing process that determines lifespan. Here, by collecting high-precision mortality statistics from large populations, we observe that interventions as diverse as changes in diet, temperature, exposure to oxidative stress, and disruption of genes including the heat shock factor hsf-1, the hypoxia-inducible factor hif-1, and the insulin/IGF-1 pathway components daf-2, age-1, and daf-16 all alter lifespan distributions by an apparent stretching or shrinking of time. To produce such temporal scaling, each intervention must alter to the same extent throughout adult life all physiological determinants of the risk of death. Organismic ageing in Caenorhabditis elegans therefore appears to involve aspects of physiology that respond in concert to a diverse set of interventions. In this way, temporal scaling identifies a novel state variable, r(t), that governs the risk of death and whose average decay dynamics involves a single effective rate constant of ageing, kr. Interventions that produce temporal scaling influence lifespan exclusively by altering kr. Such interventions, when applied transiently even in early adulthood, temporarily alter kr with an attendant transient increase or decrease in the rate of change in r and a permanent effect on remaining lifespan. The existence of an organismal ageing dynamics that is invariant across genetic and environmental contexts provides the basis for a new, quantitative framework for evaluating the manner and extent to which specific molecular processes contribute to the aspect of ageing that determines lifespan.

An insulin-to-insulin regulatory network orchestrates phenotypic specificity in development and physiology.

Insulin-like peptides (ILPs) play highly conserved roles in development and physiology. Most animal genomes encode multiple ILPs. Here we identify mechanisms for how the forty Caenorhabditis elegans ILPs coordinate diverse processes, including development, reproduction, longevity and several specific stress responses. Our systematic studies identify an ILP-based combinatorial code for these phenotypes characterized by substantial functional specificity and diversity rather than global redundancy. Notably, we show that ILPs regulate each other transcriptionally, uncovering an ILP-to-ILP regulatory network that underlies the combinatorial phenotypic coding by the ILP family. Extensive analyses of genetic interactions among ILPs reveal how their signals are integrated. A combined analysis of these functional and regulatory ILP interactions identifies local genetic circuits that act in parallel and interact by crosstalk, feedback and compensation. This organization provides emergent mechanisms for phenotypic specificity and graded regulation for the combinatorial phenotypic coding we observe. Our findings also provide insights into how large hormonal networks regulate diverse traits.

The Caenorhabditis elegans Lifespan Machine.

The measurement of lifespan pervades aging research. Because lifespan results from complex interactions between genetic, environmental and stochastic factors, it varies widely even among isogenic individuals. The actions of molecular mechanisms on lifespan are therefore visible only through their statistical effects on populations. Indeed, survival assays in Caenorhabditis elegans have provided critical insights into evolutionarily conserved determinants of aging. To enable the rapid acquisition of survival curves at an arbitrary statistical resolution, we developed a scalable imaging and analysis platform to observe nematodes over multiple weeks across square meters of agar surface at 8-µm resolution. The automated method generates a permanent visual record of individual deaths from which survival curves are constructed and validated, producing data consistent with results from the manual method of survival curve acquisition for several mutants in both standard and stressful environments. Our approach permits rapid, detailed reverse-genetic and chemical screens for effects on survival and enables quantitative investigations into the statistical structure of aging.