Covalent chemical probes are useful tools to identify potential drug targets but are rarely applied in aging drug discovery. Here we combine phenotypic screening of heterobifunctional small molecules with chemical proteomics and oxylipin profiling to identify compounds, targets, and endogenous metabolites that regulate lifespan in adult Caenorhabditis elegans. We identify two lifespan-extending aryl sulfonyl fluorides, SF-3 & SF-117, that target the undercharacterized C. elegans soluble epoxide hydrolase CEEH-1. In humans, the CEEH-1 homolog, known as soluble epoxide hydrolase (sEH), sets the balance between epoxy fatty acids and dihydroxy products. Reverse genetics revealed that CEEH-1 knockout was sufficient to extend lifespan, and oxylipin analysis revealed an increase in the ratio of epoxy/dihydroxy fatty acids. Exogenous supplementation of 5,6- and 8,9-dihydroxyeicosatrienoic acids (DiHETs) shortened lifespan through a mechanism requiring the ATF-6 arm of the UPRER. Our proteomics-enabled phenotypic screen identified a lipid-based inflammatory signaling cascade that regulates lifespan in C. elegans.

Mammalian tissue function depends on the balance between proliferative, mitotic cell populations and irreplaceable post-mitotic cells. Throughout life, aging progressively disrupts cell type balance and thus tissue function through opposing forces on different cell types. Aging promotes the aberrant proliferation or entry into pro-inflammatory senescent states of mitotic cells, while contributing to the functional erosion and, in vulnerable populations, loss of post-mitotic irreplaceable cells. The divergent age-associated vulnerabilities of mitotic and post-mitotic cells caused by aging generate a case of homeostatic conflict, in which tissue maintenance simultaneously demands the elimination of damaged mitotic cells and the preservation of irreplaceable ones. We examine the concept of homeostatic conflict using the aging brain as an example, and illustrate how the senescence-prone glia expand and acquire SASP-rich transcriptional states, while post-mitotic neurons undergo widespread loss of molecular identity and cell death, resulting in cell type imbalance and consequently tissue dysfunction. Based on the observed homeostatic conflict, we then devise potential context-sensitive therapeutic strategies by combining interventions that protect from ferroptosis to selectively protect irreplaceable cells with interventions that eliminate maladaptive ones to resolve the age-associated cell type imbalance and thus the underlying homeostatic conflict. We present homeostatic conflict as a concept that goes beyond the reductionist view in which numerous isolated processes promote aging in isolation, and provides a logical framework that integrates multiple processes and explains how their interaction drives age-associated dysfunction in a manner instructive to the development of pharmacological combination strategies.

To understand the mechanism of action of a drug and assess its clinical usefulness and viability, it is imperative that its affinity for its putative targets is determined. When coupled to mass spectrometry (MS), energetics-based protein separation (EBPS) techniques, such as a thermal shift assay, have shown great potential to identify the targets of a drug on a proteome scale. Nevertheless, the computational analyses assessing the confidence of drug-target predictions made by these methods have remained tightly tied to the protocol under which the data were produced. To identify drug targets in data sets produced using different EBPS-MS techniques, we have developed a novel flexible Bayesian inference approach named TargetSeeker-MS. We showed that TargetSeeker-MS identifies known and novel drug targets in Caenorhabditis elegans and HEK 293 samples treated with the fungicide benomyl. We also demonstrated that TargetSeeker-MS' drug-target identifications are reproducible in C. elegans samples that were processed using two different EBPS techniques (thermal shift assay and a differential precipitation of proteins, named DiffPOP). In addition, we validated a novel benomyl target by measuring its altered enzymatic activity upon drug treatment in vitro. TargetSeeker-MS, which is available as a web server (https://targetseeker.scripps.edu/), allows for the rapid, versatile, and confident identification of targets of a drug on a proteome scale, thereby providing a better understanding of its mechanisms and facilitating the evaluation of its clinical viability.

Psychotropic drugs such as antipsychotics improve symptoms of psychiatric disorders. However, they are associated with severe metabolic side effects that remodel energy balance, resulting in weight gain and increased food intake (hyperphagia). Here, we compare how antipsychotics and exogenous serotonin induce hyperphagia by remodeling energy balance. We find that the ability of serotonin and antipsychotics to remodel energy balance strictly depends on the serotonergic receptors SER-7 and SER-5, respectively. While both molecules induce hyperphagia, serotonin does so by increasing energy expenditure and reducing fat stores. In contrast, antipsychotics block the inhibitory effect of fat storage on feeding, thereby inducing hyperphagia and increasing fat stores. Thus, it is possible to manipulate energy balance to induce hyperphagia while either increasing or decreasing fat storage. Inactivation of the germline remodels energy balance similar to antipsychotic treatment, promoting hyperphagia while increasing fat storage. Consistent with overlapping mechanisms, antipsychotics are no longer able to remodel energy balance in both C. elegans and mice lacking an intact germline. Thus, our results uncouple overeating from fat storage and show that overeating can be induced by mechanisms that reduce or increase fat stores.

The degradation of cellular components through autophagy is essential for longevity and healthy aging. However, autophagy function decreases with aging, contributing to age-related diseases. In this study, we characterized a small-molecule activator of autophagy called AA-20 that enhances autophagy and lipid droplet clearance in human cells and in the nematode Caenorhabditis elegans. AA-20 reduces polyglutamine aggregation in an autophagy-dependent manner in both human cells and C. elegans, where it also promotes fitness. Consistently, we found that AA-20 extends lifespan in WT C. elegans, but not in autophagy-deficient mutants. Interestingly, our findings suggest that AA-20 acts, at least in part, through a mechanism involving the transcription factor EB, but without inhibiting the protein kinase mammalian target of rapamycin complex 1. Collectively, our results identify an autophagy activator AA-20, which may have potential therapeutic implications for aging-related proteinopathies and lipid storage disorders.

Analysis of existing lifespan-extending geroprotective compounds suggested that polypharmacological compounds are the most effective geroprotectors, specifically those that bind multiple biogenic amine receptors. To test this hypothesis, we used graph neural networks to predict polypharmacological geroprotectors and evaluated them in Caenorhabditis elegans. Over 70% of the selected compounds extended lifespan, with effect sizes in the top 5% compared to all geroprotectors recorded in the DrugAge database. Thus, our study reveals that rationally designing polypharmacological compounds enables the design of geroprotectors with exceptional efficacy.

Nucleosomes are the minimal repeating units of chromatin. Their dynamic assembly and disassembly underpins chromatin organization and genome regulation. However, it remains unclear how intrinsic nucleosome stability contributes to higher-level yet fundamental cellular and organismal properties-such as preservation of cell identity, lineage specification, stress resilience and ultimately healthy aging. To address this, we tested the impact of decreased intrinsic nucleosome stability across multiple cell, tissue and organismal models by introducing histone mutants that weaken histone-histone interactions. While nucleosome instability did not broadly alter global chromatin accessibility, DNA damage, cell proliferation or viability, it impaired lineage-specific gene expression programs, altered lineage specification and activated intrinsic inflammatory and stress pathways in a manner reminiscent of aging in mouse tissues and human cells. Consistently, nucleosome instability accelerated the onset of age-associated transcriptional alterations and functional decline in Caenorhabditis elegans and Drosophila melanogaster, and reduced cellular resilience to exogenous perturbations-including environmental, epigenetic and mitotic stress-in human cells and Saccharomyces cerevisiae. These cross-species findings identify nucleosome stability as an evolutionarily conserved epigenetic safeguard that preserves cell identity and stress resilience and supports organismal function and healthy aging.

Neurons dynamically regulate their proteome in response to sensory input, a key process underlying experience-dependent plasticity. We characterized the visual experience-dependent nascent proteome in mice within a brief, defined time window after stimulation using an optimized metabolic labeling approach. Visual experience induced cell-type-specific and age-dependent alterations in the nascent proteome, including proteostasis-related proteins. Emerin is the top activity-induced candidate plasticity protein. Activity-induced neuronal Emerin synthesis is rapid and transcription independent. Emerin broadly inhibits protein synthesis, decreasing translation regulators and synaptic proteins. Decreasing Emerin shifted the dendritic spine population from a predominantly mushroom morphology to filopodia and decreased network connectivity. Blocking visual experience-induced Emerin reduced visually evoked electrophysiological responses and impaired behaviorally assessed visual information processing. Our findings support a proteostatic model in which visual experience-induced Emerin provides a feedforward block on further protein synthesis, refining temporal control of activity-induced plasticity proteins and optimizing visual system function.

The Information Theory of Aging (ITOA) proposes that aging results from the progressive loss of epigenetic information. As the repeating units of the epigenome, nucleosomes are considered pivotal for its stability. Accordingly, the ITOA predicts that destabilization of nucleosomes will accelerate aging. However, this causal link has not been directly tested. Here, we addressed this through histone mutants that weaken histone-histone interactions. Without broadly perturbing steady-state chromatin accessibility, DNA damage, cell proliferation or viability, nucleosome instability compromised cell identity maintenance, altered lineage specification and activated intrinsic inflammatory and stress pathways in a manner reminiscent of aging in mouse tissues and human cells. Consistently, nucleosome instability accelerated age-associated transcriptional alterations and functional decline in Caenorhabditis elegans and Drosophila melanogaster, and reduced cellular resilience to exogenous perturbations-including environmental, epigenetic and mitotic stress-in human cells and Saccharomyces cerevisiae. These cross-species findings establish nucleosome stability as a fundamental requirement for preserving cell identity and stress resilience, thereby safeguarding organismal longevity.

Feeding is an essential biological process for an organism's growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans (C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Analysis of lifespan-extending compounds suggested the most effective geroprotectors target multiple biogenic amine receptors. To test this hypothesis, we used graph neural networks to predict such polypharmacological compounds and evaluated them in C. elegans. Over 70% of the selected compounds extended lifespan, with effect sizes in the top 5% compared to the DrugAge database. This reveals that rationally designing polypharmacological compounds enables the design of geroprotectors with exceptional efficacy.

Feeding is an essential biological process for an organism's growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans (C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Objective: Despite great advances in obesity therapeutics in recent years, there is still a need to identify additional therapeutic targets for the treatment of this disease. We previously discovered a signature of genes, including Chloride intracellular channel 1 (Clic1), whose expression was associated with drug-induced weight gain, and in these studies, we assess the effect of Clic1 inhibition on food intake and body weight in mice.Methods: We studied the impact of Clic1 inhibition in mouse models of binge-eating, diet-induced obese mice and genetic models of obesity (Magel2 KO mice).Results: Clic1 knockout (KO) mice ate significantly less and had a lower body weight than WT littermates when either fed chow or high fat diet. Furthermore, pharmacological inhibition of Clic1 in diet-induced obese mice resulted in suppression of food intake and promoted highly effi-cacious weight loss. Clic1 inhibition also reduced food intake in binge-eating models and hyperphagic Magel2 KO mice. We observed that chronic obesity resulted in a significant change in subcellular localization of Clic1 with an increased ratio of Clic1 in the membrane in the obese state. These observations provide a novel therapeutic strategy to block Clic1 translocation as a potential mechanism to reduce food intake and lower body weight.Conclusions: These studies attribute a novel role of Clic1 as a driver of food intake and overconsumption. In summary, we have identified hypothalamic expression of Clic1 plays a key role in food intake, providing a novel therapeutic target to treat overconsumption that is the root cause of modern obesity.(c) 2023 The Authors. Published by Elsevier GmbH.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Recent work has revealed an increasingly important role for mRNA translation in maintaining proteostasis. Here, we use chemical inhibitors targeting discrete steps of translation to compare how lowering the concentration of all or only translation initiation-dependent proteins rescues Caenorhabditis elegans from proteotoxic stress. We systematically challenge proteostasis and show that pharmacologically inhibiting translation initiation or elongation elicits a distinct protective profile. Inhibiting elongation protects from heat and proteasome dysfunction independently from HSF-1 but does not protect from age-associated protein aggregation. Conversely, inhibition of initiation protects from heat and age-associated protein aggregation and increases lifespan, dependent on hsf-1, but does not protect from proteotoxicity caused by proteasome dysfunction. Surprisingly, we find that the ability of the translation initiation machinery to control the concentration of newly synthesized proteins depends on HSF-1. Inhibition of translation initiation in wild-type animals reduces the concentration of newly synthesized proteins but increases it in hsf-1 mutants. Our findings suggest that the HSF-1 pathway is not only a downstream target of translation but also directly cooperates with the translation initiation machinery to control the concentration of newly synthesized proteins to restore proteostasis.

Energy metabolism becomes dysregulated in individuals with obesity and many of these changes persist after weight loss and likely play a role in weight regain. In these studies, we use a mouse model of diet-induced obesity and weight loss to study the transcriptional memory of obesity. We found that the ‘metabolic memory’ of obesity is predominantly localized in adipocytes. Utilizing a C. elegans -based food intake assay, we identify ‘metabolic memory’ genes that play a role in food intake regulation. We show that expression of ATP6v0a1 , a subunit of V-ATPase, is significantly induced in both obese mouse and human adipocytes that persists after weight loss. C. elegans mutants deficient in Atp6v0A1/unc32 eat less than WT controls. Adipocyte-specific Atp6v0a1 knockout mice have reduced food intake and gain less weight in response to HFD. Pharmacological disruption of V-ATPase assembly leads to decreased food intake and less weight re-gain. In summary, using a series of genetic tools from invertebrates to vertebrates, we identify ATP6v0a1 as a regulator of peripheral metabolic memory, providing a potential target for regulation of food intake, weight loss maintenance and the treatment of obesity.

To see if variations in timing of rapamycin (Rapa), administered to middle aged mice starting at 20 months, would lead to different survival outcomes, we compared three dosing regimens. Initiation of Rapa at 42 ppm increased survival significantly in both male and female mice. Exposure to Rapa for a 3-month period led to significant longevity benefit in males only. Protocols in which each month of Rapa treatment was followed by a month without Rapa exposure were also effective in both sexes, though this approach was less effective than continuous exposure in female mice. Interpretation of these results is made more complicated by unanticipated variation in patterns of weight gain, prior to the initiation of the Rapa treatment, presumably due to the use of drug-free food from two different suppliers. The experimental design included tests of four other drugs, minocycline, β-guanidinopropionic acid, MitoQ, and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), but none of these led to a change in survival in either sex.

Lifespan of model organisms can be extended by genetic, dietary and pharmacological interventions, but these effects may be negated by other factors. To understand robustness of longevity interventions within and across species, we analyzed age-dependent mortality of yeast, fruit flies, nematodes and mice subjected to thousands of genetic, pharmacological or dietary interventions, and applied the principles learned to other organisms. Across phylogeny, the accessible space of lifespan distribution functions, the “landscape of longevity”, has a distinct structure of a fiber bundle, with individual fibers given by Strehler-Mildvan degeneracy manifolds. Within species, most interventions perturb parameters of survival curves along particular degeneracy manifolds. Transitions across manifolds are difficult to achieve, but they may lead to robust lifespan-modulating effects. Analyses of intraspecific degeneracy manifolds revealed soft bounds on achievable longevity. For humans, this bound is ∼138 years.

The increasing sophistication of gene expression technologies has given rise to the idea that aging could be understood by analyzing transcriptomes. Mapping trajectories of gene expression changes in aging organisms, across different tissues and brain regions has provided insights on how biological functions change with age. However, recent publications suggest that transcriptional regulation itself deteriorates with age. Loss of transcriptional regulation will lead to non-regulated gene expression changes, but current analysis strategies were not designed to disentangle mixtures of regulated and non-regulated changes. Disentangling transcriptional data to distinguish adaptive, regulatory changes, from those that are the consequence of the age associated deterioration is likely to create an analytical challenge but promises to unlock yet poorly understood aspects of many age-associated transcriptomes.

Measuring lifespan of the model organism, Caenorhabditis elegans, in a 96-well format enables the screening of large chemical libraries to identify biologically active molecules. Furthermore, the wide availability of these animals with specific genetic mutations allows the identification of genes that influence lifespan, and by extension, age-related biological pathways. Here, we present a method for measuring the lifespan of C. elegans in 96-well microtiter plates to identify and study pharmacologically active molecules that extend lifespan. The format of this assay is readily adapted for automated liquid handling systems and imaging of phenotypes.