Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance.

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.

Proteostasis is differentially modulated by inhibition of translation initiation or elongation.

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.

Targeting Clic1 for the treatment of obesity: A novel therapeutic strategy to reduce food intake and body weight.

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 efficacious 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.

Adipocytes control food intake and weight regain via Vacuolar-type H+ ATPase.

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.

A chemical biology approach to identifying molecular pathways associated with aging.

The understanding of how aging contributes to dementia remains obscure. To address this problem, a chemical biology approach was used employing CAD031, an Alzheimer's disease (AD) drug candidate identified using a discovery platform based upon phenotypic screens that mimic toxicities associated with the aging brain. Since CAD031 has therapeutic efficacy when fed to old symptomatic transgenic AD mice, the chemical biology hypothesis is that it can be used to determine the molecular pathways associated with age-related disease by identifying those that are modified by the compound. Here we show that when CAD031 was fed to rapidly aging SAMP8 mice starting in the last quadrant of their lifespan, it reduced many of the changes in gene, protein, and small molecule expression associated with mitochondrial aging, maintaining mitochondria at the younger molecular phenotype. Network analysis integrating the metabolomics and transcription data followed by mechanistic validation showed that CAD031 targets acetyl-CoA and fatty acid metabolism via the AMPK/ACC1 pathway. Importantly, CAD031 extended the median lifespan of SAMP8 mice by about 30%. These data show that specific alterations in mitochondrial composition and metabolism highly correlate with aging, supporting the use AD drug candidates that limit physiological aging in the brain.

Rapamycin-mediated mouse lifespan extension: Late-life dosage regimes with sex-specific effects.

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, ß-guanidinopropionic acid, MitoQ, and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), but none of these led to a change in survival in either sex.

The aging transcriptome: read between the lines.

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.

Design and Analysis of Pharmacological Studies in Aging.

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.

Elevating acetyl-CoA levels reduces aspects of brain aging.

Because old age is the greatest risk factor for dementia, a successful therapy will require an understanding of the physiological changes that occur in the brain with aging. Here, two structurally distinct Alzheimer's disease (AD) drug candidates, CMS121 and J147, were used to identify a unique molecular pathway that is shared between the aging brain and AD. CMS121 and J147 reduced cognitive decline as well as metabolic and transcriptional markers of aging in the brain when administered to rapidly aging SAMP8 mice. Both compounds preserved mitochondrial homeostasis by regulating acetyl-coenzyme A (acetyl-CoA) metabolism. CMS121 and J147 increased the levels of acetyl-CoA in cell culture and mice via the inhibition of acetyl-CoA carboxylase 1 (ACC1), resulting in neuroprotection and increased acetylation of histone H3K9 in SAMP8 mice, a site linked to memory enhancement. These data show that targeting specific metabolic aspects of the aging brain could result in treatments for dementia.

Loss of genomic integrity induced by lysosphingolipid imbalance drives ageing in the heart.

Cardiac dysfunctions dramatically increase with age. Revealing a currently unknown contributor to cardiac ageing, we report the age-dependent, cardiac-specific accumulation of the lysosphingolipid sphinganine (dihydrosphingosine, DHS) as an evolutionarily conserved hallmark of the aged vertebrate heart. Mechanistically, the DHS-derivative sphinganine-1-phosphate (DHS1P) directly inhibits HDAC1, causing an aberrant elevation in histone acetylation and transcription levels, leading to DNA damage. Accordingly, the pharmacological interventions, preventing (i) the accumulation of DHS1P using SPHK2 inhibitors, (ii) the aberrant increase in histone acetylation using histone acetyltransferase (HAT) inhibitors, (iii) the DHS1P-dependent increase in transcription using an RNA polymerase II inhibitor, block DHS-induced DNA damage in human cardiomyocytes. Importantly, an increase in DHS levels in the hearts of healthy young adult mice leads to an impairment in cardiac functionality indicated by a significant reduction in left ventricular fractional shortening and ejection fraction, mimicking the functional deterioration of aged hearts. These molecular and functional defects can be partially prevented in vivo using HAT inhibitors. Together, we report an evolutionarily conserved mechanism by which increased DHS levels drive the decline in cardiac health.

Pharmacological convergence reveals a lipid pathway that regulates C. elegans lifespan.

Phenotypic screening has identified small-molecule modulators of aging, but the mechanism of compound action often remains opaque due to the complexities of mapping protein targets in whole organisms. Here, we combine a library of covalent inhibitors with activity-based protein profiling to coordinately discover bioactive compounds and protein targets that extend lifespan in Caenorhabditis elegans. We identify JZL184-an inhibitor of the mammalian endocannabinoid (eCB) hydrolase monoacylglycerol lipase (MAGL or MGLL)-as a potent inducer of longevity, a result that was initially perplexing as C. elegans does not possess an MAGL ortholog. We instead identify FAAH-4 as a principal target of JZL184 and show that this enzyme, despite lacking homology with MAGL, performs the equivalent metabolic function of degrading eCB-related monoacylglycerides in C. elegans. Small-molecule phenotypic screening thus illuminates pure pharmacological connections marking convergent metabolic functions in distantly related organisms, implicating the FAAH-4/monoacylglyceride pathway as a regulator of lifespan in C. elegans.

A phenotypic Caenorhabditis elegans screen identifies a selective suppressor of antipsychotic-induced hyperphagia.

Antipsychotic (AP) drugs are used to treat psychiatric disorders but are associated with significant weight gain and metabolic disease. Increased food intake (hyperphagia) appears to be a driving force by which APs induce weight gain but the mechanisms are poorly understood. Here we report that administration of APs to C. elegans induces hyperphagia by a mechanism that is genetically distinct from basal food intake. We exploit this finding to screen for adjuvant drugs that suppress AP-induced hyperphagia in C. elegans and mice. In mice AP-induced hyperphagia is associated with a unique hypothalamic gene expression signature that is abrogated by adjuvant drug treatment. Genetic analysis of this signature using C. elegans identifies two transcription factors, nhr-25/Nr5a2 and nfyb-1/NFYB to be required for AP-induced hyperphagia. Our study reveals that AP-induced hyperphagia can be selectively suppressed without affecting basal food intake allowing for novel drug discovery strategies to combat AP-induced metabolic side effects.

Translation attenuation by minocycline enhances longevity and proteostasis in old post-stress-responsive organisms.

Aging impairs the activation of stress signaling pathways (SSPs), preventing the induction of longevity mechanisms late in life. Here, we show that the antibiotic minocycline increases lifespan and reduces protein aggregation even in old, SSP-deficient Caenorhabditis elegans by targeting cytoplasmic ribosomes, preferentially attenuating translation of highly translated mRNAs. In contrast to most other longevity paradigms, minocycline inhibits rather than activates all major SSPs and extends lifespan in mutants deficient in the activation of SSPs, lysosomal or autophagic pathways. We propose that minocycline lowers the concentration of newly synthesized aggregation-prone proteins, resulting in a relative increase in protein-folding capacity without the necessity to induce protein-folding pathways. Our study suggests that in old individuals with incapacitated SSPs or autophagic pathways, pharmacological attenuation of cytoplasmic translation is a promising strategy to reduce protein aggregation. Altogether, it provides a geroprotecive mechanism for the many beneficial effects of tetracyclines in models of neurodegenerative disease. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Geroneuroprotectors: Effective Geroprotectors for the Brain.

Geroprotectors are compounds that slow the rate of biological aging and therefore may reduce the incidence of age-associated diseases such as Alzheimer's disease (AD). However, few have therapeutic efficacy in mammalian AD models. Here we describe the identification of geroneuroprotectors (GNPs), novel AD drug candidates that meet the criteria for geroprotectors.

The mitochondrial ATP synthase is a shared drug target for aging and dementia.

Aging is a major driving force underlying dementia, such as that caused by Alzheimer's disease (AD). While the idea of targeting aging as a therapeutic strategy is not new, it remains unclear how closely aging and age-associated diseases are coupled at the molecular level. Here, we discover a novel molecular link between aging and dementia through the identification of the molecular target for the AD drug candidate J147. J147 was developed using a series of phenotypic screening assays mimicking disease toxicities associated with the aging brain. We have previously demonstrated the therapeutic efficacy of J147 in several mouse models of AD. Here, we identify the mitochondrial α-F1 -ATP synthase (ATP5A) as a target for J147. By targeting ATP synthase, J147 causes an increase in intracellular calcium leading to sustained calcium/calmodulin-dependent protein kinase kinase ß (CAMKK2)-dependent activation of the AMPK/mTOR pathway, a canonical longevity mechanism. Accordingly, modulation of mitochondrial processes by J147 prevents age-associated drift of the hippocampal transcriptome and plasma metabolome in mice and extends lifespan in drosophila. Our results link aging and age-associated dementia through ATP synthase, a molecular drug target that can potentially be exploited for the suppression of both. These findings demonstrate that novel screens for new AD drug candidates identify compounds that act on established aging pathways, suggesting an unexpectedly close molecular relationship between the two.

Computational Analysis of Lifespan Experiment Reproducibility.

Independent reproducibility is essential to the generation of scientific knowledge. Optimizing experimental protocols to ensure reproducibility is an important aspect of scientific work. Genetic or pharmacological lifespan extensions are generally small compared to the inherent variability in mean lifespan even in isogenic populations housed under identical conditions. This variability makes reproducible detection of small but real effects experimentally challenging. In this study, we aimed to determine the reproducibility of C. elegans lifespan measurements under ideal conditions, in the absence of methodological errors or environmental or genetic background influences. To accomplish this, we generated a parametric model of C. elegans lifespan based on data collected from 5,026 wild-type N2 animals. We use this model to predict how different experimental practices, effect sizes, number of animals, and how different "shapes" of survival curves affect the ability to reproduce real longevity effects. We find that the chances of reproducing real but small effects are exceedingly low and would require substantially more animals than are commonly used. Our results indicate that many lifespan studies are underpowered to detect reported changes and that, as a consequence, stochastic variation alone can account for many failures to reproduce longevity results. As a remedy, we provide power of detection tables that can be used as guidelines to plan experiments with statistical power to reliably detect real changes in lifespan and limit spurious false positive results. These considerations will improve best-practices in designing lifespan experiment to increase reproducibility.

The DrugAge database of aging-related drugs.

Aging is a major worldwide medical challenge. Not surprisingly, identifying drugs and compounds that extend lifespan in model organisms is a growing research area. Here, we present DrugAge (http://genomics.senescence.info/drugs/), a curated database of lifespan-extending drugs and compounds. At the time of writing, DrugAge contains 1316 entries featuring 418 different compounds from studies across 27 model organisms, including worms, flies, yeast and mice. Data were manually curated from 324 publications. Using drug-gene interaction data, we also performed a functional enrichment analysis of targets of lifespan-extending drugs. Enriched terms include various functional categories related to glutathione and antioxidant activity, ion transport and metabolic processes. In addition, we found a modest but significant overlap between targets of lifespan-extending drugs and known aging-related genes, suggesting that some but not most aging-related pathways have been targeted pharmacologically in longevity studies. DrugAge is freely available online for the scientific community and will be an important resource for biogerontologists.

C. elegans as Model for Drug Discovery.

Small molecule screens using C. elegans as a model are becoming increasingly popular as the number of high-throughput methodologies has steadily increased over the years. Here we focus on the biology that underlies this increased popularity and outline the reasons that make C. elegans an attractive model for drug discovery. We discuss successful C. elegans based drug discovery projects in the literature and future challenges ahead.

Metabolic drift in the aging brain.

Brain function is highly dependent upon controlled energy metabolism whose loss heralds cognitive impairments. This is particularly notable in the aged individuals and in age-related neurodegenerative diseases. However, how metabolic homeostasis is disrupted in the aging brain is still poorly understood. Here we performed global, metabolomic and proteomic analyses across different anatomical regions of mouse brain at different stages of its adult lifespan. Interestingly, while severe proteomic imbalance was absent, global-untargeted metabolomics revealed an energymetabolic drift or significant imbalance in core metabolite levels in aged mouse brains. Metabolic imbalance was characterized by compromised cellular energy status (NAD decline, increased AMP/ATP, purine/pyrimidine accumulation) and significantly altered oxidative phosphorylation and nucleotide biosynthesis and degradation. The central energy metabolic drift suggests a failure of the cellular machinery to restore metabostasis (metabolite homeostasis) in the aged brain and therefore an inability to respond properly to external stimuli, likely driving the alterations in signaling activity and thus in neuronal function and communication.