Apfeld, Javier et al. (2009) International Worm Meeting "Relating cellular and organismal aging phenotypes in C. elegans."

Apfeld, Javier, Fontana, Walter

[International Worm Meeting, 2009]

Aging appears to be a universal phenomenon in living systems leading to functional changes affecting the onset of disease and the timing of death. Living systems comprise multiple structural and organizational levels, from sub-cellular to whole organism. Aging may occur at any of these levels. In the past two decades aging research has begun to map how organismal lifespan depends on genetic and environmental factors. Yet, the relationship between cellular and organismal aging phenotypes is rarely investigated and poorly understood. We have developed and deployed indicators of cellular state to probe causality between processes leading to failure in individual neurons and processes that determine organismal lifespan in C. elegans. We perform longitudinal measurements in individual neurons utilizing fluorescent probes in both wild-type animals and transgenic animals commonly used as models of neurodegenerative disease. We find that the states of certain neurons become more uncertain as animals get older. In addition, time-dependent correlations between some pairs of neurons develop from initially uncorrelated states. The existence of correlations between specific states of different cells suggests the existence of processes of inter-cellular communication mediating those dependencies. Notably, these neuronal aging processes are independent of processes that determine organismal lifespan. We find that mutations in components of the insulin signaling pathway that increase lifespan promote neuronal aging processes in some neurons yet inhibit them in others. The action of insulin signaling in these neuronal processes is independent of its influence on organismal lifespan. Together, these results show that C. elegans undergoes independent aging processes at different organismal levels.

Servello, Francesco et al. (2021) International Worm Meeting "The heat shock transcription factor HSF-1 protects Caenorhabditis elegans from peroxide stress"

Apfeld, Javier, Servello, Francesco

[International Worm Meeting, 2021]

Cells induce conserved defense mechanisms that protect them from oxidative stress. How these defenses are regulated in multicellular organisms is incompletely understood. Here, we show that the heat shock transcription factor HSF-1 protects the nematode Caenorhabditis elegans from the oxidative stress caused by environmental peroxide. In response to a heat shock or a mild temperature increase, HSF-1 protects the nematodes from subsequent peroxide stress in a manner that depends on HSF-1's transactivation domain. At constant temperature, HSF-1 protects the nematodes from peroxide stress independently of its transactivation domain, likely by inducing the expression of asp-4/cathepsin and dapk-1/dapk. Thus, two distinct HSF-1-dependent processes protect C. elegans from peroxide stress.

Apfeld J et al. (1996) West Coast Worm Meeting "A GENETIC SCREEN FOR LONG-LIVED WORMS."

Dorman JB, Hsin H, Apfeld J, Kenyon CJ, Albinder B

[West Coast Worm Meeting, 1996]

We are interested in finding genes that control lifespan in C. elegans. We are currently doing a clonal screen for mutations that lead to an increase in lifespan. We mutagenize a temperature-sensitive sterile strain, clone out F2 progeny, allow these worms to self fertilize, clone some of their progeny into a new plate at the restrictive temperature, and screen these for increased lifespan. So far we have screened 360 F2 clones and are in the process of retesting 3 of them. Javier Apfeld is also studying the gene daf-2. The e1370 allele of this gene has multiple phenotypes: at 25°C daf-2(e1370) worms develop into dauer larvae (a developmentally arrested, stress resistant, long lived alternative larval form); at 20°C daf-2(e1370) worms do not form dauer larvae, but live 2.5 times longer than wild-type (N2) worms. Javier wants to know where daf-2 functions and how it acts, and is currently doing mosaic analysis to address these questions.

Xu, Yuyan et al. (2021) International Worm Meeting "Signals from the germline act systemically to regulate cytosolic protein oxidation in somatic cells in C. elegans"

Xu, Yuyan, Apfeld, Javier

[International Worm Meeting, 2021]

The reversible formation of disulfide bonds between cysteine residues is an important post-translational modification that modulates the activity of hundreds of cytosolic proteins involved in a wide variety of cellular processes. Little is known about the intercellular signaling pathways that can sway the oxidation of these proteins in a concerted manner. The primary determinant of cysteine oxidation across the proteome is the glutathione redox potential (EGSH). The mechanisms that regulate this potential in vivo remained largely unexplored until the development of the EGSH -specific, reduction-oxidation-sensitive Green Fluorescent Protein (roGFP) family of genetically-encoded biosensors. These GFP-derived biosensors include two cysteines that form a (reversible) intramolecular disulfide bond upon oxidation, resulting in spectral changes that can be quantified via fluorescence-ratio microscopy. Using these biosensors, we determined the extent to which the germline regulates protein oxidation in different somatic tissues C. elegans. We found that ablation of the germline lowers cytosolic protein oxidation in multiple tissues, including the intestine, pharyngeal muscles, and vulval muscles. Within the pharynx, germline ablation affects both the overall level of cytosolic protein oxidation and the spatial pattern of protein oxidation within the tissue. The effects of germline ablation on pharyngeal muscle protein oxidation are mediated in part by the partially redundant action of the DAF-16/FOXO and SKN-1/NRF transcription factors. We have begun identifying the specific germline cell types that modulate protein oxidation in different tissues. Our finding suggest that the germline sends signals that act systemically to promote protein oxidation in somatic cells in C. elegans.

Stroustrup, Nicholas et al. (2011) International Worm Meeting "The Lifespan Machine comes of age: Time-dependent changes in the action of insulin signaling are revealed by high-resolution mortality data."

Fontana, Walter, Apfeld, Javier, Stroustrup, Nicholas

[International Worm Meeting, 2011]

Aging C. elegans populations show characteristic changes in mortality rate and tolerance to stresses including exposure to high temperature and oxidizing agents. We are interested in how individual genes determine the variability and temporal dynamics of these age-dependent phenotypes. We have developed an automated, high-throughput microscopy platform (the "lifespan machine") capable of assaying several thousand worms for movement on agar plates every ten minutes over several weeks. In addition to acquiring high-resolution lifespan distributions at 20 deg C and 25 deg C, our method also produces consistent survival curves under stress regimes including 35 deg C thermotolerance assays and tert-butyl hydroperoxide resistance assays. By subjecting mutant populations to these stressors starting at different times of adulthood, we probe how individual genes affect the age-dependency of stress resistance. In isogenic populations, thermotolerance and oxidant resistance can be quite variable between individuals, reminiscent of the wide variation observed in lifespan under standard conditions. We find that genetic determinants of the mean and variation in lifespan also affect the temporal dynamics of stress resistance as animals age. Strikingly, we find that mutations in several pathway components of insulin signaling known to affect lifespan only affect thermotolerance and resistance to oxidative stress at certain times during adulthood. These findings indicate that worms experience age-dependent changes in the action of insulin signaling.

Romero, Catalina et al. (2011) International Worm Meeting "Investigating the spatial and temporal dynamics of protein oxidation in C. elegans."

Romero, Catalina, Fontana, Walter, Apfeld, Javier

[International Worm Meeting, 2011]

Increased protein oxidation is a phenomenon common to aging in animals in diverse phyla. Protein oxidation is often associated with loss of protein function, which may, in turn, impair cellular protein homeostasis and contribute to the organism's functional decline with age. We are interested in investigating the temporal and spatial dynamics of protein oxidation in different tissues during aging, a question that is difficult to tackle through biochemical approaches. To achieve this goal, we have created transgenic animals expressing a genetically encoded redox sensor, which reports the level of oxidation of specific cysteine residues via changes in fluorescence. We have validated our approach by showing that: i) the sensor responds quickly and reversibly to exposure of the worm to cell-permeable oxidants and reductants; ii) the sensor correctly reports differences in the oxidative environments of specific cellular compartments (nucleus, cytosol and endoplasmic reticulum). We are currently investigating whether mutations that affect lifespan also affect the level of protein oxidation, under normal and oxidative-stress conditions.

Stroustrup, Nicholas et al. (2009) International Worm Meeting "The Lifespan Machine: Automated C. elegans lifespan acquisition on agar plates."

Stroustrup, Nicholas, Fontana, Walter, Apfeld, Javier

[International Worm Meeting, 2009]

The current method for identifying individual death times within C. elegans populations is both labor-intensive and repetitive, limiting the quality and scope of demographic aging data available to the C. elegans research community. In response, we have developed a scalable and low-cost imaging platform to allow fully-automated acquisition of nematode mortality data. Our system captures month-long, time-lapse videos of entire C. elegans populations, with images taken once every two hours at 8 mm resolution. Animals are maintained on temperature-controlled NGM agar plates seeded with E. coli, allowing straightforward integration of machine-acquired results with mortality and behavioral data collected via traditional manual techniques. Through utilization of consumer-grade optical equipment, we have produced an inexpensive system suitable for both small, single-researcher installations and large, high-throughput clusters capable of monitoring tens of thousands of worms simultaneously. To complement the new hardware, we have developed a software package that analyzes time-lapse videos to extract survival curves in a fully-automated fashion. We present survival curves acquired with our technique of large populations (>500 animals each) at high temporal resolution (observations made every two hours). We use this high-resolution mortality data as a basis for the quantitative comparison of aging demographics between various long-lived mutant strains. We also demonstrate the suitability of our method for performing RNAi screens. We hope to share this technology as a platform for the collection and analysis of more and higher quality demographic aging data in the C. elegans community.

Romero, Catalina et al. (2009) International Worm Meeting "In vivo determination of mitochondrial state during the C. elegans aging process."

Apfeld, Javier, Romero, Catalina, Fontana, Walter

[International Worm Meeting, 2009]

Several mutations and RNAi knockdowns of mitochondrial genes affect the lifespan of C. elegans. Furthermore, environmental and genetic perturbations that increase lifespan, such as caloric restriction and reduction in insulin signaling, increase mitochondrial activity (Bishop & Guarente, Nature 447, 545-9, 2007; Houthoofd et al., Aging Cell 4, 87-95, 2005). We are interested in studying how alterations in mitochondrial activity lead to metabolic and physiological changes that result in an extended lifespan. We are quantifying mitochondrial matrix pH in vivo by targeting a pH-sensitive GFP variant to the mitochondrial matrix of muscle cells. This allows us to follow changes in mitochondrial function and morphology in single cells over time. We are currently investigating how (i) differences between individuals and (ii) differences between cells within each individual correlate with the large variation on lifespan that is observed in isogenic populations of worms. We hope that these studies will allow us to quantify the dependencies between changes in mitochondrial activity at the cellular level and the aging phenotype at the organismic level, both in wild type animals and in mutants with extended lifespans.

Stroustrup, Nicholas et al. (2013) International Worm Meeting "Life in the hot seat: Comparing aging and stress resistance."

Apfeld, Javier, Fontana, Walter, Stroustrup, Nicholas, Nash, Zachary

[International Worm Meeting, 2013]

To address the current limitations of manual survival assays with regard to throughput, statistical power, and reproducibility, we developed an automated system for the acquisition and analysis of C. elegans lifespan data. Our technology -- dubbed the "Lifespan Machine" - provides a standardized method for accurate measurement of lifespan, while being low-cost and scalable to any desired statistical resolution. Our lab's installation uses an array of fifty flatbed scanners to monitor up to 30,000 individuals across 2.5 square meters of agar lawn distributed across 800 plates every fifteen minutes at 8 mm optical resolution. We have developed software that analyzes this large volume of image data to identify the death times of individual worms based on their spontaneous movement, with a focus on subtle, late-life postural changes. Our toolset permits the rapid visual validation of automatic measurements, which constitutes a crucial step for the routine production of rigorous mortality statistics. By using standard agar Petri plates, the lifespan machine can automate a variety of assays including feeding RNAi experiments, stress- and pathogen-resistance assays, as well as compound testing. The statistical and temporal resolution afforded by automation allowed us to take a first look at the time-dependent hazard rate of C. elegans populations dying across the full temperature range from 20 deg C to 36.5 deg C at fraction of degree intervals. We identified striking similarities between the hazard functions of animals dying over 14 hours at 33 deg C and those dying over two weeks at 25 deg C. This calls into question the separation between stress resistance (time to death at high temperatures) and aging (time to death at room temperature), widely considered as qualitatively distinct phenomena. Our finding that the shape of the hazard function is universal across temperature is especially surprising when considering that the observed scaling relationship (mean lifespan vs temperature) suggest that distinct physiological transitions occur as temperature increases.

Stroustrup, Nicholas et al. (2015) International Worm Meeting "Slowing Down: Diverse interventions extend C. elegans lifespan through temporal scaling."

Anthony, Winston, Apfeld, Javier, Stroustrup, Nicholas, Fontana, Walter

[International Worm Meeting, 2015]

Many interventions alter C. elegans lifespan: changes in diet, temperature, exposure to xenobiotics, and the disruption of many genes. Yet, individuals vary enormously in their lifespan even within isogenic populations in controlled environments. To probe this stochastic component of aging, we used the Lifespan Machine, our automated system for capturing lifespan data, to collect multiple replicates of high-resolution survival curves across different classes of interventions. We find that most but not all interventions alter lifespan distributions through what appears to be a perfect temporal scaling: the simple stretching of survival curves along the time axis.This temporal scaling provides a strong hint as to the physiological consequences of the interventions studied. Quantitatively, a "stretching" of survival curves seems to require that a "stretching" occurs in each individual's physiology, that every physiologic process's contribution to lifespan is altered in unison. Indeed, we observe that exposure to moderate increases in temperature during just the first three days of adulthood shortens lifespan to the extent expected if animals were uniformly "sped-up" during exposure. The magnitude of this youthful, transient, apparent acceleration in aging corresponds closely to that predicted by the temporal scaling of survival curves across temperatures.Previous work has demonstrated that the rates of decline in feeding behaviors, stress resistance, fertility, and other "healthspan" phenotypes are decoupled by lifespan-extending perturbations. Yet, our results suggest that the specific subset of aging processes causal to death must in fact be altered to exactly the same extent, in young and old animals alike, across a broad class of interventions including changes in diet, temperature, oxidative stress, and disruption of daf-2, daf-16, hsf-1, and hif-1.