The C. elegans Observatory: High-throughput exploration of behavioral aging.

Organisms undergo a variety of characteristic changes as they age, suggesting a substantial commonality in the mechanistic basis of aging. Experiments in model organisms have revealed a variety of cellular systems that impact lifespan, but technical challenges have prevented a comprehensive evaluation of how these components impact the trajectory of aging, and many components likely remain undiscovered. To facilitate the deeper exploration of aging trajectories at a sufficient scale to enable primary screening, we have created the Caenorhabditis elegans Observatory, an automated system for monitoring the behavior of group-housed C. elegans throughout their lifespans. One Observatory consists of a set of computers running custom software to control an incubator containing custom imaging and motion-control hardware. In its standard configuration, the Observatory cycles through trays of standard 6 cm plates, running four assays per day on up to 576 plates per incubator. High-speed image processing captures a range of behavioral metrics, including movement speed and stimulus-induced turning, and a data processing pipeline continuously computes summary statistics. The Observatory software includes a web interface that allows the user to input metadata and view graphs of the trajectory of behavioral aging as the experiment unfolds. Compared to the manual use of a plate-based C. elegans tracker, the Observatory reduces the effort required by close to two orders of magnitude. Within the Observatory, reducing the function of known lifespan genes with RNA interference (RNAi) gives the expected phenotypic changes, including extended motility in daf-2(RNAi) and progeria in hsf-1(RNAi). Lifespans scored manually from worms raised in conventional conditions match those scored from images captured by the Observatory. We have used the Observatory for a small candidate-gene screen and identified an extended youthful vigor phenotype for tank-1(RNAi) and a progeric phenotype for cdc-42(RNAi). By utilizing the Observatory, it is now feasible to conduct whole-genome screens for an aging-trajectory phenotype, thus greatly increasing our ability to discover and analyze new components of the aging program.

Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity.

Partial pluripotent reprogramming can reverse features of aging in mammalian cells, but the impact on somatic identity and the necessity of individual reprogramming factors remain unknown. Here, we used single-cell genomics to map the identity trajectory induced by partial reprogramming in multiple murine cell types and dissected the influence of each factor by screening all Yamanaka Factor subsets with pooled single-cell screens. We found that partial reprogramming restored youthful expression in adipogenic and mesenchymal stem cells but also temporarily suppressed somatic identity programs. Our pooled screens revealed that many subsets of the Yamanaka Factors both restore youthful expression and suppress somatic identity, but these effects were not tightly entangled. We also found that a multipotent reprogramming strategy inspired by amphibian regeneration restored youthful expression in myogenic cells. Our results suggest that various sets of reprogramming factors can restore youthful expression with varying degrees of somatic identity suppression. A record of this paper's Transparent Peer Review process is included in the supplemental information.

Reversible Age-Related Phenotypes Induced during Larval Quiescence in C. elegans.

Cells can enter quiescent states in which cell cycling and growth are suspended. We find that during a long developmental arrest (quiescence) induced by starvation, newly hatched C. elegans acquire features associated with impaired proteostasis and aging: mitochondrial fission, ROS production, protein aggregation, decreased proteotoxic-stress resistance, and at the organismal level, decline of mobility and high mortality. All signs of aging but one, the presence of protein aggregates, were reversed upon return to development induced by feeding. The endoplasmic reticulum receptor IRE-1 is completely required for recovery, and the downstream transcription factor XBP-1, as well as a protein kinase, KGB-1, are partially required. Interestingly, kgb-1(-) mutants that do recover fail to reverse aging-like mitochondrial phenotypes and have a short adult lifespan. Our study describes the first pathway that reverses phenotypes of aging at the exit of prolonged quiescence.

A screen for genes involved in respiration control and longevity in Schizosaccharomyces pombe.

We present results showing that glucose signaling has proaging effects in the yeast Schizosaccharomyces pombe. Deletion of the receptor that senses extracellular glucose (Git3) increases the life span of S. pombe, while constitutive activation of the Galpha subunit acting downstream of this receptor (Gpa2) shortens its life span. The latter mutant is also impaired for growth under respiration conditions. We have used this phenotype in a selection strategy to identify genes that when overexpressed can rescue the respiratory defect of constitutively active Galpha subunit mutants. Here, we report an extended version of the work we presented at the IABG meeting and the results of this screen. This strategy allowed us to isolate four genes: psp1(+)/moc1(+), cka1(+), adh1(+), and rpb10(+). Interestingly, the overexpression of these genes was also capable of increasing the chronological life span of wild-type yeast cells.

Fission yeast and other yeasts as emergent models to unravel cellular aging in eukaryotes.

In the past years, simple organisms such as yeasts and worms have contributed a great deal to aging research. Studies pioneered in Saccharomyces cerevisiae were useful to elucidate a significant number of molecular mechanisms underlying cellular aging and to discover novel longevity genes. Importantly, these genes proved many times to be conserved in multicellular eukaryotes. Consequently, such discovery approaches are being extended to other yeast models, such as Schizosaccharomyces pombe, Candida albicans, Kluyveromyces lactis, and Cryptococcus neoformans. In fission yeast, researchers have found links between asymmetrical cell division and nutrient signaling pathways with aging. In this review, we discuss the state of knowledge on the mechanisms controlling both replicative and chronological aging in S pombe and the other emergent yeast models.

Pro-aging effects of glucose signaling through a G protein-coupled glucose receptor in fission yeast.

Glucose is the preferred carbon and energy source in prokaryotes, unicellular eukaryotes, and metazoans. However, excess of glucose has been associated with several diseases, including diabetes and the less understood process of aging. On the contrary, limiting glucose (i.e., calorie restriction) slows aging and age-related diseases in most species. Understanding the mechanism by which glucose limits life span is therefore important for any attempt to control aging and age-related diseases. Here, we use the yeast Schizosaccharomyces pombe as a model to study the regulation of chronological life span by glucose. Growth of S. pombe at a reduced concentration of glucose increased life span and oxidative stress resistance as reported before for many other organisms. Surprisingly, loss of the Git3 glucose receptor, a G protein-coupled receptor, also increased life span in conditions where glucose consumption was not affected. These results suggest a role for glucose-signaling pathways in life span regulation. In agreement, constitutive activation of the Galpha subunit acting downstream of Git3 accelerated aging in S. pombe and inhibited the effects of calorie restriction. A similar pro-aging effect of glucose was documented in mutants of hexokinase, which cannot metabolize glucose and, therefore, are exposed to constitutive glucose signaling. The pro-aging effect of glucose signaling on life span correlated with an increase in reactive oxygen species and a decrease in oxidative stress resistance and respiration rate. Likewise, the anti-aging effect of both calorie restriction and the Deltagit3 mutation was accompanied by increased respiration and lower reactive oxygen species production. Altogether, our data suggest an important role for glucose signaling through the Git3/PKA pathway to regulate S. pombe life span.

Regulation of chronological aging in Schizosaccharomyces pombe by the protein kinases Pka1 and Sck2.

Budding yeast shows a progressive decline in viability after entering stationary phase, a phenomenon known as chronological aging. We show here that the fission yeast Schizosaccharomyces pombe also undergoes chronological aging and that the process is regulated by genes controlling two related nutrient signalling pathways. The first pathway includes the serine/threonine cAMP-activated protein kinase Pka1 and the second pathway comprises the serine/threonine kinase Sck2, a homologue of Saccharomyces cerevisiae SCH9. A double mutant for pka1 and sck2 displayed an additive effect on prolonging the fission yeast lifespan, suggesting that these genes regulate related but independent pathways. These long-lived mutants also accumulated less reactive oxygen species and had a delayed initiation of apoptosis compared with wild-type cells. We also found that strains carrying pka1 deletion but not those with sck2 deletion gained resistance to oxidative stress due to exposure to H(2)O(2) or menadione. On the other hand, the additional increase in lifespan shown by the Deltapka1Deltasck2 double-mutant strain correlated with an increased resistance to both oxidative stress and heat shock. These results underscore the importance of nutrient signalling pathways and reactive oxygen species on organismal lifespan and establish S. pombe as a new model organism to study the molecular mechanisms underlying aging.