Aging is a multifaceted biological process marked by the decline in both mitotic and postmitotic cellular function, often central to the development of age-related diseases. In the pursuit of slowing or even reversing the aging process, a prominent strategy of significant interest is calorie restriction (CR), also known as dietary restriction, and the potential influence of a drug called rapamycin (RM). Both CR and RM have demonstrated the capacity to extend healthspan and lifespan across a diverse array of species, including yeast, worms, flies, and mice. Nevertheless, their individual and combined effects on mitotic and postmitotic cells, as well as their comparative analysis, remain areas that demand a thorough investigation. In this study, we employ RNA-sequencing methodologies to comprehensively analyze the impact of CR, RM, and their combination (CR + RM) on gene expression in yeast cells. Our analysis uncovers distinctive, overlapping, and even contrasting patterns of gene regulation, illuminating the unique and shared effects of CR and RM. Furthermore, the transcriptional synergistic interaction of CR + RM is validated in extending the lifespan of both yeast and human cells.

CRISPR/Cas9 technology is an important tool for functional genomics and crop improvement. It can be used to generate mutations at precise positions in the genome. Base editors consist of deaminase components and Cas9 to specify the type of mutation, such as C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. Available adenine base editor vectors usually make use of canonical Cas9, which limits their use to 5’-NGG-3’ containing targets. We combined a relaxed variant of SpCas9 that uses 5’-NG-3’ containing targets with the adenine base editor containing TadA7.10 or TadA8e to make a set of vectors. By using a phenotype-based screen, we found that our vectors efficiently induce A-to-G somatic mutations in a 5’-NGG-3’ PAM context in Arabidopsis thaliana up to 81% efficiency. Such mutations are inheritable at the homozygous stage in T2. Among tested vectors, pECNUS4 (Addgene #184887), which carries TadA8e, showed highest efficiency at generating a stable A-to-G transition in a 5’-NGH-3’ PAM context in the gene DM3 . Using this vector, we were able to recreate a naturally occurring allele of DM3 in two generations without the transgene. pECNUS4 is a new component of the CRISPR toolbox to be used for introducing desired adenine base transitions with an expanded target window for functional genomic research and trait improvement.

Gerontology research on anti-aging interventions with drugs could be an answer to age-related diseases, aiming at closing the gap between lifespan and healthspan. Here, we present two methods for assaying chronological lifespan in human cells: (1) a version of the classical outgrowth assay with quantitative assessment of surviving cells and (2) a version of the PICLS method (propidium iodide fluorescent-based measurement of cell death). Both methods are fast, simple to conduct, cost-effective, produce quantitative data for further analysis and can be used with diverse human cell lines. Whereas the first method is ideal for validation and testing the post-intervention reproductive potential of surviving cells, the second method has true high-throughput screening potential. The new technologies were validated with known anti-aging compounds (2,5-anhydro- d -mannitol and rapamycin). Using the high-throughput screening method, we screened a library of 162 chemical entities and identified three compounds that extend the longevity of human cells.

Uncovering the regulators of cellular aging will unravel the complexity of aging biology and identify potential therapeutic interventions to delay the onset and progress of chronic, aging-related diseases. In this work, we systematically compared genesets involved in regulating the lifespan of Saccharomyces cerevisiae (a powerful model organism to study the cellular aging of humans) and those with expression changes under rapamycin treatment. Among the functionally uncharacterized genes in the overlap set, YBR238C stood out as the only one downregulated by rapamycin and with an increased chronological and replicative lifespan upon deletion. We show that YBR238C and its paralog RMD9 oppositely affect mitochondria and aging. YBR238C deletion increases the cellular lifespan by enhancing mitochondrial function. Its overexpression accelerates cellular aging via mitochondrial dysfunction. We find that the phenotypic effect of YBR238C is largely explained by HAP4 - and RMD9 -dependent mechanisms. Furthermore, we find that genetic- or chemical-based induction of mitochondrial dysfunction increases TORC1 (Target of Rapamycin Complex 1) activity that, subsequently, accelerates cellular aging. Notably, TORC1 inhibition by rapamycin (or deletion of YBR238C ) improves the shortened lifespan under these mitochondrial dysfunction conditions in yeast and human cells. The growth of mutant cells (a proxy of TORC1 activity) with enhanced mitochondrial function is sensitive to rapamycin whereas the growth of defective mitochondrial mutants is largely resistant to rapamycin compared to wild type. Our findings demonstrate a feedback loop between TORC1 and mitochondria (the TO RC1– MI tochondria– TO RC1 (TOMITO) signaling process) that regulates cellular aging processes. Hereby, YBR238C is an effector of TORC1 modulating mitochondrial function.

Genetic incompatibility occurs when a mismatched pair of plant immune components mounts autoimmune responses in hybrids. Highly diversified NLR receptors are main culprits of the genetic conflict, recognizing host proteins from different origin as immune trigger. Here, we report the molecular mechanism underlying a DANGEROUS MIX (DM) autoimmunity, comprising DM2h/RPP1 NLR and its incompatible partner DM3, an alpha/beta hydrolase. Cryo-electron microscopy reveals the oligomeric nature of two natural DM3 variants in a trimer of dimer configuration. The polymorphism triggering autoimmunity is located at the dimer interface, resulting in drastic structural differences such that dimerizing helix and loop reinforcing the interface is lost and disordered. Structure-function analysis shows that integrity of the dimer interface, but neither maintenance of hexamer nor its enzymatic activity, is the key factor contributing to autoimmunity. Our finding pinpoints checkpoints embedded in the oligomeric configuration of a host enzyme that controls the switching mechanism of NLR activity. ### Competing Interest Statement J.S. is a co-founder and CTO of Epinogen. N.C. is an employee of Merck Pte Ltd.

Aging is an inevitable biological process that contributes to the onset of age-related diseases, often as a result of mitochondrial dysfunction. Understanding the mechanisms behind aging is crucial for developing therapeutic interventions. This study investigates the effects of curcumin on postmitotic cellular lifespan (PoMiCL) during chronological aging in yeast, a widely used model for human postmitotic cellular aging. Our findings reveal that curcumin significantly prolongs the PoMiCL of wildtype yeast cells, with the most pronounced effects observed at lower concentrations, indicating a hormetic response. Importantly, curcumin also extends the lifespan of postmitotic cells with mitochondrial deficiencies, although the hormetic effect is absent in these defective cells. Mechanistically, curcumin inhibits TORC1 activity, enhances ATP levels, and induces oxidative stress. These results suggest that curcumin has the potential to modulate aging and offer therapeutic insights into age-related diseases, highlighting the importance of context in its effects.

The quest to understand and manipulate the mechanisms of cellular aging has far-reaching implications for improving human health and longevity. Our comprehensive effort has led to the discovery of the intriguing anti-aging potential of hemin, an FDA-approved drug primarily used for the treatment of acute intermittent porphyria. Leveraging both yeast and human cell models, we investigate the multifaceted effects of hemin on extending cellular lifespan. Intriguingly, the involvement of the AMPK pathway emerges as a pivotal mechanism underlying hemin’s anti-aging effects. The exploration of hemin’s impact on cellular functionality further uncovers its influence on mitochondrial processes. Notably, both mitochondrial-dependent and -independent mechanisms are implicated in hemin’s ability to extend cellular lifespan, with autophagy playing a significant role in the latter. Additionally, a striking synergy between hemin and the TORC1 inhibitor rapamycin is unveiled, underlining the complexity of cellular signaling networks involved in lifespan extension. Translating these findings to human cells, hemin demonstrates an analogous ability to induce mitochondrial biogenesis, reduce proinflammatory cytokine expression, and enhance antioxidant response. The conservation of hemin’s anti-aging effects across species holds promise for therapeutic applications in addressing age-related diseases and promoting healthier aging.

Abstract Gerontology research on anti-aging interventions with drugs could be an answer to age-related diseases, aiming at closing the gap between lifespan and healthspan. Here, we present two methods for assaying chronological lifespan in human cells: 1) a version of the classical outgrowth assay with quantitative assessment of surviving cells and 2) a version of the PICLS method (propidium iodide fluorescent-based measurement of cell death). Both methods are fast, simple to conduct, cost-effective, produce quantitative data for further analysis and can be used with diverse human cell lines. Whereas the first method is ideal for validation and testing the post-intervention reproductive potential of surviving cells, the second method has true high-throughput screening potential. The new technologies were validated with known anti-aging compounds (2,5-anhydro-D-mannitol and rapamycin). Using the HTS method, we screened a library of 162 chemical entities and identified three novel anti-aging compounds that extend the cellular longevity of human cells.

Aging is an inevitable biological process intricately linked to age-related diseases, including cardiovascular diseases, neurodegeneration, sarcopenia, and age-related macular degeneration. These ailments are often exacerbated by mitochondrial dysfunction, which plays a pivotal role in postmitotic cells. Curcumin, a natural compound, is explored for its anti-aging potential. This study explores the influence of curcumin on the postmitotic cellular lifespan (PoMiCL) of yeast during chronological aging, examining its potential implications for age-related diseases. Our findings reveal that curcumin significantly extends the lifespan of postmitotic wildtype yeast cells, with maximal effects observed at lower concentrations, displaying a hormetic response. Importantly, curcumin mitigates accelerated aging in cells afflicted by mitochondrial dysfunction. Intriguingly, the hormetic effect is absent under these conditions. Mechanistically, curcumin enhances ATP levels but induces oxidative stress and inhibits TORC1. These findings shed light on curcumin’s potential as an anti-aging modulator and its relevance to age-related diseases, offering insights into novel therapeutic approaches for healthy aging while highlighting the context-dependent nature of its effects.

Target of Rapamycin Complex 1 (TORC1) is a conserved eukaryotic protein complex that links the presence of nutrients with cell growth. In Saccharomyces cerevisiae, TORC1 activity is positively regulated by the pres-ence of amino acids and glucose in the medium. However, the mechanisms underlying nutrient-induced TORC1 activation remain poorly understood. By utilizing an in vivo TORC1 activation assay, we demonstrate that differential metabolism of glucose activates TORC1 through three distinct pathways in yeast. The first "ca-nonical Rag guanosine triphosphatase (GTPase)-dependent pathway"requires conversion of glucose to fruc-tose 1,6-bisphosphate, which activates TORC1 via the Rag GTPase heterodimer Gtr1GTP-Gtr2GDP. The second "non-canonical Rag GTPase-dependent pathway"requires conversion of glucose to glucose 6-phosphate, which activates TORC1 via a process that involves Gtr1GTP-Gtr2GTP and mitochondrial function. The third "Rag GTPase-independent pathway"requires complete glycolysis and vacuolar ATPase reassembly for TORC1 activation. We have established a roadmap to deconstruct the link between glucose metabolism and TORC1 activation.

AbstractUncovering the regulators of cellular aging will unravel the complexity of aging biology and identify potential therapeutic interventions to delay the onset and progress of chronic, aging-related diseases. In this work, we systematically compared gene sets involved in regulating the lifespan ofSaccharomyces cerevisiae(a powerful model organism to study the cellular aging of humans) and those with expression changes under rapamycin treatment. Among the functionally uncharacterized genes in the overlap set,YBR238Cstood out as the only one downregulated by rapamycin and with an increased chronological and replicative lifespan upon deletion. We show thatYBR238Cand its paralogueRMD9oppositely affect mitochondria and aging.YBR238Cdeletion increases the cellular lifespan by enhancing mitochondrial function. Its overexpression accelerates cellular aging via mitochondrial dysfunction. We find that the phenotypic effect ofYBR238Cis largely explained byHAP4- andRMD9-dependent mechanisms. Further, we find that genetic or chemical-based induction of mitochondrial dysfunction increases TORC1 (Target of Rapamycin Complex 1) activity that, subsequently, accelerates cellular aging. Notably, TORC1 inhibition by rapamycin (or deletion ofYBR238C) improves the shortened lifespan under these mitochondrial dysfunction conditions in yeast and human cells. The growth of mutant cells (a proxy of TORC1 activity) with enhanced mitochondrial function is sensitive to rapamycin whereas the growth of defective mitochondrial mutants is largely resistant to rapamycin compared to wild type. Our findings demonstrate a feedback loop between TORC1 and mitochondria (theTORC1-MItochondria-TORC1 (TOMITO) signaling process) that regulates cellular aging processes. Hereby,YBR238Cis an effector of TORC1 modulating mitochondrial function.