Periodic Reporting for period 4 - NUAGE (Nucleolar regulation of longevity)
Periodo di rendicontazione: 2024-07-01 al 2025-12-31
Over the past century, advances in public health have markedly increased human life expectancy. However, this has also led to a growing prevalence of age-related diseases, creating major challenges for society. Because aging is the primary risk factor for conditions such as cardiovascular disease, diabetes, cancer, and neurodegeneration, targeting the aging process itself could simultaneously reduce multiple morbidities. Achieving this goal requires reliable biomarkers of aging and a deeper understanding of the molecular mechanisms that promote resilience and prevent functional decline. Notably, several evolutionarily conserved metabolic signaling pathways and dietary interventions extend healthspan and lifespan in model organisms. Whether these pathways converge on common downstream mechanisms of resilience remains largely unknown. Our research aims to identify such convergent mechanisms to promote healthy aging.
One key mechanism centers on the nucleolus, a nuclear subcompartment responsible for production of ribosomes, which carry out cellular protein synthesis. Because protein synthesis is highly energy-intensive, nucleolar activity strongly influences cellular metabolic rate. We previously found that longevity-promoting metabolic pathways reduce nucleolar size from worms to mammals, while mutations that enlarge the nucleolus, such as ncl-1 in C. elegans, shorten lifespan. However, the mechanisms linking nucleolar activity to longevity were unclear.
In the NUAGE project, we investigated the role of the nucleolus in aging using integrated systems approaches and candidate pathway analyses in C. elegans, mice, and human tissues. We identified downstream mechanisms through which nucleolar function affects lifespan. In particular, we found that improved alignment of ribosomal component stoichiometry promotes longevity and that nucleolar-mitochondrial communication plays a key role in maintaining metabolic health.
Our findings also suggest that the nucleolus may serve as an evolutionarily conserved biomarker of inflammation and longevity. Through various studies, we found that increased nucleolar size correlates with increased kidney injury, and poorer hematopoietic stem cell performance in mouse models. We also found that nucleolar size increases with age in various tissues, and is linked to reproductive senescence in C. elegans. Similarly, levels of the nucleolar protein fibrillarin rise in human lymphocytes with age. We are currently evaluating nucleolar markers in blood cells as potential biomarkers of frailty in individuals over 64 and for disease states associated with chronic inflammation.
One key mechanism centers on the nucleolus, a nuclear subcompartment responsible for production of ribosomes, which carry out cellular protein synthesis. Because protein synthesis is highly energy-intensive, nucleolar activity strongly influences cellular metabolic rate. We previously found that longevity-promoting metabolic pathways reduce nucleolar size from worms to mammals, while mutations that enlarge the nucleolus, such as ncl-1 in C. elegans, shorten lifespan. However, the mechanisms linking nucleolar activity to longevity were unclear.
In the NUAGE project, we investigated the role of the nucleolus in aging using integrated systems approaches and candidate pathway analyses in C. elegans, mice, and human tissues. We identified downstream mechanisms through which nucleolar function affects lifespan. In particular, we found that improved alignment of ribosomal component stoichiometry promotes longevity and that nucleolar-mitochondrial communication plays a key role in maintaining metabolic health.
Our findings also suggest that the nucleolus may serve as an evolutionarily conserved biomarker of inflammation and longevity. Through various studies, we found that increased nucleolar size correlates with increased kidney injury, and poorer hematopoietic stem cell performance in mouse models. We also found that nucleolar size increases with age in various tissues, and is linked to reproductive senescence in C. elegans. Similarly, levels of the nucleolar protein fibrillarin rise in human lymphocytes with age. We are currently evaluating nucleolar markers in blood cells as potential biomarkers of frailty in individuals over 64 and for disease states associated with chronic inflammation.
We used both unbiased and candidate approaches to identify regulators of nucleolar size and function. First, we performed EMS mutagenesis in C. elegans and screened for mutants with enlarged nucleoli using the nucleolar marker FIB-1::GFP. As expected, we recovered new mutations in ncl-1, which we previously showed expands nucleoli and reduces longevity. We also identified several epigenetic regulators that altered reporter expression but did not affect nucleolar size or aging. We therefore focused on how ncl-1 regulates lifespan by integrating transcriptomic, proteomic, and phenotypic analyses, primarily comparing long-lived glp-1 mutants with short-lived glp-1 ncl-1 double mutants.
We found that mutation of ncl-1 induces accelerated aging, characterized by premature senescence markers and reduced mobility. Molecularly, glp-1 ncl-1 mutants show dysregulated ribosome biogenesis: rRNA transcription in the nucleolus is increased while mature rRNA levels decline. mRNA and protein levels of ribosomal components become uncoupled, disrupting ribosomal stoichiometry, impairing translation efficiency, and increasing protein aggregation (Martinez-Miguel, Popkes-van Oepen et al., 2026, BioRxiv). An unbiased RNAi screen revealed that knockdown of the mitochondrial ribosomal protein mrps-16 and the RNase P/MRP component popl-1 restores ribosomal balance and rescues longevity in glp-1 ncl-1 mutants, identifying key components that can bypass nucleolar dysfunction. Ongoing work investigates how mitochondrial–nucleolar cross-talk links metabolism to aging.
In a candidate approach, we examined the argonaute protein NRDE-3, a nuclear RNAi factor that interacts with NCL-1 in yeast two-hybrid assays. Similar to ncl-1, double mutants of glp-1; nrde-3 are short-lived, although nrde-3 appears to regulate lifespan independently of nucleolar function. Current RNAi screens aim to identify the mechanisms involved.
To assess nucleolar function in mammals, we generated mice with a heterozygous deletion of nucleolar fibrillarin and are conducting integrative systems biology and phenotypic analyses, including lifespan and metabolic studies. In parallel, we showed that nucleolar size may be a viable biomarker of aging and inflammation in mice and humans. Enlarged nucleoli correlate with reduced stem cell potential in hematopoietic stem cells in mice (Lengefeld et al., 2021, Science Advances) and with reproductive senescence in C. elegans germline stem cells (Nonninger, Mak, Gerisch et al., 2025, Nature Aging). In mouse kidney injury models, increased nucleolar size correlates with tissue damage but can be prevented by dietary restriction (Koehler et al., 2022, Translational Research). In human blood samples, lymphocytes from older donors show increased fibrillarin levels. Moreover, nucleoli enlarge during infection and inflammation in clinical cohorts and in patients with periodic fever syndromes (Steiner et al., 2023, Rheumatology). Ongoing longitudinal studies aim to establish nucleolar size as a biomarker of inflammation and disease activity.
We found that mutation of ncl-1 induces accelerated aging, characterized by premature senescence markers and reduced mobility. Molecularly, glp-1 ncl-1 mutants show dysregulated ribosome biogenesis: rRNA transcription in the nucleolus is increased while mature rRNA levels decline. mRNA and protein levels of ribosomal components become uncoupled, disrupting ribosomal stoichiometry, impairing translation efficiency, and increasing protein aggregation (Martinez-Miguel, Popkes-van Oepen et al., 2026, BioRxiv). An unbiased RNAi screen revealed that knockdown of the mitochondrial ribosomal protein mrps-16 and the RNase P/MRP component popl-1 restores ribosomal balance and rescues longevity in glp-1 ncl-1 mutants, identifying key components that can bypass nucleolar dysfunction. Ongoing work investigates how mitochondrial–nucleolar cross-talk links metabolism to aging.
In a candidate approach, we examined the argonaute protein NRDE-3, a nuclear RNAi factor that interacts with NCL-1 in yeast two-hybrid assays. Similar to ncl-1, double mutants of glp-1; nrde-3 are short-lived, although nrde-3 appears to regulate lifespan independently of nucleolar function. Current RNAi screens aim to identify the mechanisms involved.
To assess nucleolar function in mammals, we generated mice with a heterozygous deletion of nucleolar fibrillarin and are conducting integrative systems biology and phenotypic analyses, including lifespan and metabolic studies. In parallel, we showed that nucleolar size may be a viable biomarker of aging and inflammation in mice and humans. Enlarged nucleoli correlate with reduced stem cell potential in hematopoietic stem cells in mice (Lengefeld et al., 2021, Science Advances) and with reproductive senescence in C. elegans germline stem cells (Nonninger, Mak, Gerisch et al., 2025, Nature Aging). In mouse kidney injury models, increased nucleolar size correlates with tissue damage but can be prevented by dietary restriction (Koehler et al., 2022, Translational Research). In human blood samples, lymphocytes from older donors show increased fibrillarin levels. Moreover, nucleoli enlarge during infection and inflammation in clinical cohorts and in patients with periodic fever syndromes (Steiner et al., 2023, Rheumatology). Ongoing longitudinal studies aim to establish nucleolar size as a biomarker of inflammation and disease activity.
While previous studies have linked nucleolar enlargement to shortened life span, we have now demonstrated that nucleolar size increases with chronological age in C. elegans and humans and may serve as a useful aging biomarker.
Even though nucleolar size increases, protein synthesis and ribosome abundance progressively decline during aging, and paradoxically, genetic or pharmacological attenuation of translation extends lifespan across species. In our study in C. elegans, we unexpectedly found that improving ribosome homeostasis and restoring ribosome stoichiometry rescues the lifespan of glp-1 ncl-1 mutant worms independent of the absolute levels of ribosomal proteins, rRNA and overall translation. We identify age-associated ribosome dysfunction as a qualitative failure of ribosomal biogenesis and show that restoring ribosomal homeostasis, rather than translational inhibition per se, improves proteostasis and extends lifespan. Furthermore, we are excited to pursue the outcome of our heterozygous fibrillarin deletion mice on metabolism and lifespan, since this project tests the idea that direct modulation of nucleolar function can affect health outcomes.
The nucleolus as a biomarker for biological age and health is an extremely exciting avenue to explore. Current biomarkers and assessments of biological age, frailty or for disease state and successful interventions are often expensive to measure (-omics approaches), time-consuming to record (patient questionnaires and extensive examination), difficult to access (measuring calprotectin as an inflammatory marker), or not well correlated with disease state (antibody levels for autoimmune diseases), while the nucleolus can be examined microscopically from low volumes of readily available blood samples. We have successfully streamlined workflows for isolating and freezing peripheral blood mononuclear cells (PBMCs) from patients, and for reproducibly measuring nucleolar size and protein levels in defined cell types by flow cytometry, imaging flow cytometry and confocal microscopy. We have imaged samples from over 1000 geriatric patients (older than 64 years), for whom detailed clinical data, and extensive baseline as well as follow-up frailty and outcome assessments are available through participation in a clinical trial. Computational analysis will be exploited to link nucleolar features with frailty and outcome measures as well as different molecular readouts that are being generated by our collaboration partners. Simultaneously, we are imaging samples from autoimmune patients to correlate nucleolar size with disease state, for which currently no reliable, easily accessible biomarker is available. Predicting frailty, patient outcome or disease state from nucleolar readouts would be a significant breakthrough in the field.
Even though nucleolar size increases, protein synthesis and ribosome abundance progressively decline during aging, and paradoxically, genetic or pharmacological attenuation of translation extends lifespan across species. In our study in C. elegans, we unexpectedly found that improving ribosome homeostasis and restoring ribosome stoichiometry rescues the lifespan of glp-1 ncl-1 mutant worms independent of the absolute levels of ribosomal proteins, rRNA and overall translation. We identify age-associated ribosome dysfunction as a qualitative failure of ribosomal biogenesis and show that restoring ribosomal homeostasis, rather than translational inhibition per se, improves proteostasis and extends lifespan. Furthermore, we are excited to pursue the outcome of our heterozygous fibrillarin deletion mice on metabolism and lifespan, since this project tests the idea that direct modulation of nucleolar function can affect health outcomes.
The nucleolus as a biomarker for biological age and health is an extremely exciting avenue to explore. Current biomarkers and assessments of biological age, frailty or for disease state and successful interventions are often expensive to measure (-omics approaches), time-consuming to record (patient questionnaires and extensive examination), difficult to access (measuring calprotectin as an inflammatory marker), or not well correlated with disease state (antibody levels for autoimmune diseases), while the nucleolus can be examined microscopically from low volumes of readily available blood samples. We have successfully streamlined workflows for isolating and freezing peripheral blood mononuclear cells (PBMCs) from patients, and for reproducibly measuring nucleolar size and protein levels in defined cell types by flow cytometry, imaging flow cytometry and confocal microscopy. We have imaged samples from over 1000 geriatric patients (older than 64 years), for whom detailed clinical data, and extensive baseline as well as follow-up frailty and outcome assessments are available through participation in a clinical trial. Computational analysis will be exploited to link nucleolar features with frailty and outcome measures as well as different molecular readouts that are being generated by our collaboration partners. Simultaneously, we are imaging samples from autoimmune patients to correlate nucleolar size with disease state, for which currently no reliable, easily accessible biomarker is available. Predicting frailty, patient outcome or disease state from nucleolar readouts would be a significant breakthrough in the field.