Periodic Reporting for period 3 - NUAGE (Nucleolar regulation of longevity)
Période du rapport: 2023-01-01 au 2024-06-30
Ageing is a major unsolved biological question: with increasing life expectancy over the last decades, the proportion of elderly with age-related disease has also increased, posing significant challenges for individuals, families, and society. Modulating the process of ageing holds the possibility of tackling the source of age-related morbidities at once, and could thus increase health span into old age. A necessary prerequisite for interfering with the ageing process is a rigorous understanding of the underlying mechanisms. Previous research has shown that ageing entails a progressive decline in cellular performance and organ function over time, eventually leading to organismal demise. These processes are regulated by evolutionarily conserved metabolic and signaling pathways. However, whether these pathways converge on common regulators or shared downstream mechanisms has remained largely unknown. This important question comprises the main focus of our recent research, because it gets to the heart of longevity.
In particular, we previously discovered the nucleolus as a convergence point of all major longevity pathways, with long-lived mutants representing these pathways showing reduced nucleolar size. The nucleolus is a fascinating nuclear subcompartment best known as the site of ribosomal RNA (rRNA) synthesis and ribogenesis, but it has also been shown to respond to various signaling and metabolic pathways and to stress, and can be thought of as a central hub of protein and RNA quality control and assembly.
In the NUAGE project, we set out to determine the role of the nucleolus in the context of ageing more comprehensively, combining unbiased systems approaches and the analysis of candidate nucleolar processes using the nematode C. elegans, the killifish N. furzeri, as well as mouse and human tissue samples. We aim to discover the upstream regulators and downstream mediators of nucleolar function with regards to the nucleolus’ effect on lifespan, and to map out which aspects of nucleolar function in longevity are evolutionarily conserved. Collectively, the objective of this work is to gain a better understanding of the basic mechanisms that link the nucleolus to ageing, and also to determine whether the nucleolus may be used as a simple cellular readout to give important clues about animal health and longevity.
In particular, we previously discovered the nucleolus as a convergence point of all major longevity pathways, with long-lived mutants representing these pathways showing reduced nucleolar size. The nucleolus is a fascinating nuclear subcompartment best known as the site of ribosomal RNA (rRNA) synthesis and ribogenesis, but it has also been shown to respond to various signaling and metabolic pathways and to stress, and can be thought of as a central hub of protein and RNA quality control and assembly.
In the NUAGE project, we set out to determine the role of the nucleolus in the context of ageing more comprehensively, combining unbiased systems approaches and the analysis of candidate nucleolar processes using the nematode C. elegans, the killifish N. furzeri, as well as mouse and human tissue samples. We aim to discover the upstream regulators and downstream mediators of nucleolar function with regards to the nucleolus’ effect on lifespan, and to map out which aspects of nucleolar function in longevity are evolutionarily conserved. Collectively, the objective of this work is to gain a better understanding of the basic mechanisms that link the nucleolus to ageing, and also to determine whether the nucleolus may be used as a simple cellular readout to give important clues about animal health and longevity.
During the first period of the NUAGE project, we performed both unbiased and candidate-approaches to identify upstream and downstream regulators of nucleolar size, and determine whether these also impact ageing. We used an unbiased, high-throughput mutagenesis approach in C. elegans to screen 60,000 individual worms and search for mutants with altered nucleolar size. Using whole-genome sequencing we mapped the mutations in these animals and found multiple variants in genes related to small regulatory RNAs. In initial follow-up experiments we verified that reduced expression of these genes increased nucleolar size and we are currently assessing their impact on ageing. In parallel experiments, we also studied the role of nrde-3, an argonaute-protein involved in RNAi-mediated gene silencing, and a previously published putative regulator of ribosomal RNA expression and nucleolar size. We verified that nrde-3 mutants had reduced nucleolar size and altered lifespan, providing an additional link between small regulatory RNAs, nucleolar size and lifespan regulation.
To determine the regulatory mechanisms downstream of the nucleolus we performed in-depth transcriptomic and proteomic analysis of wild-type worms, long-lived glp-1 mutant animals (with small nucleoli) and short-lived glp-1 ncl-1 animals (with large nucleoli). Based on the systematic analysis of the results we identified 70 potential candidates, which we tested for suppression of glp-1 ncl-1 shortevity. 6 genes significantly extended lifespan, and we are now following up on these genes to understand what mechanisms they regulate. To complement this approach, we studied multiple candidate processes that had been previously linked to the nucleolus, and tested whether they affected or were affected by nucleolar size. Interestingly, multiple experiments pointed towards a link between protein homeostasis and nucleolar size.
Collectively, using a number of independent approaches we have identified multiple likely upstream regulators and downstream mediators linking nucleolar size and lifespan in C. elegans. Having identified these candidates, detailed follow-up experiments have been initiated to pin down the exact mechanism and contribution of the genes and proteins.
To evaluate the evolutionary conservation of nucleolar function in longevity we have generated multiple transgenic killifish lines expressing the ortholog of the ncl-1 gene as a transgene. We also established novel protocols to measure nucleolar size using small blood samples from killifish. Unfortunately, the obtained results were inconsistent, both with regards to lifespan and nucleolar size, most likely due to the different integration sites of the transgene. Work on these lines has therefore been stopped and we are establishing novel methods for robust, reproducible transgene expression in killifish. We will repeat our analysis once these new lines have been generated.
Separately, we studied nucleolar size in tissue samples from mice and humans. In a collaboration with the Amon Lab we found that increased nucleolar size correlated with increased cell size in mouse hematopoietic stem cells, which the Amon Lab showed to correlate with loss of stem cell potential and reduced proliferation (published in Lengefeld et al (2021) Sci Adv 7(46)). We further established nucleolar staining protocols in fixed mouse solid tissues and human blood samples. We measured nucleolar size in different mouse cohorts that underwent life-extending treatments and identified multiple treatments that reduced nucleolar size in kidneys. In initial experiments using human blood samples from young and old blood donors, we found significant nucleolar size differences in different peripheral blood mononuclear cell types (PBMCs) with age. Our current work focuses on verifying whether changes in nucleolar size are consistent across different tissues in mice, and studying the correlation between nucleolar size and other markers of health and healthspan in the animals. We are also poised to measure nucleolar size in PBMCs from different long-lived human cohorts.
To determine the regulatory mechanisms downstream of the nucleolus we performed in-depth transcriptomic and proteomic analysis of wild-type worms, long-lived glp-1 mutant animals (with small nucleoli) and short-lived glp-1 ncl-1 animals (with large nucleoli). Based on the systematic analysis of the results we identified 70 potential candidates, which we tested for suppression of glp-1 ncl-1 shortevity. 6 genes significantly extended lifespan, and we are now following up on these genes to understand what mechanisms they regulate. To complement this approach, we studied multiple candidate processes that had been previously linked to the nucleolus, and tested whether they affected or were affected by nucleolar size. Interestingly, multiple experiments pointed towards a link between protein homeostasis and nucleolar size.
Collectively, using a number of independent approaches we have identified multiple likely upstream regulators and downstream mediators linking nucleolar size and lifespan in C. elegans. Having identified these candidates, detailed follow-up experiments have been initiated to pin down the exact mechanism and contribution of the genes and proteins.
To evaluate the evolutionary conservation of nucleolar function in longevity we have generated multiple transgenic killifish lines expressing the ortholog of the ncl-1 gene as a transgene. We also established novel protocols to measure nucleolar size using small blood samples from killifish. Unfortunately, the obtained results were inconsistent, both with regards to lifespan and nucleolar size, most likely due to the different integration sites of the transgene. Work on these lines has therefore been stopped and we are establishing novel methods for robust, reproducible transgene expression in killifish. We will repeat our analysis once these new lines have been generated.
Separately, we studied nucleolar size in tissue samples from mice and humans. In a collaboration with the Amon Lab we found that increased nucleolar size correlated with increased cell size in mouse hematopoietic stem cells, which the Amon Lab showed to correlate with loss of stem cell potential and reduced proliferation (published in Lengefeld et al (2021) Sci Adv 7(46)). We further established nucleolar staining protocols in fixed mouse solid tissues and human blood samples. We measured nucleolar size in different mouse cohorts that underwent life-extending treatments and identified multiple treatments that reduced nucleolar size in kidneys. In initial experiments using human blood samples from young and old blood donors, we found significant nucleolar size differences in different peripheral blood mononuclear cell types (PBMCs) with age. Our current work focuses on verifying whether changes in nucleolar size are consistent across different tissues in mice, and studying the correlation between nucleolar size and other markers of health and healthspan in the animals. We are also poised to measure nucleolar size in PBMCs from different long-lived human cohorts.
Using different, independent techniques we have uncovered multiple candidate genes and pathways upstream and downstream of nucleolar size regulation. Their detailed study will enable us to place the nucleolus in a larger framework of cellular pathways and map out points of organellar communication that influence ageing. We anticipate that by the end of the project our work will reveal how the nucleolus acts as a convergent hub for longevity, and will thereby also identify novel points of intervention.
In addition, we have established a set of novel methods to study the nucleolus in killifish and human PBMCs. Particularly in killifish, method development goes beyond the framework of this project, and helps consolidate the role of killifish as a model organism for ageing research. Furthermore, by using killifish, mouse and human samples to test our findings from C.elegans we are developing a unique pipeline from a high-throughput nematode model system to vertebrate models. By the end of the project, we expect to have elucidated whether nucleolar size is a robust determinant of healthy ageing in these vertebrate systems, including humans.
In addition, we have established a set of novel methods to study the nucleolus in killifish and human PBMCs. Particularly in killifish, method development goes beyond the framework of this project, and helps consolidate the role of killifish as a model organism for ageing research. Furthermore, by using killifish, mouse and human samples to test our findings from C.elegans we are developing a unique pipeline from a high-throughput nematode model system to vertebrate models. By the end of the project, we expect to have elucidated whether nucleolar size is a robust determinant of healthy ageing in these vertebrate systems, including humans.