Periodic Reporting for period 4 - DeAge (Deconstructing Ageing: from molecular mechanisms to intervention strategies)
Reporting period: 2022-03-01 to 2023-08-31
Over many years, our research group has explored the complex relationship between cancer and aging. As part of this work, we have generated mouse models that are protected from cancer but exhibit accelerated aging. These models allowed us to unveil novel mechanisms of normal and pathological aging, to discover two new human progeroid syndromes, and to develop therapies for the Hutchinson-Gilford progeria syndrome, now in clinical trials. We also integrated data from many laboratories to define the Hallmarks of Aging and the metabolic basis of longevity. During this project, we have explored the relative relevance of cell-intrinsic and -extrinsic mechanisms of aging. For this purpose, we pursued three major aims: 1) to characterize cell-intrinsic alterations that drive aging; 2) to investigate aging as a systemic process; and 3) to design intervention strategies aimed at expanding longevity. Using both hypothesis-driven and unbiased approaches, we have provided new insights into the molecular and evolutionary mechanisms of aging. Thus, we assembled and analyzed the genomes of immortal, long-lived and short-lived organisms and we formulated wide-range hypotheses on the adaptations affecting biochemical pathways, such as DNA damage response to address the challenges posed by an extended longevity. In addition, to study cell-intrinsic mechanisms relevant to human aging, we developed genome-editing screenings which allowed us to single out novel genes regulating cellular senescence. For the validation of hypotheses on the role of intrinsic hallmarks of aging, we generated murine models targeting the regulation of mitochondrial function and proteostasis. We have also used these models to assess the role of cell-extrinsic factors, such as microbiota, diet and NAD+ levels, in normal and pathological aging. Finally, we explored these and other cell-extrinsic interventions as tools to extend longevity and healthspan. In summary, this project has leveraged both new and established tools to mine high-throughput data and draw conclusions on novel cell-intrinsic and cell-extrinsic factors that may serve as the basis for innovative advances in age-related healthcare.
In line with the approaches proposed for the study of cell-intrinsic mechanisms that drive aging (Aim 1), we have investigated the molecular features underlying rejuvenation in the “immortal jellyfish”, Turritopsis dohrnii, which undergoes life-cycle reversal conferring biological immortality. We have also performed a genomic study of a giant tortoise, Lonesome George, the last representative of Chelonoidis abingdonii (published in Nature Ecol&Evol) identifying evolutionary strategies linked to lifespan and expanding our understanding of the determinants of aging. Together with genomic studies on long- and short-lived species of fish and sea urchin, these results gave us a glimpse of the evolutionary basis of longevity. We have set up genome-editing screenings to identify genes with the ability to reverse cell senescence, identifying candidates to reverse the senescent phenotype. Among them, we found well-known regulators of senescence, such as the tumor suppressors p53 and p21, which demonstrates the soundness of the screen. To confirm these results, we have individually validated the selected candidates through gain- or loss-of-function assays. In relation to the proposed Lonp1 transgenesis project, we found dual effects on healthspan of overexpression of this mitochondrial protease. First, in a p53-deficient background, mice overexpressing Lonp1 had higher mortality while in old animals, the health status of transgenic mice appeared to be improved. As for the studies on proteostasis in aging, we have generated a new mouse model overexpressing AIRAPL to study its role in cancer and longevity. Surprisingly, elevated levels of this proteostasis factor do not confer health benefits, suggesting the need of proper expression levels.
To investigate cell-extrinsic factors involved in aging (Aim 2), we have generated a new mouse model aimed at boosting cell proliferation in the stromal compartment of progeroid animals. However, as the genetic strategy involved did not work as expected, we used alternative approaches to this objective. Thus, we focused on immune modulators produced in progeroid mice in response to the accumulation of damage. To this end, we generated a new LmnaG609G model null for Klrk1, since natural killer cells sense stressed or damaged cells with a major contribution of the signaling mediated by this receptor. Likewise, we have investigated the role of RANKL (Tnfs11) in the aging process by crossing Zmpste24—/— animals with mice harboring a floxed Tnfsf11 allele and mice expressing Cre recombinase in osteocytes, to specifically eliminate RANKL in these cells. These mice showed a reversion of the osteoporotic phenotype and increased survival. In addition, we performed the first large-scale profiling of the plasma proteome of two progeroid mouse models (LmnaG609G/G609G and Zmpste24—/— mice). This work (published in Aging Cell) provided an extensive list of plasma proteins as potential biomarkers and/or therapeutic targets for further exploration in both physiological and premature aging. We have also characterized microbiota alterations in accelerated aging, finding intestinal dysbiosis in human patients and two different mouse models of progeria. Remarkably, transplantation of microbiota from wild-type into progeroid mice improved their healthspan and lifespan, pointing to a link between aging and the gut microbiota and supporting the anti-aging potential of correcting dysbiosis (Barcena et al., Nat Med 2019).
Regarding the design of intervention strategies aimed at expanding longevity (Aim 3), we have found that restoration of NAD+ levels in Zmpste24—/— mice delays aging and ameliorates osteoporosis and metabolic alterations. Our research on dietary interventions also demonstrated that methionine restriction extends healthspan and lifespan in progeroid mice (Barcena et al., Cell Rep 2018; Autophagy 2018). Finally, we have evaluated CRISPR/Cas9-based gene editing as a therapy for progeria, using the LmnaG609G/G609G mice as a model of this disease. We showed that in vivo delivery of this system produced an amelioration of progeroid mice and an extension of their lifespan, suggesting that CRISPR/Cas9-based approaches represent a potential treatment for progeria (Santiago-Fernández, Nat Med 2019). We are currently testing prime editors to precisely correct the pathogenic mutation and, in parallel, developing lipid nanoparticle-based delivery systems to avoid the risks associated to viral vectors.
To investigate cell-extrinsic factors involved in aging (Aim 2), we have generated a new mouse model aimed at boosting cell proliferation in the stromal compartment of progeroid animals. However, as the genetic strategy involved did not work as expected, we used alternative approaches to this objective. Thus, we focused on immune modulators produced in progeroid mice in response to the accumulation of damage. To this end, we generated a new LmnaG609G model null for Klrk1, since natural killer cells sense stressed or damaged cells with a major contribution of the signaling mediated by this receptor. Likewise, we have investigated the role of RANKL (Tnfs11) in the aging process by crossing Zmpste24—/— animals with mice harboring a floxed Tnfsf11 allele and mice expressing Cre recombinase in osteocytes, to specifically eliminate RANKL in these cells. These mice showed a reversion of the osteoporotic phenotype and increased survival. In addition, we performed the first large-scale profiling of the plasma proteome of two progeroid mouse models (LmnaG609G/G609G and Zmpste24—/— mice). This work (published in Aging Cell) provided an extensive list of plasma proteins as potential biomarkers and/or therapeutic targets for further exploration in both physiological and premature aging. We have also characterized microbiota alterations in accelerated aging, finding intestinal dysbiosis in human patients and two different mouse models of progeria. Remarkably, transplantation of microbiota from wild-type into progeroid mice improved their healthspan and lifespan, pointing to a link between aging and the gut microbiota and supporting the anti-aging potential of correcting dysbiosis (Barcena et al., Nat Med 2019).
Regarding the design of intervention strategies aimed at expanding longevity (Aim 3), we have found that restoration of NAD+ levels in Zmpste24—/— mice delays aging and ameliorates osteoporosis and metabolic alterations. Our research on dietary interventions also demonstrated that methionine restriction extends healthspan and lifespan in progeroid mice (Barcena et al., Cell Rep 2018; Autophagy 2018). Finally, we have evaluated CRISPR/Cas9-based gene editing as a therapy for progeria, using the LmnaG609G/G609G mice as a model of this disease. We showed that in vivo delivery of this system produced an amelioration of progeroid mice and an extension of their lifespan, suggesting that CRISPR/Cas9-based approaches represent a potential treatment for progeria (Santiago-Fernández, Nat Med 2019). We are currently testing prime editors to precisely correct the pathogenic mutation and, in parallel, developing lipid nanoparticle-based delivery systems to avoid the risks associated to viral vectors.
The comparative genomic studies on different species revealed new genes and pathways involved in aging and proved the value of supervised gene annotation as a complement to automatic algorithms. The CRISPR-based screenings identified novel potential targets of interventions to revert cell senescence, as well as factors whose inhibition may have senolytic effect. The plasma proteome of progeroid mice yielded unexpected candidates that we will characterize to determine their relevance in aging and healthspan improvement. Finally, we have proved in mice the anti-progeria potential of dietary interventions, microbiota transplantation and in vivo gene editing, which represents, to our knowledge, the first application of this approach to a multiorganic genetic condition.