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Deconstructing Ageing: from molecular mechanisms to intervention strategies

Periodic Reporting for period 3 - DeAge (Deconstructing Ageing: from molecular mechanisms to intervention strategies)

Reporting period: 2020-09-01 to 2022-02-28

Over many years, our research group has explored the complex relationship between cancer and ageing. As part of this work, we have generated mouse models of protease deficiency which are protected from cancer but exhibit accelerated ageing. Further studies with these mice have allowed us to unveil novel mechanisms of both normal and pathological ageing, to discover two new human progeroid syndromes, and to develop therapies for the Hutchinson-Gilford progeria syndrome, now in clinical trials. We have also integrated data from many laboratories to first define The hallmarks of ageing and the current possibilities for metabolic control of longevity. Now, we propose to leverage our extensive experience in this field to further explore the relative relevance of cell-intrinsic and -extrinsic mechanisms of ageing. Our central hypothesis is that ageing derives from the combination of both systemic and cell-autonomous deficiencies which lead to the characteristic loss of fitness associated with this process. Accordingly, it is necessary to integrate multiple approaches to understand the mechanisms underlying ageing. This project has three major aims: 1) to characterize critical cell-intrinsic alterations which drive ageing; 2) to investigate ageing as a systemic process; and 3) to design intervention strategies aimed at expanding longevity. To fully address these objectives, we will use both hypothesis-driven and unbiased approaches, including next-generation sequencing, genome editing, and cell reprogramming. We will also perform in vivo experiments with mouse models of premature ageing, genomic and metagenomic studies with short- and long-lived organisms, and functional analyses with human samples from both progeria patients and centenarians. The information derived from this project will provide new insights into the molecular mechanisms of ageing and may lead to discover new opportunities to extend human healthspan.
Regarding the identification and characterization of cell-intrinsic mechanisms that drive ageing:

- We are studying gene expression changes in Oikopleura dioica populations with different lifespan due to different feeding conditions. Regarding other invertebrate models, we have sequenced the genome of two hydrozoans: Turritopsis dohrnii which undergoes life cycle reversal when exposed to stress, and T. rubra which hardly ever does. We have also analyzed the molecular changes during life cycle reversal in T. dohrnii. Moreover, we have started comparative genomics of sea urchin species with large lifespan differences.

- We have performed an in-depth genomic study of a giant tortoise from the Galápagos Islands, the iconic Lonesome George, the last representative of Chelonoidis abingdonii and renowned emblem of both extreme longevity and species extinction. Our study (published in Nature Ecol&Evol) has identified specific evolutionary strategies linked to increased lifespan and has expanded our understanding of the genomic determinants of ageing.

- We have set-up a series of genome-editing screenings in cultured cells to identify genes with ability to reverse the aging-related state of cell senescence. The first candidate genes identified by these approaches are currently under functional validation.

- The Lonp1 transgenesis project has shown dual effects of overexpression of this mitochondrial protease in healthspan. Increased LONP1 levels trigger an improvement of healthspan due to their positive effects on some tissues, but may also increase cancer incidence in older animals.

- We have generated a new mouse model overexpressing a proteostasis factor to study its role in cancer and longevity, and we are now evaluating putative phenotypic changes in these mice. We are also exploring the role of proteostasis improvement in an Alzheimer’s mouse model, by assessing neuronal loss, inflammation, amyloid accumulation, and behavioural changes.

As for the study of the systemic mechanisms responsible for the effects of ageing:

- We have generated new mouse models to explore cell-extrinsic mechanisms involved in ageing. These models include mice harboring three genetically-modified alleles (Zmpste24-/-/hPDGFRAD842V/Col1a2-ERT); LmnaG609G mice in a background null for NK cell activating receptor (Klrk1-/- mice); and Zmpste24-/- mice lacking Tnfsf11 in osteocytes.

- We have characterized microbiota alterations in accelerated aging, and we have used progeroid mice to investigate the role of these changes in the aging process and to explore microbiome-based anti-aging interventions. This work has been reported in Nature Medicine.

Regarding the design of intervention strategies aimed at expanding longevity:

- We have performed nicotinamide riboside supplementation experiments in Zmpste24-/- mice and found that restoration of NAD+ levels delays aging onset and ameliorates aging phenotypes such as osteoporosis and metabolic alterations.

- We have tested dietary interventions in two mouse models of Hutchinson-Gilford progeria syndrome, and found that methionine restriction extends healthspan and lifespan, attenuating alterations in inflammation, DNA damage response, and metabolome. We have reported these findings in Cell Reports.

- We have initiated a study to explore how fasting diets can affect to hematological neoplasms using a new model for these diseases.

- Finally, we have evaluated the use of CRISPR/Cas9-based gene editing as a therapy for Hutchinson-Gilford progeria syndrome in a mouse model of this disease. This work has been reported in Nature Medicine.
We expect to determine the key genes involved in lifespan extension in O. dioica. Regarding Turritopsis, we will specify the key genes involved in life cycle reversal by genomic and transcriptomic approaches. Finally, we will detect gene amplifications and specific gene changes potentially responsible for the large lifespan differences in the sea urchin species analyzed in the project.

The CRISPR/based screening of senescence targets has shed light on the role of DOT1L in aging, since this enzyme is necessary for survival of senescent cells. This may suggest that inhibition of DOT1L could have a senolytic effect.

In the Lonp1 transgenic project, we expect to describe the effects of Lonp1 overexpression in different tissues to understand its dual effects on lifespan. Also, we will perform a complete mitochondrial functional analysis to define the molecular mechanisms responsible for changes in healthspan and cancer incidence.

We plan to complete the characterization of mice with increased levels of the proteostasis factor AIRAPL. We will also finish the behavioural tests in the Alzheimer disease mouse model and explore their proteostasis network though proteomic and transcriptomic analyses. Finally, after evaluating the hematologic alterations in fasted and control mice, we will analyze the metabolic pathways underlying them.

We expect to identify secreted factors, acting either locally or systemically, that may modulate the ageing process, and to explore their value for healthspan improvement.

As for the NAD+ project in progeroid mice, we expect to further evaluate the benefits of NR supplementation to Zmpste24-/- mice. Finally, we will analyze gene expression changes and metabolic adaptations underlying the benefits of this intervention.
Integrated objectives of the DeAge project