Periodic Reporting for period 4 - UPRmt (The Mitochondrial Unfolded Protein Response)
Periodo di rendicontazione: 2023-05-01 al 2024-03-31
Aging, defined as the time-dependent general molecular and physiological decline, ultimately leading to death, is a pervasive property of biological systems. By 2050, older people (>60 yrs) are predicted to outnumber young people (<24 yrs) worldwide, posing significant medical and societal challenges, as increased lifespan does not necessarily translate into an increase in healthspan, the duration of life spent in good health. Prolonging lifespan without improving healthspan causes a rise in age-associated non-communicable diseases (NCDs). Most healthspan studies in vertebrates have been restricted to animals on a single genetic background, and previous studies in rodent genetic reference populations (GRPs) only measured a limited number of clinical or molecular traits, limiting their ability to model the factors that govern healthspan in humans. Our ERC-funded (AdG-787702) longitudinally-designed healthspan study in a large mouse GRP consisting of ~4000 female mice from 82 strains aimed to study the impact of the mitochondrial unfolded protein response (UPRmt) on healthspan. the mouse population used was termed “hybrid diversity panel” (HDP), which we have now renamed “healthspan diversity project” to reflect its biological interpretation rather than purely the population structure. The HDP addressed these pitfalls by incorporating longitudinal cardiometabolic phenotyping in adult (4-8 mo) and aged (16-20 mo) mice, roughly corresponding to 30-40 and 50-70 yrs in humans. In addition, we established a biobank of more than 100’000 samples from young (2 mo), adult (8 mo) and old (18 and 20 mo) mice that enables deep molecular phenotyping. The biochemical and computational analysis of this dataset mechanistically refined the molecular features of aging and how stimulating the mitochondrial unfolded protein response (UPRmt) can influence these features. In addition, our genetic analyses have identified many of the single genes andgene networks––from mouse to human––that control mitochondrial stress and aging. It will as such provide the next step in finding ways of interfering with the aging process and translate them into human therapies.
Our plan for the HDP project was centered on achieving 4 Specific Aims, and the outcome of each Aim is briefly discussed below.
Aim 1: Map mammalian UPRmt genes and molecular and mitochondrial networks in vivo after the induction of the UPRmt in a large murine GRP at 3 different time-points throughout life with 2 different inducers – referred to as the tissue collection pipeline.
Mice were housed with no stressors for 2, 8 or 18 months, roughly corresponding to 20, 40 and 70 years in humans, then treated with UPRmt inducers Doxycycline (Dox), the NAD+ precursor, nicotinamide riboside (NR) or vehicle control by gavage 24 and 4 hours before sacrifice (Figure 1A). The tissue collection in pipeline proceeded without any major issues, and we have now gathered a biobank of >100K samples which are undergoing further analysis.
Aim 2: Integrate these UPRmt networks with a large set of clinical, mitochondrial, and molecular phenotypes collected throughout life to establish links between the UPRmt and homeostasis, health- and lifespan. Referred to as the phenotyping pipeline.
We evaluated a wide set of physiological traits collected under basal, non-stressed conditions in ~85 HDP strains in adult and old mice; this constituted the phenotyping pipeline, which concluded in Q3 2023 (Figure 1B, Figure 2A-C)., This life-long in cage activity, collected with the Tecniplast Digital Ventilated Cages (DVC) system which represents >200 years of combined data, allows us to analyze circadian rhythms, response to external stress and a range of other behaviors (Figure 2D-E).
Aim 3: Mechanistically validate new UPRmt genes and networks, using loss-of-function studies in cells, worms and mice.
We have completed a large phenotyping and multi-omic experiment on a GRP composed of 85 C. elegans Recombinant Inbred Advanced Intercross Lines (RIAILs) derived from a cross of the N2 strain and the Hawaiian CB4856 under control and Dox treated conditions. The first part of this work is now a pre-print (PMID: 38293129) and under revision at Cell Genomics, focusing on the genetics of lifespan and healthspan in basal conditions in the RIAILs strains, and revealing several lifespan regulators validated across species. A second manuscript, focusing on the effects of UPRmt (induced by Dox) on healthspan in these RIAILs strains is currently in preparation (Figure 3).
Aim 4: Clinically translate the most promising UPRmt regulators and systems using joint genetic association studies with human cohorts.
Although the mouse studies of the HDP have not yet reached the stage of human validation, we have not been idle on improving our analysis tools of human datasets. Our suite of tools for candidate validation is now fully on line and has been used to validate cross-species regulators of C. elegans healthspan (PMID: 38293129), as well as metabolic health (Cell systems, accepted), and diet-induced colon inflammation (PMID: 37855835) in the BXD. As part of this ERC project, we have also devised ways to better quantify healthspan in the UK Biobank, China Kadoorie, Estonian Biobank and All of Us, by using longitudinal metrics and cox survival models, rather than simply the first occurrence of NCDs.
Aim 1: Map mammalian UPRmt genes and molecular and mitochondrial networks in vivo after the induction of the UPRmt in a large murine GRP at 3 different time-points throughout life with 2 different inducers – referred to as the tissue collection pipeline.
Mice were housed with no stressors for 2, 8 or 18 months, roughly corresponding to 20, 40 and 70 years in humans, then treated with UPRmt inducers Doxycycline (Dox), the NAD+ precursor, nicotinamide riboside (NR) or vehicle control by gavage 24 and 4 hours before sacrifice (Figure 1A). The tissue collection in pipeline proceeded without any major issues, and we have now gathered a biobank of >100K samples which are undergoing further analysis.
Aim 2: Integrate these UPRmt networks with a large set of clinical, mitochondrial, and molecular phenotypes collected throughout life to establish links between the UPRmt and homeostasis, health- and lifespan. Referred to as the phenotyping pipeline.
We evaluated a wide set of physiological traits collected under basal, non-stressed conditions in ~85 HDP strains in adult and old mice; this constituted the phenotyping pipeline, which concluded in Q3 2023 (Figure 1B, Figure 2A-C)., This life-long in cage activity, collected with the Tecniplast Digital Ventilated Cages (DVC) system which represents >200 years of combined data, allows us to analyze circadian rhythms, response to external stress and a range of other behaviors (Figure 2D-E).
Aim 3: Mechanistically validate new UPRmt genes and networks, using loss-of-function studies in cells, worms and mice.
We have completed a large phenotyping and multi-omic experiment on a GRP composed of 85 C. elegans Recombinant Inbred Advanced Intercross Lines (RIAILs) derived from a cross of the N2 strain and the Hawaiian CB4856 under control and Dox treated conditions. The first part of this work is now a pre-print (PMID: 38293129) and under revision at Cell Genomics, focusing on the genetics of lifespan and healthspan in basal conditions in the RIAILs strains, and revealing several lifespan regulators validated across species. A second manuscript, focusing on the effects of UPRmt (induced by Dox) on healthspan in these RIAILs strains is currently in preparation (Figure 3).
Aim 4: Clinically translate the most promising UPRmt regulators and systems using joint genetic association studies with human cohorts.
Although the mouse studies of the HDP have not yet reached the stage of human validation, we have not been idle on improving our analysis tools of human datasets. Our suite of tools for candidate validation is now fully on line and has been used to validate cross-species regulators of C. elegans healthspan (PMID: 38293129), as well as metabolic health (Cell systems, accepted), and diet-induced colon inflammation (PMID: 37855835) in the BXD. As part of this ERC project, we have also devised ways to better quantify healthspan in the UK Biobank, China Kadoorie, Estonian Biobank and All of Us, by using longitudinal metrics and cox survival models, rather than simply the first occurrence of NCDs.
Overall, the HDP project was a success, although a number of delays, largely due to the COVID crisis caused the in vivo part of the project to finish nearly a year later than anticipated. This delayed some of the planned multi-omic measurements (Aim 1) which will be completed in the next months . As a result of this project, we have built an unprecedented database and biobank that we will use to study aging, healthspan and the influence of mitochondria on both. Our database contains thousands of measurements on mice covering the main axes of aging and health, such as metabolic health and metabolic syndrome, neuromuscular health and frailty as well as behavior and cognitive decline. These tests are the equivalent of a very complete health checkup as could be done in humans, as indeed many of these tests are the direct equivalents of diagnostic tests performed in humans (see Figure 1B for the direct human equivalents). This complete health checkup was performed twice on every mouse, at young and old age, allowing to capture the health decline with age. In addition, the lifelong follow-up with the DVC system can be compared to continuous data from wearables such as smart watches, except unlike in humans we have access to this data over the entire lifespan of the mouse, capturing the evolution from youth to old age. We are now combining this clinical data with the molecular features of different organs across age. This allows us to causally link molecular manifestations of aging such as mitochondrial decline or somatic mutations with the health decline seen at the level of the whole organism. This combination is something that greatly exceeds the current state of the art of the aging field. We have already published a number of findings resulting from this study, from new longevity genes to regulators of metabolism, but we expect to go much further as we combine the different scales of aging research, from the molecular to the clinical.