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.
