Periodic Reporting for period 1 - ElucidAge (Elucidating and targeting the mechanisms encoded in the genome of long-lived individuals to improve healthy ageing)
Reporting period: 2022-08-01 to 2025-01-31
Advancing age is the major risk factor for many serious illnesses, including cancer, cardiovascular disease, and dementia. The rising number of older individuals is thus causing a major burden of ill health. However, individuals that reach an exceptional old age often seem to escape or delay age-related diseases, and part of this trait seems to be encoded in their genome. Hence, by studying the genome of long-lived individuals, we may be able to identify mechanisms that could be harnessed to foster healthy ageing in the general population. Our previous work suggests that large genome-wide association studies (GWAS) of long-lived individuals can be used to identify genetic variants involved in longevity. However, the common genetic variants thus far identified using GWAS only explain a minor part of the genetic component of longevity. This trait, therefore, may well be mainly determined by rare genetic variants, which can be detected using whole-genome or exome sequencing of long-lived families or exceptionally long-lived individuals. The aim of the proposed project is to establish the effect of genetic variants identified in genetic studies of long-lived individuals on general health and lifespan using cellular models and, subsequently, model organisms. To this end, we use CRISPR/Cas9 genome editing to generate cell lines and mice that harbour genetic variants identified in GWAS and sequencing studies of long-lived families and exceptionally long-lived individuals. Our initial focus is on rare genetic variants in genes involved in the insulin/insulin-like growth factor 1 (IIS)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling pathways, given the well-known role of these pathways in ageing in pre-clinical model organisms. We subsequently perform detailed characterisation of the generated cell lines, where we look at the effect of the variants on IIS/mTOR and MAPK/ERK signalling as well as general cellular health. In the generated mouse lines we study effects on lifespan and general health. The phenotypic characterisation is performed at three stages during the life of the mice, i.e. young, middle, and old age and we also collect tissues of the mice for molecular analyses at the same timepoints. In addition, we aim to design a high-throughput screening platform to identify compounds that can pharmacologically recapitulate the effects we observed in our generated cell lines.
Using a newly established protocol, we were able to create >10 mouse embryonic stem cell (mESC) lines harbouring genetic variants in multiple genes involved in the IIS/mTOR and MAPK/ERK signalling pathways. The majority of these variants were identified in long-lived families from the Leiden Longevity Study (LLS), but we also included variants identified in exceptional long-lived individuals from the German Longevity Study (GLS). The detailed characterisation of these cell lines is ongoing, but we already found that the downstream effects of the variants seem to diverge from a shared path into two branches. The part of the path that is shared between all variants is associated with altered IIS/mTOR and MAPK/ERK signalling in a manner that has previously been associated with increased lifespan in model organisms. However, the two branches also show some diverging, or even opposing, effects on, for example, resistance to cellular stressors, which indicates these branches may represent alternative strategies for regulation of stem cell health. We also generated some additional mESC lines harbouring variants identified through alternative bioinformatic approaches using data from both the LLS and GLS and are currently investigating the effect of these variants on the implicated proteins and downstream targets.
Based on our initial findings from the mESCs, we decided to create four mouse lines harbouring variants in different genes representing both cellular branches. The first striking observation we made was that two of the mouse lines show an increase in litter size compared to wildtype mice, which we are currently exploring in more detail. For two of the mouse lines the phenotypic characterisation is almost complete and, although the effects are small, we observed some interesting differences between our mutant and wildtype mice in body composition and motor coordination, which we are currently following up molecularly.
To identify compounds that can pharmacologically recapitulate the effects we observed in our mESCs, we decided to use CRISPR/Cas9 to endogenously tag the proteins that are best representing the two observed branches (i.e. showing strong differential regulation when compared to wildtype mESCs). We already managed to generate several mESCs with tagged proteins and, as a next step, we plan to perform a high-throughput screen to see if there are any compounds that recapitulate the effects observed for the mESCs harbouring our variants of interest.
Based on our initial findings from the mESCs, we decided to create four mouse lines harbouring variants in different genes representing both cellular branches. The first striking observation we made was that two of the mouse lines show an increase in litter size compared to wildtype mice, which we are currently exploring in more detail. For two of the mouse lines the phenotypic characterisation is almost complete and, although the effects are small, we observed some interesting differences between our mutant and wildtype mice in body composition and motor coordination, which we are currently following up molecularly.
To identify compounds that can pharmacologically recapitulate the effects we observed in our mESCs, we decided to use CRISPR/Cas9 to endogenously tag the proteins that are best representing the two observed branches (i.e. showing strong differential regulation when compared to wildtype mESCs). We already managed to generate several mESCs with tagged proteins and, as a next step, we plan to perform a high-throughput screen to see if there are any compounds that recapitulate the effects observed for the mESCs harbouring our variants of interest.
The most striking observation we have made thus far is that the mESC lines harbouring our variants of interest cluster in two main groups. Although we expected that some of the variants would show shared downstream mechanisms, we did not expect this would be so obvious and that some genes would have variants represented in both groups. As a next step we want to determine if we can recapitulate these effects using pharmacological compounds. If successful, this would be the first human-informed drug screening platform for longevity, which could help enhance the efficiency and precision of drug discovery for healthy ageing. We are already in contact with several industrial stakeholders to discuss the possibility of commercialisation of our screening platform.
The creation of a pipeline for the generation and characterisation of mESCs lines harbouring genetic variants linked to longevity and healthy ageing has already resulted in several small side projects in which we look at variants in additional genes that have not yet been linked to human longevity. Moreover, we have started a collaboration with a group in the United States to try to expand our work to human cell lines, which will help to make our results more comprehensive.
Another aspect of our research that is considered beyond the state-of-the-art is the fact that we have created mouse lines harbouring rare genetic variants identified in long-lived individuals. Although similar approaches have been used for other diseases, no such mouse model exists for longevity or healthy ageing. Hence, we are already in contact with several new collaborators that would be interested in using (tissues of) our mouse lines to look at effects on their mechanism of interest. However, the usability of our mouse lines for other researchers will of course depend on the lifespan and phenotypic effects they will display.
The creation of a pipeline for the generation and characterisation of mESCs lines harbouring genetic variants linked to longevity and healthy ageing has already resulted in several small side projects in which we look at variants in additional genes that have not yet been linked to human longevity. Moreover, we have started a collaboration with a group in the United States to try to expand our work to human cell lines, which will help to make our results more comprehensive.
Another aspect of our research that is considered beyond the state-of-the-art is the fact that we have created mouse lines harbouring rare genetic variants identified in long-lived individuals. Although similar approaches have been used for other diseases, no such mouse model exists for longevity or healthy ageing. Hence, we are already in contact with several new collaborators that would be interested in using (tissues of) our mouse lines to look at effects on their mechanism of interest. However, the usability of our mouse lines for other researchers will of course depend on the lifespan and phenotypic effects they will display.