Periodic Reporting for period 4 - GeroProtect (Developing Geroprotectors to Prevent Polymorbidity)
Reporting period: 2022-06-01 to 2022-11-30
Advancing age is the major risk factor for many diseases, including cancer, cardiovascular disease and neurodegeration. Increasing life expectancy in Europe is posing major medical, social and economic challenges. There is therefore an urgent need to find ways of compressing late-life morbidity and disability. Ageing has proved malleable to genetic and pharmacological interventions in laboratory animals, and many of the mechanisms are conserved in evolution. Specifically, reduced activity of the nutrient-sensing insulin/insulin-like growth factor/TOR signalling network can increase healthspan, with growing evidence of its relevance to human ageing. Pharmacological intervention could therefore prevent more than one ageing-related condition, rather than tackling diseases one by one and as they arise. The aim the research in this programme was to evaluate the potential for pharmacological prevention of ageing-related decline in humans with a polypill targeting the nutrient-sensing network.
We have investigated the geroprotective effects of three licensed drugs: lithium, rapamycin and trametinib, which inhibit different nodes in the nutrient-sensing network. We aimed to understand mechanisms of geroprotection, and whether a polypill of these drugs could be more effective than any of them singly. Rapamycin is the most established geroprotector in mice. We found that in both Drosophila and mice, the drug acts by inducing a long-term increase in autophagy in the gut and that it acts differently in males and in females. Rapamycin suppresses activity of S6 kinase, and in Drosophila this decreased the inflammation that occurs during ageing. We investigated whether lithium was geroprotective in mice, but with increasing dose the first effect seen was mild kidney toxicity. In contrast, trametinib extended lifespan in both female and male mice, and we are assessing health data. Remarkably, in mice rapamycin and trametinib together increased lifespan and health much more than did either drug alone.
We have also extended our investigations to humans, by examining genetic information from the long-lived families in the Leiden Longevity Study (LLS) to determine if there is a signal from the RAS pathway. We have followed up the candidate mutants that we identified in tissue culture, Drosophila and mice to determine if they have evolutionarily conserved effects on healthspan. The outputs of the research will inform future clinical trials in humans.
We have investigated the geroprotective effects of three licensed drugs: lithium, rapamycin and trametinib, which inhibit different nodes in the nutrient-sensing network. We aimed to understand mechanisms of geroprotection, and whether a polypill of these drugs could be more effective than any of them singly. Rapamycin is the most established geroprotector in mice. We found that in both Drosophila and mice, the drug acts by inducing a long-term increase in autophagy in the gut and that it acts differently in males and in females. Rapamycin suppresses activity of S6 kinase, and in Drosophila this decreased the inflammation that occurs during ageing. We investigated whether lithium was geroprotective in mice, but with increasing dose the first effect seen was mild kidney toxicity. In contrast, trametinib extended lifespan in both female and male mice, and we are assessing health data. Remarkably, in mice rapamycin and trametinib together increased lifespan and health much more than did either drug alone.
We have also extended our investigations to humans, by examining genetic information from the long-lived families in the Leiden Longevity Study (LLS) to determine if there is a signal from the RAS pathway. We have followed up the candidate mutants that we identified in tissue culture, Drosophila and mice to determine if they have evolutionarily conserved effects on healthspan. The outputs of the research will inform future clinical trials in humans.
Rapamycin increases lifespan only in female flies. We found higher baseline autophagy in the gut in males, and showed that gut cell sexual identity dictates basal autophagy, gut health and lifespan and their responses to rapamycin. Rapamycin treatment increased histone protein levels in gut, and this altered expression of specific autophagy genes. Remarkably, brief rapamycin treatment induced long-term elevated autophagy in gut, which increased gut health and extended lifespan. Because lithium did not extend lifespan in mice (Aim 2), we discontinued work with it. We instead showed that rigosertib extends fly lifespan. Trametinib reduced gut dysplasia in aged flies, and also markedly reduced inflammation during ageing. However, reduced inflammation seems not to be causal for increased lifespan, and we are continuing work on the exact mechanisms at work.
We found remarkably persistent effects of earlier treatment with rapamycin on gut health in middle-aged mice. We have discovered that lifespan is also extended by earlier treatment, as much as a year after the drug has been withdrawn. Intermittent treatment with the drug also extends lifespan, although less than chronic treatment, but with fewer side-effects. Trametinib extended lifespan in both female and male mice, with the health and pathology data currently under analysis. The effects of RAS pathway activity on ageing are thus conserved in mice. Although it extended fly lifespan, rigosertib did not extend mouse lifespan at the dose tested.
This part was carried out in collaboration with Prof. Eline Slagboom (Leiden University Medical Center), and was the highest risk part of the project. The Leiden team identified protein altering variants in genes from the Ras Mek Erk pathway in long lived subjects from the LLS and their long-lived siblings, after whole-genome sequencing and stringent filtering and validation by Sanger sequencing. We generated cell lines harboring these variants and assessed resulting functional changes. A cell line harboring a variant in Nf1 shows significantly decreased p-Erk/Erk ratio, decreased protein and gene expression levels of Nf1 and decreased ER stress resistance in response to tunicamycin. A variant in Raf1 showed the opposite, i.e. a significantly increased p-Erk/Erk ratio, with increased protein and gene expression levels of Raf1 and increased ER stress resistance in response to tunicamycin. We are following up these findings in transgenic flies and mice.
We found remarkably persistent effects of earlier treatment with rapamycin on gut health in middle-aged mice. We have discovered that lifespan is also extended by earlier treatment, as much as a year after the drug has been withdrawn. Intermittent treatment with the drug also extends lifespan, although less than chronic treatment, but with fewer side-effects. Trametinib extended lifespan in both female and male mice, with the health and pathology data currently under analysis. The effects of RAS pathway activity on ageing are thus conserved in mice. Although it extended fly lifespan, rigosertib did not extend mouse lifespan at the dose tested.
This part was carried out in collaboration with Prof. Eline Slagboom (Leiden University Medical Center), and was the highest risk part of the project. The Leiden team identified protein altering variants in genes from the Ras Mek Erk pathway in long lived subjects from the LLS and their long-lived siblings, after whole-genome sequencing and stringent filtering and validation by Sanger sequencing. We generated cell lines harboring these variants and assessed resulting functional changes. A cell line harboring a variant in Nf1 shows significantly decreased p-Erk/Erk ratio, decreased protein and gene expression levels of Nf1 and decreased ER stress resistance in response to tunicamycin. A variant in Raf1 showed the opposite, i.e. a significantly increased p-Erk/Erk ratio, with increased protein and gene expression levels of Raf1 and increased ER stress resistance in response to tunicamycin. We are following up these findings in transgenic flies and mice.
IAim 1: Unexpected results from Drosophila have opened up the field. Increased lifespan from rapamycin treatment requires suppressed activity of S6 kinase, but the mechanisms at work have proved highly elusive. We found that suppression is required specifically in fat body (fly equivalent of mammalian liver and white adipose tissue), and that it results in decreased inflammation during ageing. This work is under revision for publication. It will be important to extend these findings to mice, where deletion of S6 kinase 1 has already been shown to extend lifespan, in females only, by unidentified mechanisms.
Aim 2: Remarkably, we have found that a combined treatment with rapamycin and trametinib produces an additive effect on lifespan in male and female mice, with each drug enhancing the effect of the other and producing major health imporvements. This is an important finding because it points to the potential efficacy of polypharmacy in the nutrient-sensing network for geroprotection. We are following up with analysis of molecular mechanisms, health and pathology, and we are also putting in an application to the Intervention Testing Programme at the US National Institutes of Aging to test these drugs singly and in combination. It will also be important to discover how these two drugs act additively to extend lifespan in Drosophila.
Aim 3: This was the highest risk part of the programme, and it was carried out in collaboration with Professor Eline Slagboom at Leiden University. Our Scientific Advisory Board highlighted this effort to link directly work in humans and in animal models as a major achievement of the 6-year review period for the Institute.
The Leiden team investigated how molecular profiles, including the RAS pathway, responded to a lifestyle intervention in older individuals in conjunction with changes in health parameters. This response was captured by the blood metabolome and an algorithm was generated that can be used as a biomarker. The responses were also captured by the blood, fat and muscle transcriptome, explored from a whole genome perspective and analysed by focusing on Ras Mek Erk pathway genes. The effect of the intervention was strongest in postprandial muscle in both sexes, and those for fat are stronger in women than in men. 40 Ras Mek Erk genes showed at least one significant association with health marker changes and changes in DXA scans.
Aim 2: Remarkably, we have found that a combined treatment with rapamycin and trametinib produces an additive effect on lifespan in male and female mice, with each drug enhancing the effect of the other and producing major health imporvements. This is an important finding because it points to the potential efficacy of polypharmacy in the nutrient-sensing network for geroprotection. We are following up with analysis of molecular mechanisms, health and pathology, and we are also putting in an application to the Intervention Testing Programme at the US National Institutes of Aging to test these drugs singly and in combination. It will also be important to discover how these two drugs act additively to extend lifespan in Drosophila.
Aim 3: This was the highest risk part of the programme, and it was carried out in collaboration with Professor Eline Slagboom at Leiden University. Our Scientific Advisory Board highlighted this effort to link directly work in humans and in animal models as a major achievement of the 6-year review period for the Institute.
The Leiden team investigated how molecular profiles, including the RAS pathway, responded to a lifestyle intervention in older individuals in conjunction with changes in health parameters. This response was captured by the blood metabolome and an algorithm was generated that can be used as a biomarker. The responses were also captured by the blood, fat and muscle transcriptome, explored from a whole genome perspective and analysed by focusing on Ras Mek Erk pathway genes. The effect of the intervention was strongest in postprandial muscle in both sexes, and those for fat are stronger in women than in men. 40 Ras Mek Erk genes showed at least one significant association with health marker changes and changes in DXA scans.