Gene expression analysis of mTOR pathway: association with human longevity

Willemijn M. Passtoors,1 Marian Beekman,1,2 Joris Deelen,1,2 Ruud van der Breggen,1 Andrea B. Maier,3 Bruno Guigas,4 Evelyna Derhovanessian,5 Diana van Heemst,3 Anton J. M. de Craen,3 David A. Gunn,6 Graham Pawelec5 and Pieternella E. Slagboom1,2

(mTORC1) and mTOR complex 2 (mTORC2), each with different functions (Polak & Hall, 2009), although their exact respective roles remain to be elucidated. The main known functions of mTORC1 include translation initiation, protein synthesis and autophagy, while those of mTORC2 include cytoskeletal organization (Laplante & Sabatini, 2009). In addition, the two complexes are connected in that mTORC2 regulates Akt phosphorylation, part of an upstream pathway that controls activation of mTORC1 (Stanfel et al., 2009). Inhibition of the mTOR signalling pathway in yeast (Kaeberlein et al., 2005), worms (Vellai et al., 2003), flies (Kapahi et al., 2004) and mice (Harrison et al., 2009) results in lifespan extension. In mouse models, inhibition of the mTOR signalling pathway provides cardiovascular benefits and improved metabolic function (Stanfel et al., 2009). Additionally, a primary hallmark of calorie restriction (CR) in rodents, which leads to inhibition of the mTORC1 pathway, is a dramatic reduction in age-associated cancer incidence and growth rate (Spindler, 2005), implying that enhanced mTOR signalling plays a central role in cancer progression. In rats, the mTOR pathway has also been implicated in both Type 1 and Type 2 diabetes, with chronic activity of mTORC1 contributing to obesity and insulin insensitivity (Newgard et al., 2009). Because reduced mTOR signalling in model organisms extends lifespan and is associated with better metabolic health, we postulated that a similar reduction in this signalling pathway may also contribute to longevity and healthy aging in humans.

1Section of Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands 2Netherlands Consortium for Healthy Ageing, Leiden, The Netherlands 3Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, The Netherlands 4Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands 5Center for Medical Research, University of Tu¨bingen, Tu¨bingen, Germany 6Unilever Discover, Colworth, Sharnbrook, Bedfordshire, UK

Summary

mTOR signalling is implicated in the development of disease and in lifespan extension in model organisms. This pathway has been associated with human diseases such as diabetes and cancer, but has not been investigated for its impact on longevity per se. Here, we investigated whether transcriptional variation within the mTOR pathway is associated with human longevity using whole- blood samples from the Leiden Longevity Study. This is a unique cohort of Dutch families with extended survival across genera- tions, decreased morbidity and beneficial metabolic profiles in middle-age. By comparing mRNA levels of nonagenarians and middle-aged controls, the mTOR signalling gene set was found to associate with old age (P = 4.6 3 10(cid:1)7). Single gene analysis showed that seven of 40 mTOR pathway genes had a significant differential expression of at least 5%. Of these, the RPTOR (Raptor) gene was found to be differentially expressed also when the offspring of nonagenarians was compared with their spouses, indicating association with familial longevity in mid- dle-age. This association was not explained by variation between the groups in the prevalence of type 2 diabetes and cancer or glucose levels. Thus, the mTOR pathway not only plays a role in the regulation of disease and aging in animal models, but also in human health and longevity.

Thus far, mTOR signalling has been investigated in humans mainly by studying disease. Its activity has been found to be elevated in different cancers including lymphomas, melanomas, breast and prostate (Guertin & Sabatini, 2005; Stanfel et al., 2009). Elevated mTOR signalling in human tumours is correlated with poor tumour prognosis, and inhibitors of this pathway are showing promising results in clinical trials (Le et al., 2008; Strimpakos et al., 2008; Yap et al., 2008). Additionally, interme- diate phenotype insulin resistance in human diabetes can be prevented by mTOR inhibitors (Krebs et al., 2007). Metformin, a commonly prescribed anti-diabetic drug which inhibits mTORC1 downstream pathways through activation of AMP kinase, has also been shown to reduce tumour growth (Dowling et al., 2007). Human CR studies used as model for down-regulation of mTOR are much more short term in comparison with animal studies, but nevertheless, CR may reduce the risk of age-related diseases even in non-obese humans (Fontana et al., 2004; Holloszy & Fontana, 2007). Thus, accumulating evidence is showing that mTOR signalling can play an important role in human disease. In contrast, it has not been investigated whether natural variation in mTOR signalling could also contribute to disease risk.

Introduction

Mammalian target of rapamycin (mTOR) is an evolutionarily conserved nutrient-sensing protein kinase that belongs to the phosphoinositide 3- kinase (PI3K)-related protein kinase family and acts as a central regulator of growth and cell division (Stanfel et al., 2009). The core kinase mTOR is