Recently, we have identified 360 genes, the expression of which was associated with human longevity (Passtoors et al., 2012). MTOR (also known as FRAP1) was among these, suggesting a role for the mTOR signalling pathway in human longevity. In the current study, we investigated whether common natural genetic and transcriptional variation among the 40 genes of the mTOR pathway (Fig. 1) was associated with human longevity. To do this, we took advantage of the Leiden Longevity Study (LLS), a Dutch cohort in which families are selected on the basis of nonagenarian sibling pairs (Schoenmaker et al., 2006). The middle-aged offspring of these nonagenarians have a

Professor Eline Slagboom, Department of Molecular Epidemiology, Leiden University Medical Center, Postzone S-

-P, PO Box

,

RC Leiden, The Netherlands. Tel.: +

; fax: +

; e-mail: P. Slagboom@lumc.nl

                                                                                                                              Hypowa TT Cytoskeletal organisationcounter TS¢t a RhoA | | PKCaDNA damage a \ /Wht signaling mTORGDP ane Deptor | | mLSTeSok k_——_ | RICTORAmino acids Rheb {GTP mSIN1 ProtormTOR | mTORC2Ra DeGAB jeptor | | mLSTB FOXOISRagC/D RAPTORpi4 | MP4 | translocation PRAS40HIF1: > VEGF signalinpis mTORC1 a Sramfom] Stress resistance.ae SREBP FOXO13 | [—* _ apoptosisULK1 4EBP1 S6K1 [SREBP |ATGI3 IL /r™~ [oe 1 I — Lipid synthesisFIP200 elF4E | [ elF4B |[ SKAR || $6 _LPPARy |ATG101| mRNA translation, protein synthesis POCta MitochondrialRibosome biogenesis Yvt [-* __ biogenesisAutophagy    

Fig. 1 mTOR signalling pathway of which genetic variation and expression of 40 genes are investigated for their association with human aging and longevity. mTORC1 receives signals from growth factors, nutrients, energy status and a range of stressors and positively regulates cell growth and proliferation by promoting many anabolic processes, including biosynthesis of proteins, lipids and organelles, and by limiting catabolic processes such as autophagy (Sengupta et al., 2010). mTORC2 phosphorylates and activates Akt, serum- and glucocorticoid-regulated kinase (sGK), RhoA and protein kinase C (PKC), which regulate cell survival, cell cycle progression and cytoskeleton organization (Sarbassov et al., 2005; Garcia-Martinez & Alessi, 2008; Ikenoue et al., 2008).

First, we selected the two mTOR complexes mTORC1 and mTORC2 including their downstream targets often described in the literature (Laplante & Sabatini, 2009; Polak & Hall, 2009). Next, we included the direct activators and inhibitors of these two complexes. Figure 1 shows the mTOR pathway as we investigated.

decreased prevalence of myocardial infarction and type 2 diabetes (Westendorp et al., 2009) and display healthier intrinsic metabolism reflected by lipid profiles (Barzilai et al., 2001; Heijmans et al., 2006), glucose metabolism and preservation of insulin sensitivity (Rozing et al., 2009; Wijsman et al., 2011) than age-matched controls. They also have different immune profiles in that they fail to show a lower number of CD8+ naive T cells and a higher number of CD8+ late-stage differentiated memory cells dependent on cytomegalovirus infection often seen as a hallmark of immune aging (Derhovanessian et al., 2010). To investigate whether expression of mTOR signalling genes is associated with familial longevity, we compared the levels of mRNA for these genes in peripheral blood of nonagenarians from families enriched for longevity and younger controls. In addition, genetic variation in mTOR pathway genes of LLS nonagenarians is compared with that of younger controls. Next, mRNA levels of the top longevity- associated genes were compared between the offspring of the nonagenarians and similarly aged controls to exclude the possibility that differential expression of these genes did not simply mark old age, but are a defining characteristic of the long-lived families in middle-age. Finally, we examined whether the association of the expression of mTOR signalling genes with longevity is independent of metabolic health parameters, namely plasma glucose levels, and the prevalence of type 2 diabetes and cancer, because these factors are known characteristics of the long-lived families and are suggested to be influenced by mTOR signalling.

Gene expression in whole blood: nonagenarians vs. controls

We measured gene expression levels for each of the 40 mTOR signalling genes using RT-qPCR on whole blood samples from 87 LLS nonagenar- ians, 337 of their middle-aged offspring and 321 middle-aged LLS controls (Table 1). When comparing long-lived individuals to younger controls differentially expressed genes may associate either with calendar age, familial longevity or both. We tested the total gene set for such association using the globaltest method (Goeman & Oosting, 2011). We observed that the gene expression levels of the mTOR signalling gene set did associate significantly with age and/or familial longevity in these families (P = 4.6 9 10(cid:1)7).

Second, to investigate which genes are primarily responsible for this association, single gene analysis using linear regression was performed on all 40 mTOR signalling genes (Table 2). After Bonferroni correction for multiple testing, seven genes showed significant differential expression with at least a 5% expression difference: FOXO1 and RPTOR

Table 1 Analysed samples: gene expression

Results

Samples Number Mean age Age range Men/women Long-lived individuals 87 94.3 89–102 40/47 Offspring 337 61.3 34–78 194/143 Controls 321 61.2 32–81 146/175

Selection of mTOR pathway genes

For the mTOR pathway, we selected 40 genes encoding proteins that belong to the well-described core of the mammalian mTOR pathway.

Table 2 Linear regression results of gene expression of long-lived individuals compared with controls