In our study, we found RPTOR, AKT1S1 and EIF4EBP1 to be associated with old age and/or familial longevity, and the directions of association indicate a transcriptional down-regulation of mTORC1. Even in middle- age, a down-regulation of RPTOR mRNA was associated with familial longevity, providing another example of the highly conserved role mTOR signalling has in lifespan regulation across different species.

Because glucose levels differed between offspring and controls (Supplementary Table S5), we investigated whether the mRNA differ- ences of RPTOR and PRR5L depended on glucose levels in non-diabetic participants. The association of familial longevity with RPTOR was not affected by adjustment for glucose levels, and the association with PRR5L expression gained significance (Supplementary Table S7). Thus, the expression level of the RPTOR gene in blood mark familial longevity independent of the prevalence of type 2 diabetes and cancer and glucose levels. Due to the influence of T-cell differentiation, glucose levels and diabetes prevalence on its gene expression, the association of PRRL5 with familial longevity is more complex.

Not only mTORC1, but also mTORC2 activity has been linked health and lifespan. Rictor, an important component of contributes to glucose homeostasis in murine muscle tissue et al., 2008), TORC2 C. elegans mutants show modulation of by affecting feeding and metabolism of different diets (Soukas et 2009), TORC2 is a mediator of proliferative and survival signals cancer cells (Fang et al., 2012) and a study in mouse fibroblasts showed that mTORC2 regulates the TLR-mediated matory response via FoxO1 (Brown et al., 2011). In addition, which is part of mTORC2, has been suggested to play a role apoptosis in HeLa cells, but whether it is pro-apoptotic or apoptotic remains unclear (Thedieck et al., 2007). Recently, et al. showed that disruption of mTORC2 can result in intolerance and insulin resistance in rodents and may be relevant the pathogenesis of type 2 diabetes and metabolic syndrome et al., 2012). Our results regarding the association of PRR5L, and FOXO1 with old age and/or familial longevity suggest a tional up-regulation of mTORC2, which would be in concordance these earlier findings in animal models. As we provide the first that mTORC2 is beneficial for healthy aging in humans, research is required to replicate these findings.

Discussion

By comparing mRNA levels in blood of nonagenarians and middle-aged controls from the LLS, we found that the mTOR signalling gene is differentially expressed in old age. Single gene analysis showed that seven of 40 mTOR pathway genes had a significant differential expression of at least 5%, up to 1.81-fold. The expression of the genes EIF4EBP1, LAMTOR2, AKT1S1, PRR5L and RHOA were higher in nonagenarians, whereas FOXO1 and RPTOR were lower. The directions of these differential gene expressions indicate separate effects on mTORC1 and mTORC2 complexes. Two of the seven differentially expressed genes, RPTOR and PRR5L, were expressed to a lower level in

We observed that PRR5L expression was higher in nonagenarians, but lower in their offspring as compared to the controls. If expression of a gene is associated with familial longevity in this population, we would expect to find a similar direction in both long-lived nonagenarians as well as their offspring. Late-differentiated memory T-cells accumulate with age and this hallmark feature of immunosenescence is believed to contribute to the weakened immune status in the elderly. It has also been shown that offspring of the long-lived nonagenarians have less accumulation of these effector memory T cells in comparison with controls (Derhovanessian et al., 2010). Therefore, varying proportions of T-cell subsets might influence observed PRR5L expression levels in the different groups of the LLS. When testing for association with cell subtype measurements in a subset of middle-aged LLS participants, we found that PRR5L expression is positively associated with the amount of CCR7(cid:1)CD45RA(cid:1)CD27(cid:1)CD28(cid:1) late-differentiated effector memory cells. Data on T-cell subset counts are not available for the LLS nonagenarians, but Koch and colleagues showed the same subset of effector memory cells to be increased in the elderly (Koch et al., 2008). This may explain the higher PRR5L expression levels we found in the nonagenarians, while lower amounts of effector memory cells (Derhov- anessian et al., 2010) and also lower PRR5L expression was found in the middle-aged offspring compared with controls. From this, we conclude that PRR5L expression may be a marker for the differentiation state of T cells, indicating a more late-differentiated T-cell compartment in nonagenarians as compared to younger controls, but a less-differenti- ated phenotype in the offspring in middle-age. The link between the mTOR pathway and T-cell activation and differentiation is perhaps not surprising when it is considered that rapamycin was first identified and exploited as a T-cell-directed immunosuppressive agent in transplanta- tion (Morath et al., 2007). The differential distribution of T-cell pheno- types contributed approximately to 60% of the PRR5L effect, suggesting that PRR5L might have another role in longevity other than marking/ influencing T-cell population subtypes; further research is required to identify such a role.

Selection of mTOR pathway

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

Sample collection and RNA preparation