FIG 2. Western blot analysis of calcium/calmodulin-dependent protein kinase IV (CAMKIV). (A) Western blot analysis of CAMKIV revealing higher CAMKIV levels in mononuclear cells of subjects expressing wild-type CAMKIV (CAMKIV WT) compared to subjects homozygous for the CAMKIV minor allele (CAMKIV rs10491334). CAMKIV levels were normalized to actin (corrected densitometry units, CDU; n ¼10). (B and C) Western blot analyses of HT29 cells expressing CAMKIV revealing a significant increase of phosphorylated FOXO3A, AKT, and SIRT1 compared with cells infected with empty vector (MOCK). Data from immunoblots were quantified by densitometric analysis. Statistics were performed with analysis of

the higher frequency of wild-type CAMKIV genotypes de- tected among LLIs, supports a prosurvival role for CAMKIV.

CAMKIV is activated by and is a key component of the cAMP signaling cascade that induces CREB-dependent transcription modulating synaptic plasticity and nerve growth factor (NGF)-induced sympathetic neuron survival.45,46 Fur- thermore, the innate response to Gram-negative lipopolysac- charides induces dendritic cell survival triggered by increased CAMKIV-mediated expression of antiapoptotic B cell lym- phoma 2 (Bcl-2) protein.47 Moreover, CAMKIV was previously linked to aging in a transcriptional profiling study conducted on aging cortex in mice, rhesus macaques, and humans. The greatest conserved change was age-dependent down- regulation of CAMKIV, indicating a potential involvement of CAMKIV in age-related decline of synaptic function.48

tected among LLIs, supports a prosurvival role for CAMKIV. Then, we tested whether CAMKIV could induce phosphor- ylation of SIRT1 (at Ser47), AKT (at Ser473), and FOXO3A (at Ser253). To this end, we expressed wild-type CAMKIV in HT29 cells, a cell line that does not express CAMKIV, with the use of a lentiviral vector. We found that CAMKIV induces phosphory- lation of the three prosurvival proteins, thus proposing CAM- KIV as a key regulator of survival genes (Fig. 2B,C).

Discussion

Longevity genes can be discovered by genetic association studies conducted on LLIs. This type of study identifies gene variants that have been selected by demographic pressure and, therefore, that are in some way significant for human health. The identification of these gene variants is important in that they could represent potential targets for fighting aging-associated illness.

Here, we report an association between the presence of wild-type CAMKIV and the propensity for exceptional lon- gevity, and a correlation between the presence of the rs10491334 variant of CAMKIV and the reduction of CAM- KIV protein expression in mononuclear cells. We demon- strate, moreover, that CAMKIV is important in the activation of the prosurvival proteins analyzed, namely AKT, SIRT1, and FOXO3A. Therefore, the abundance of native CAMKIV protein probably has a role in slowing the aging process, in addition to CAMKIV’s known crucial role in osteoclasto- genesis, long-term memory, and neuron survival.46,49,50

Many genes capable of regulating aging in lower eu- karyotes and, more recently, in mammals have been identi- fied to date.17 Of these, only APOE and FOXO3A have been consistently replicated across populations, even if some of the population-specific genetic findings have been function- ally validated, such as Klotho and IGF-1 receptor genes.16,36 Therefore it is plausible that population-specific environmental conditions do not exercise similar demographic pressures across populations, with consequently inconsistent results.

We replicated the genetic finding in an independent set of LLIs and controls, thus strengthening our evidence of asso- ciation. However, a limitation of our study is the small population sample size used for replication that reached a 60% power to replicate rs10491334 and the male/female ratio that did not overlap with the ratio of the screening set, even if no gender effect was observed by regression analysis in the original association. Nonetheless, the functional validation of CAMKIV, demonstrating the ability of CAMKIV to regulate the key aging proteins SIRT1, AKT, and FOXO3A, is a strength of the study.

The use of younger populations as controls exposes studies to potential bias due to the comparison of two pop- ulations that belong to different generations and, possibly, to different genetic backgrounds caused by recent immigration. This was excluded by analysis of genetic structure.

The fine-tuning of key genes allows the cell to fight the accumulation of radical oxygen species and genotoxic mol- ecules, and to regulate the balance between proliferation, cell cycle arrest, and apoptosis. The expression of these genes is triggered by oxidative stress, growth factors, nutrients, and chronic stresses, such as caloric restriction and endurance training. For example, the phenols resveratrol and n-tyrosol, contained in red and white wine, respectively, promote cell survival through the activation of SIRT1, AKT, and FOXO3A.15 SIRT1 is activated by the low-energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is activated in turn by caloric restriction.37 AMPK also induces endothelial nitric oxide synthase (eNOS), responsible for cardioprotection.38,39 Interestingly, despite the strong evidence in favor of a prosurvival role for SIRT1, none of the genetic variants of this gene, or of its homolog sirtuin 3 (SIRT3), have been associated with longevity.40,41 Therefore it is possible that genes that regulate SIRT1, rather than SIRT1 itself, are under demographic pressure and, hence, better targets to delay aging.