The Effects of the FOXO3 Longevity Associated Variant on Biomarkers of Aging and Stress

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The Forkhead Box O3 (FOXO3) protein is one of four transcription factors that make up the FOXO family. The FOXOs are the mammalian homologues of the Caenorhabditis elegans daf-16 gene which has been associated with longevity. The four mammalian isoforms, FOXO1, -3, -4, and -6 are differentially expressed in various tissues. FOXO3 is expressed throughout the body, most significantly in the heart, brain, liver, muscle, spleen, testes, ovaries, and resident stem cells. Previous studies have shown that FOXO3 is positioned as a key regulator in multiple longevity regulatory pathways although the specific mechanisms are not well understood. We developed studies to explore the possible effect of the longevity-associated FOXO3 rs2802292 allele on age related change in telomere length, telomerase activity, FOXO3 gene expression, and plasma pro- and anti-inflammatory cytokine levels. The general hypothesis is that the FOXO3 longevity-variant is associated with increased FOXO3 expression, elevated levels of established healthy aging biomarkers, and reduction in biomarkers of stress response. In the initial study, Okinawa Study #1, telomere length and telomerase activity were assessed with respect to age as a function of both FOXO3 rs2802292 longevity associated variants and APOE variants in a population of Okinawan-Japanese. 121 subjects from Okinawa, Japan, ranging in age from 21-95 years, were recruited during annual physical appointments, where after consent, peripheral circulatory blood was collected and sent to the John A. Burns School of Medicine at the University of Mānoa for work-up and analysis. Telomere length was assessed in mononuclear cells using Southern blot analysis and telomerase activity was analyzed using the telomere repeat amplification protocol (TRAP). FOXO3 genotyping of the rs2802292 variant was performed using amplification-refractory mutation system allele-specific PCR while genotyping of the Apolipoprotein E variant done by standard PCR amplification and restriction enzyme digestion. We found that carriage of the FOXO3 G-allele was associated with protection against telomere loss with respect to age compared to those lacking the G-allele (TT-carriers). Importantly, this effect was only observed in subjects of middle age (arbitrary cut off of 50) and older. We did not detect an effect of carriage of the G-allele on telomerase activity in this preliminary study. In contrast, carriage of neither the ϵ4 -allele (risk allele) nor the ϵ2 -allele (protective allele) of APOE were associated with an effect on telomere attrition with respect to age. To further our analysis from our first study, we initiated a second larger Okinawa study (Okinawa Study #2), to assess telomere length, telomerase activity, FOXO3 gene expression, and circulatory plasma cytokines IL-1β, IL-2, IL-6, IL-10, and TNFα. We recruited 325 Okinawan Japanese subjects with roughly equal numbers of men and women (ages 19-104 years; 49:51 M:F ratio). FOXO3 genotyping, and telomerase activity was assessed using the same protocols as in the first study. Telomere length was analyzed using a monochrome multiplex quantitative PCR, FOXO3 gene expression was analyzed by quantitative PCR, and cytokine levels were assessed using a Milliplex MAP Human High Sensitivity T Cell Panel on a Luminex 200 system. In agreement with our previous findings, the FOXO3 longevity variant conferred protection against telomere shortening in peripheral blood mononuclear cells from adult aged 55 and older in both men and women (P<0.001). This was accompanied by higher levels of telomerase activity in carriers of the longevity-associated FOXO3 G-allele (P=0.015). Carriage of the FOXO3 G-allele was associated with a modest increase in FOXO3 gene expression in both young and old groups compared to decreased gene expression in TT-carriers, the difference in trend being most significant in the young population (P=0.02). We also observed two sex-specific protective effects of the FOXO3 rs2802292 G-allele in the older population with respect to inflammatory cytokine levels. In women, carriers of the G-allele displayed a modest decline in levels of pro-inflammatory cytokine IL-6 with respect to age in contrast to increasing levels with age in TT-carrier counterparts (P=0.07). This protective effect in maintaining lower levels of pro-inflammatory cytokines was highly significant in comparison to G-carrier men (P=0.0006). For the anti-inflammatory cytokine IL-10, G-carrier men in the old population retained IL-10 levels with age better than TT-carriers (P=0.04), and even more significantly compared to G-carrier women (P=0.007). Finally, in a collaborative study with Exeter University, we analyzed the expression of the FOXO3 isoform levels in peripheral blood using TaqMan PCR. Using a limited subset of RNA samples from the Okinawa Study#2 population, we were unable to find a significant effect of the longevity associated FOXO3 rs2802292 genotype on moderating levels of FOXO3-FL (full length) and FOXO3-TR (truncated) isoforms, although a trend towards increased expression in G-allele carriers was observed. Collaborators at the University of Exeter analyzed peripheral blood samples from the UK BioBank with respect to the FOXO3 rs13217795 longevity-associated SNP that is in linkage disequilibrium with the rs2802292 SNP. They found that carriage of the longevity-associated “C”-allele was associated with increased expression of the full-length isoform in peripheral blood as compared to TT-carriers. Truncated isoform expression was not observed in peripheral blood samples in either the Okinawan or UK BioBank cohorts. Through these findings, we believe that we have developed a foundation for FOXO3 and aging research that will provide an infrastructure to further life science and medical research in numerous specialties that will ultimately improve our understanding of healthy aging and further the development and technologies available to help patients in doing so.