Figure5. SpontaneouslocomotoractivityfordifferentagesinWT,HT,andKOmicerearedinaDEandanEE.Valuescorrespond to means ((cid:8)SE) and correspond to average activity over 90 min. The genotype-by-age interaction on locomotor activity was significant(F(cid:1)3.61,p(cid:7)0.001,(cid:4) 2(cid:1)0.034)withoverallsignificantlyloweractivitylevelsforKOmicethanforWTorHTmice p (p   0.05). The interaction of environment by age with locomotor activity was also significant (F “weesF=) 2.77, p   0.017,   2  ““~ p 0.013); overall, DE mice were significantly more active than EE mice (p wayr<C 0.05). However, since animals in an EE decrease in spontaneouslocomotoractivity(Garlandetal.,2011)andwedidnotrecord24hactivitylevels,wecannotcompareactivitylevels acrossthetwoenvironmentalconditions.

creasedinanEEforHT(9%increase)andWT(16%increase)mice, the longevity of DRD4 KO mice did not differ between DE and EE rearing conditions (p  0.15; Fig. 4, inset).

(LDD test). The pattern of LD decay surrounding the DRD4 7R allele, together with the highest LD near the VNTR decaying in both directions, suggests that the DRD4 7R VNTR is the “target” of this Darwinian selection, with a recent evolutionary origin Ma(~4 ( 40,000–50,000 years ago). Consistent with this recent evolu- tionary origin and the known biochemical differences noted above, we have speculated that a “response-ready” adaptation influenced by the DRD4 7R allele might have played a role in the Out-of-Africa human exodus (Ding et al., 2002; Wang et al.,

Discussion

We have shown that a human DRD4 variant associated with ac- tivity in younger individuals is at higher observed frequency in the oldest-old (Figs. 1, 2, 3), and that the DRD4 gene in mice moderated the beneficial effects of an enriched environment on lifespan (Figs. 4, 5). These results are consistent with human studies showing that sensitivity to environmental factors, both

associated with higher BMI (Guo et al., 2007; Fuemmeler et al., 2008), except in African American populations, but few studies on DRD4/activity have been conducted except for individuals diag- nosed with ADHD (Swanson et al., 2000). Measurements of the 7s7 ~0 heritability of physical activity vary widely, with a value of 0.30 often reported (Pe´russe et al., 1989; Horimoto et al., 2011). The proportionofthevarianceinphysicalactivityexplainedbyDRD47R in the oldest-old population (Fig. 3) is r2  0.023, or mn)r~7 7.7% of the varianceattributabletogenesmeasuredinheritabilitystudies.How- ever,heritabilitystudiesassumethatmultipleadditivegenescontrib- utetothephenotype,butaninteractinggenemodel,asproposedfor the DRD4 7R/ADHD association (Grady et al., 2003), would make the contribution of DRD4 7R to the variance in activity level signif- icantlygreater(Zuketal.,2012).Clearly,furtherstudiesofDRD47R contributions to the variance of activity level in other populations should be pursed. Unfortunately, there are no genetic markers on commercially available genotyping arrays that are in strong LD with the DRD4 7R VNTR (Saccone et al., 2009), making direct genotyp- ing/sequencing essential.

In further discussing our findings, it is important to note their limitations. The ideal control for the genotype/haplotype frequen- cies of DRD4 alleles would have been the nonliving birth cohorts of our oldest-old population, which is clearly not possible. However, human gene frequencies cannot change radically in a few genera- tions, except following extreme population bottlenecks, which have not occurred in the last 100 years in European ancestry populations. A possible selective force that could have introduced intergenera- tional variation is the high mortality due to infectious disease expe- rienced early in life by this oldest-old cohort, which has been largely eliminated through the use of immunization and antibiotics in the younger cohort (Fig. 1). Thus, any hypothetical “resistance” allele might exhibit higher frequencies in the oldest-old population than currently observed, due to this environmental intervention. How- ever, it is unlikely that the DRD4 gene (or any of the three closest genesinmodestLDwithit:DEAF1,SCT,andMUPCDH)(Interna- tional Human Genome Sequencing Consortium, 2004) would con- fer such resistance. Moreover, allele frequencies at 20 randomly chosen polymorphisms in nine unrelated genomic regions (CFTR, ERCC8, ERBB4, FANCC, HTERT, NRF1, RTN1, SLC6A3, and UCP3) did not differ significantly among the oldest-old individuals, younger control individuals, or HapMap European ancestry popu- lations(InternationalHapMapConsortiumetal,2007);norwasthe observed DRD4 7R male/female allele frequency bias (Fig. 2) found at any of these randomly chosen sites. It is difficult to conceive of a mechanismthatexplainsthevariationsinallelefrequenciesbetween males and females except for one that involves the functional signif- icanceoftheunderlyingvariantlocus.Wecannot,however,ruleout alternative behavioral possibilities to explain why 7R/x individuals are disproportionately found in our specific cohort, such as the abil- ity/desire to live in a planned retirement community with good ac- cess to leisure activities [see the discussion of the potential impact of socioeconomic status (SES)]. Clearly, further studies of the allele frequencies of DRD4 throughout the lifespan, rather than just at the extremes (Fig. 1) are warranted.

Forgeneticstudiesofcomplexphenotypes,mostassociatedvariants observed to have relatively small effect sizes ((cid:7) 1%), as expected for populations similar to the oldest-old population studied here 25%;Herskind al., 1996; Mitchell et al., 2001). Given the estimated effect size for the 7R/longevity association ((cid:3)2 whens 0.026; Fig. 2), we can estimate ndt~l 10.2% of the variance attributable to genes, measured in herita- 0.03–0.04;Fig.4).Thesevaluesarequite for a phenotype expected to be associated with many genes, and that variants of DRD4 make a significant impact on longevity,