Results

To test our hypothesis, we determined DRD4 exon 3 VNTR ge- notypes/sequences for 310 90(cid:2) study individuals (the “oldest- old”; mean age, 95.2 years; age range, 90–109 years; 91 males) >t~] (Fig. 1). These individuals represent 1.4–2.9% of their initial birth cohort and, by definition, should have altered allele fre- quencies of genes that impact health and longevity. When com- pared with matched European ancestry controls of individuals VES 50–90 years younger (N 2902; age range, 7–45 years), the oldest-old had a 66% increase in observed 7R/x genotype (Fig. 2) (0.364 vs 0.219, (cid:2)2  33.28, p  3.5 wivxX 10(cid:4)9; (cid:3)2  0.026). This extremely small p value would be significant at a genome-wide level following Bonferroni correction. Comparing allele fre- quency rather than genotype, the frequency of the 7R allele is greater in the oldest-old population by 72% (0.205 vs 0.119, (cid:2)2  38.27, p aa NAS 6.15 aoe 10(cid:4)10). This increase was preferentially ob- served in females (0.393 vs 0.297 males, (cid:2)2  9.65, p )ac.( 0.002), among whom the difference was significant at a genome-wide level(0.393vs0.219,(cid:2)2(cid:1) 34.66,p(cid:1) 1.7(cid:3) 10(cid:4)9).Incontrast,the difference in males, while significant, did not reach genome-wide level significance (0.297 vs 0.219, (cid:2)2 i 3.11, p  0.034) (Fig. 2).

To test the association of the DRD4 gene with longevity and re- sponses to the environment directly, we compared the lifespans of mice that expressed DRD4 (HT and WT) versus those of KO mice (Rubinstein et al., 1997) that lacked the DRD4 gene, when reared in eitheraDE(standardlaboratorycage)orEE(groupcageswithmul- tiple options for physical activity and exploration) environment (Ullman-Cullere´ andFoltz,1999;Quimbyetal.,2010).DRD4geno- type significantly affected the lifespan (F  5.74, p ye A 0.004, (cid:4)2 pa  0.03; Fig. 4). DRD4 KO mice (N 68) had a 9.7% shorter lifespan oNSe (mean and SEs: 94.8 2.08 weeks; 95% CI, 91–99) than HT mice    aa} *<< (N 57; 104 1.82 weeks; 95% CI, 100–108; p 0.05), and 7% shorter than WT mice (N oy aH 117; 101.4   2.38 weeks; 95% CI, 97–106;p(cid:7) 0.05).Therearingenvironmentalsoinfluencedlongev- ity (F   10.58, p   0.001, (cid:4)2  0.03; Fig. 4). Mice in EE (N   111; It | 102.2 1.76weeks;95%CI,98–106)lived5.7%longerthanmicein DE (N   131; 96.7 rane—- 1.54 weeks; 95% CI, 94–100; p Ae<< ( 0.05), but these effects were moderated by the DRD4 genotype, as shown by a significant environment ow genotype interaction (F eos 8.05, p ‘Le<< 0.001, (cid:4)2  0.04; Fig. 4). Specifically, whereas the lifespan was in-

positive and negative, is modulated by the DRD4 7R variant (Sheese et al., 2007; Belsky et al., 2009; Das et al., 2011; Olsson et al., 2011). The reduced spontaneous lo- comotor activity observed in DRD4 KO mice compared with WT and HT mice (Fig. 5) supports this interpretation, as well as the higher reported physical activ- ity levels in oldest-old individuals with a DRD4 7R variant (Fig. 3). However, from our experimental design it cannot be de- termined whether the beneficial effects of an enriched environment on longevity in WT and HT mice is moderated through social factors rather than increased loco- motor activity per se.

Survival30% 420% +10% 5 0%     Survival in WeeksWT-DE WT-EE HT-DE -HT-EE -KO-DE__KO-EE30 40 50 60 70 80 90 100 110 120 130 140 150Time (weeks)—KO-EE — HT-EE ——WT-EE - - KO-DE - - HT-DE - - WT-DE

The mechanism by which the DRD4 variants would differentially influence envi- ronmental responses is unclear. The variant DRD47Rproteinhasabluntedresponsefor cAMP reduction compared with the 4R protein(Asgharietal.,1995;Jovanovicetal., 1999; Swanson et al., 2000; Wang et al., 2004). Also, the DRD4 7R protein, unlike the DRD4 4R protein, does not form func- tional heteromers with the short presynap- tic isoform of the dopamine D2 receptor in the striatum (Borroto-Escuela et al., 2011; Gonza´lezetal.,2012).DysfunctionalD2-D4 7R heteromers may impair dopaminergic control of corticostriatal glutamatergic neurotransmission. Thus, it is plausible that either a dopamine signaling deficit or en- hanced glutamate release, mediated by DRD4 7R, could enhance reactivity to envi- ronmental stimuli.

Figure4. Kaplan–MeiersurvivalcurvesofmiceasafunctionofgenotypeandexposuretoanEEorDE.Insert,Lifespanpergroup (meanandSE).WTandHTmicerearedinanEElivedsignificantlylongerthanwhenrearedinaDE.Incontrast,survivalofDRD4KO micewasnotincreasedwhenrearedinanEEcomparedwithaDEandwasshorterthanthatofWTandHTmice.

800 KO-EE HT-EE WT-EEGenotype/Environment   KOE —O—HT-EE —e— Wee 3g3g550 MH -KO-DE - MN -HT-DE - M -WT-DE oon28500 600550MonthKO-DE HT-DE = WT-DEGenotype/Environment

While the biochemical/physiological significance of DRD4 variants is under continuing investigation, there is solid molecular evolutionary evidence that functional differences exist at this locus. Extensive linkage disequilibrium (LD) surrounds the DRD4 7R allele in individ- uals sampled worldwide (compared with the random LD at the ancestral 4R allele), as determined by direct DNA resequenc- ing of the entire gene and genotyping of polymorphisms extending 100 kb in both directions (Ding et al., 2002; Wang et al., 2004, 2006). The high frequency of an al- lele coupled with extensive LD is a power- ful indicator of recent adaptive selection (Wang et al., 2006; Hawks et al., 2007)