Functional CRH variation increases stress-induced alcohol consumption in primates

Abstract

Corticotropin-releasing factor (CRF), encoded by the CRH gene, is a key integrator of stress responses, and, as such, CRH gene variation may contribute to individual differences in susceptibility to stress-related pathology. In rhesus macaques, a single nucleotide polymorphism (SNP) is found within the CRH promoter (-248C--> T). Here, we assessed whether this variant influenced stress responding and, because increased CRF system activity drives alcohol drinking in rodents, we examined whether it predicted voluntary alcohol consumption as a function of prior stress exposure. Using a hypothalamic nuclear extract, we showed that the -248 T allele resulted in increased DNA protein interactions relative to the C allele. In vitro, the T allele resulted in CRH promoter activity that was higher following both stimulation with forskolin and treatment with dexamethasone. Endocrine and behavioral responses to social separation stress (release of ACTH and cortisol, and suppression of environmental exploration, respectively) were higher among carriers of the T allele, particularly among those exposed to early adversity in the form of peer rearing. We also found that T allele carriers with a history of early life adversity consumed more alcohol in a limited-access paradigm. Our data suggest that CRH promoter variation that confers increased stress reactivity increases the risk for alcohol use disorders in stress-exposed individuals.

Fig. 1.

Comparative genome information for the CRH gene from the UCSC Genome Browser Gateway (http://genome.ucsc.edu). (A) Coding regions for CRH are shown in addition to vertebrate conservation and Indel-based Conservation for the region −1 KB to + 1.6 KB from the transcription start site. (B) Conserved transcription factor-binding sites, regulatory potential and vertebrate conservation are shown for the CRH proximal promoter (−350 KB - +1). Also shown is the location of the rhesus −248 C → T SNP and a human SNP reported in this region (−201 C → T, rs28364015).

Fig. 2.

Location of the CRH −248 C → T SNP and effects on DNA-protein interactions. (A) Schematic of CRH promoter regulation and the transcription factors whose interactions are essential for regulatory control of CRH transcription, as reproduced from Nicholson et al., 2004 (46). The location of −248 C → T within this regulatory region is indicated. (B) Gel shift assay result, showing altered DNA-protein interactions with −248 T allele probes in experiments performed using nuclear extract from a hypothalamic cell line. Arrows to the right of the gel image indicate complexes that differ as a function of the −248 T allele.

Fig. 3.

Functional consequences of the CRH −248 C → T SNP. Reporter assay result, comparing promoter activity of −248 C and T constructs at baseline and following treatment with forskolin, forskolin + dexamethasone, and TPA. Constructs were co-transfected with a GFP reporter, and an expression value was obtained by dividing intensity of pDsRed by that for GFP. GFP and dsRed images were merged by overlaying the GFP image with the dsRed image (both at 40% transparency). *, P < 0.05.

Fig. 4.

Interaction between rhCRH genotype (C/C vs. C/T) and early rearing history (MR, mother-reared, vs. PR, peer-reared) on ACTH and cortisol responses to stress. Peer-reared T allele carriers exhibited higher plasma levels of ACTH and cortisol (mean percent change + SEM, MR C/C = 100, MR C/T = 4, PR C/C = 42, PR C/T = 3). ***, P < 0.0001; **, P < 0.01.

Fig. 5.

Interaction between rhCRH genotype (C/C vs. C/T) and early rearing history (MR, mother-reared, vs. PR, peer-reared) on levels of environmental exploration during periods of social separation stress. Peer-reared T allele carriers exhibited lower levels of environmental exploration during stress than did other groups of study (mean + SEM, MR C/C = 135, MR C/T = 7, PR C/C = 64, PR C/T = 4). *, P < 0.05.

Fig. 6.

Interaction between rhCRH genotype (C/C vs. C/T) and rearing condition (MR, mother-reared, vs. PR, peer-reared) on levels of voluntary alcohol consumption. When given simultaneous access to alcohol (8.4% vol/vol) and sweetened vehicle in a limited access paradigm, PR animals with the C/T genotype consumed higher levels of alcohol (mean + SEM, MR C/C = 101, MR C/T = 5, PR C/C = 74, PR C/T = 5). ***, P < 0.001.