Functional NPY variation as a factor in stress resilience and alcohol consumption in rhesus macaques

Abstract

Context: Neuropeptide Y (NPY) counters stress and is involved in neuroadaptations that drive escalated alcohol drinking in rodents. In humans, low NPY expression predicts amygdala response and emotional reactivity. Genetic variation that affects the NPY system could moderate stress resilience and susceptibility to alcohol dependence.

Objective: To determine whether functional NPY variation influences behavioral adaptation to stress and alcohol consumption in a nonhuman primate model of early adversity (peer rearing).

Design: We sequenced the rhesus macaque NPY locus (rhNPY) and performed in silico analysis to identify functional variants. We performed gel shift assays using nuclear extract from testes, brain, and hypothalamus. Levels of NPY in cerebrospinal fluid were measured by radioimmunoassay, and messenger RNA levels were assessed in the amygdala using real-time polymerase chain reaction. Animals were exposed to repeated social separation stress and tested for individual differences in alcohol consumption. Animals were genotyped for -1002 T > G, and the data were analyzed using analysis of variance.

Setting: National Institutes of Health Animal Center. Subjects Ninety-six rhesus macaques. Main Outcome Measure Behavior arousal during social separation stress and ethanol consumption.

Results: The G allele altered binding of regulatory proteins in all nuclear extracts tested, and -1002 T > G resulted in lower levels of NPY expression in the amygdala. Macaques exposed to adversity had lower cerebrospinal fluid NPY levels and exhibited higher levels of arousal during stress, but only as a function of the G allele. We also found that stress-exposed G allele carriers consumed more alcohol and exhibited an escalation in intake over cycles of alcohol availability and deprivation.

Conclusions: Our results suggest a role for NPY promoter variation in the susceptibility to alcohol use disorders and point to NPY as a candidate for examining gene x environment interactions in humans.

Figure 1. rhNPY −1002 T>G is present in a conserved portion of a NPY repressor and results in altered DNA-protein interactions and decreased amygdala NPY expression

A. Schematic of the NPY gene and regulatory region and SNPs detected by sequencing of genomic DNA. B. Region 40-bp up and downstream of the −1002 T>G SNP. The precise locations of the −1002 T>G SNP (in bold) and the oligonucleotide sequence used in the gel shift assays (underlined) are indicated. Predicted sites for transcription factor binding (above) and sequence conservation among primates (below, black = conserved) are shown. Binding sites in dashed lines (Sox5 and a preferred glucocorticoid response element half site, GR) were predicted to be disrupted by the −1002 T>G SNP. C. Gel shift assay results from experiments performed using nuclear extracts from Whole Brain, osteosarcoma cells (MG-63), and glucocorticoid-treated hypothalamic cells (IVB + Dex). Relative migrations of the protein molecular weight standards are shown to the left (kDa). Open arrows indicate bands that increase with G allele probes, closed arrows indicate that which shows a relative increase with T allele probes. D. NPY mRNA expression in amygdala as a function of the −1002 T>G allele (P = 0.0008; T/T, N = 4, G carrier, N = 8). *** P < 0.001.

Figure 2. Interaction between rhNPY genotype (T/T, T/G, G/G) and early rearing history (MR, mother- reared, vs. PR, peer-reared) on CSF levels of NPY

There was an interaction between genotype and rearing (F(2, 66) = 4.2, P < 0.02). The G allele dose-dependently decreased levels of NPY measured in a cisternal CSF sample among stress-exposed monkeys (Tukey-Kramer, P < 0.05) (MR T/T = 17, MR T/G = 14, MR G/G = 4; PR T/T = 16, PR T/G = 13, PR G/G = 8). Genotype accounted for 28 % of the variance in PR subjects. Values shown are mean pg/ml ± SEM. * P < 0.05.

Figure 3. Interaction between rhNPY genotype (T/T, T/G, G/G) and early rearing history (MR, mother- reared, vs. PR, peer-reared) on arousal during periods of A. Acute (1 H) and B. Chronic (96 H) separation stress

During acute separation stress, there were main effects of both rearing (F(1, 96) = 6.4, P = 0.01) and genotype (F(2, 96) = 3.2, P = 0.04) on “Arousal”, with genotype accounting for 7% of the variance. During chronic stress, there was an interaction between rearing and genotype (F(2, 96) = 4.2, P = 0.02). Although PR T/T subjects responded no differently than did MR animals, PR G allele carriers exhibited higher levels of arousal (T/G and G/G vs. T/T, Tukey-Kramer, P < 0.05), with genotype accounting for 10% of the variance in these subjects. (MR T/T = 35, MR T/G = 27, MR G/G = 10; PR T/T = 9, PR T/G = 15, PR G/G = 6). Values shown are “Arousal” factors scores ± SEM. * P < 0.05.

Figure 4. Interaction between rhNPY genotype (T/T, T/G, G/G) and early rearing history (MR, mother- reared, vs. PR, peer-reared) on levels of voluntary alcohol consumption (mean ± SEM)

There was an interaction between rearing condition and genotype on alcohol consumption (F(2, 85) = 3.3, P = 0.04). When given simultaneous access to alcohol (8.4% v/v) and sweetened vehicle in a limited access paradigm, PR monkeys who were carriers of the G allele consumed higher levels of alcohol than did non-stress-exposed (MR) subjects genotype (Tukey-Kramer, P < 0.05). Genotype accounted for 12.5% of the variance in PR monkeys. (MR T/T = 29, MR T/G = 25, MR G/G = 8; PR T/T = 10, PR T/G = 11, PR G/G = 8). Values shown are G/KG alcohol consumed in a 1-hour session (G/KG/H) ± SEM. * P < 0.05.

Figure 5. Interaction between rhNPY genotype (T/T, T/G, G/G) and early rearing history (MR, mother- reared, vs. PR, peer-reared) on alcohol consumption over repeated weeks of alcohol deprivation (WEEKS 1, 2, 3, and 4)

There were both genotype by time (F(6, 204) = 3.02, P < 0.008) and genotype by time by rearing time (F(6, 204) = 2.2, P < 0.05) interactions. In PR monkeys, alcohol intake decreased across over time in individuals with the T/T genotype, but an escalation in consumption was observed in those carrying G allele. There was no significant effect of genotype in MR monkeys. The interaction between time and genotype accounted for 19% of the variance in PR monkeys. (MR T/T = 22, MR T/G = 19, MR G/G = 7; PR T/T = 9, PR T/G = 10, PR G/G = 7). Values shown are G/KG alcohol consumed in a 1-hour session (G/KG/H) ± SEM following a 3-day period of deprivation.