Post-traumatic stress disorder is associated with PACAP and the PAC1 receptor

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to broadly regulate the cellular stress response. In contrast, it is unclear if the PACAP-PAC1 receptor pathway has a role in human psychological stress responses, such as post-traumatic stress disorder (PTSD). Here we find, in heavily traumatized subjects, a sex-specific association of PACAP blood levels with fear physiology, PTSD diagnosis and symptoms in females. We examined 44 single nucleotide polymorphisms (SNPs) spanning the PACAP (encoded by ADCYAP1) and PAC1 (encoded by ADCYAP1R1) genes, demonstrating a sex-specific association with PTSD. A single SNP in a putative oestrogen response element within ADCYAP1R1, rs2267735, predicts PTSD diagnosis and symptoms in females only. This SNP also associates with fear discrimination and with ADCYAP1R1 messenger RNA expression in human brain. Methylation of ADCYAP1R1 in peripheral blood is also associated with PTSD. Complementing these human data, ADCYAP1R1 mRNA is induced with fear conditioning or oestrogen replacement in rodent models. These data suggest that perturbations in the PACAP-PAC1 pathway are involved in abnormal stress responses underlying PTSD. These sex-specific effects may occur via oestrogen regulation of ADCYAP1R1. PACAP levels and ADCYAP1R1 SNPs may serve as useful biomarkers to further our mechanistic understanding of PTSD.

Figure 1. PACAP blood levels predict PTSD symptoms in females

A) PTSD Symptoms (scale range 0–51), relative to Plasma PACAP38 blood levels (pM); (N=34 females; r = 0.497, p ≤ 0.005). B) Total PTSD symptoms graphed relative to sex and levels of plasma PACAP38 (N=64, low: < 20pM, high: > 20pM); females with high PACAP blood levels have increased symptoms (**, p<0.0005). C) PACAP levels (low vs. high) are also differentially associated with PTSD Intrusive, Avoidance, and Hyperarousal symptoms in females (N = 64,** p < 0.005). D) PACAP levels (low vs. high) were examined in a replication sample of highly traumatized women, with differential association in hyperarousal symptoms (left, N=74,** p=0.002) and in percent of subjects with significant symptoms (right, χ²=4.9, p<0.05). E) Acoustic startle reflex (EMG) relative to the fear conditioning trial in subjects without (non-PTSD) vs with PTSD. Habituation is seen in non-PTSD subjects during late acquisition (bar). F) Startle magnitude during the late acquisition period vs. trial type (noise alone, CS+, and CS−), showing that females with high PACAP levels show enhanced startle responses to both fear cues (CS+, p = 0.02) and safety cues (CS−, p = 0.005) (N=27; 16 male, 11 female). Bars represent mean ± SEM., N’s for each group at bottom of bar graphs.

Figure 2. Genetic Association of PAC1 Receptor (ADCYAP1R1) with PTSD

A) 30 single nucleotide polymorphisms (SNPs) spanning the ADCYAP1R1 gene (x-axis), with the–log(p-value) of logistic regression for each SNP association with PTSD (diagnosis based on DSM-IV criteria from PTSD Symptom Scale). Subjects were analyzed with logistic regression in females only (N=503) or males only (N=295). Horizontal lines represent the nominal p = 0.05 or the corrected p-value, p = 0.0011 (44 SNPs, correcting for 30 ADCYAP1R1 SNPs and 14 ADCYAP1 SNPS (Supp. Fig. 1)). rs2267735 is the only SNP remaining significant after multiple corrections (p = 0.0002). B) Table of p-values resulting from the association of each genotyped, ADCYAP1R1 SNP with PTSD diagnosis (by gender). The location on Chromosome 7 (GRCh37) for each SNP including the distance (bp) between the SNPs is given. The average distance between SNPs is 2.2 kb. SNP rs2267735 is located in an intron of ADCYAP1R1, and is not in LD with other SNPS (for African Americans in our population, data not shown). C) rs2267735 (C/G), in red, is located within a canonical estrogen response element (ERE) binding site (Capitol letters – conserved canonical ERE nucleotides; blue letters – mismatches with the ADCYAP1R1 gene and canonical ERE; Reverse strand shown).

Figure 3. Association of ADCYAP1R1 rs2267735 with PTSD symptoms and physiological fear responses

A) Table demonstrating the N, Wald χ², and p-value, in males and females, in the original, replication, and combined samples for logistic regression of rs2267735 with PTSD diagnosis. B) Total PTSD symptoms (PSS total) are differentially associated with rs2267735 genotype in females (p ≤ 0.001). C) Hyperarousal is the most robustly associated symptom with rs2267735 genotype (p = 0.0009). D) In a dominant/recessive model, even after controlling for childhood trauma, adult trauma, and age, genotype predicts total PTSD symptoms (p ≤ 0.001) and (E) hyperarousal symptoms (p ≤ 0.0001). F) Fear Discrimination (CS+ Startle minus CS− Startle) is impaired in females with rs2267735 ‘CC’ genotype. G) Dark enhanced startle (Startle^Dark – Startle^Light) is significantly increased in females with rs2267735 ‘CC’ genotype. (N’s are shown at base of each bar, bars represent mean ± SEM. N’s are slightly different across analyses due to differences in number of subjects across measures. * p<0.05; ** p<0.001; *** p<0.0002)

Figure 4. ADCYAP1R1 Methylation and mRNA expression

A) Methylation within the first CpG island of ADCYAP1R1 (beta value, Illumina #cg27076139) is positively correlated with total PTSD symptoms (both sexes; N=107; r = 0.354, p < 0.0005). B) Subjects with PTSD have higher levels of ADCYAP1R1 methylation (median split, N = 107; chi-squared analyses, p < 0.005). C) ADCYAP1 mRNA levels are inversely correlated with ADCYAP1R1 mRNA levels in cortex (from prior dataset13)(r=−0.219; p<0.001). D) ADCYAP1R1 mRNA levels are differentially expressed in females compared to males based on imputed ADCYAP1R1 rs2267735 genotype (from prior dataset13)(*p<0.05 male vs. female CC carriers, # p<0.05, one-tailed, CC vs G-carriers). Bars represent mean ± SEM, N’s for each group at bottom of each graph.

Figure 5. Regulation of ADCYAP1R1 and ADCYAP1 mRNA in rodent models

A) Percentage of time freezing, in mice, to the conditioned tone (CS+) following tone-shock pairings during the conditioned fear trials. B) rtPCR analyses of mRNA levels within mouse amygdala and mPFC 2 hrs after fear conditioning or in control handling conditions, showing a significant increase in amygdala ADCYAP1R1 mRNA (N = 15, 1.47 fold, p < 0.05) and a non-significant trend in mPFC (1.19 fold change). C) Correlation between average amygdala and PFC ACYAP1R1 mRNA and % freezing at trial 4, demonstrating an association between ADCYAP1R1 mRNA with rate of fear learning (r²=0.49, p<0.05). D) ADCYAP1 mRNA in rat BNST in female rats (N = 12/group) following ovarectomy and estradiol implant vs. vehicle replacements. ADCYAP1 mRNA is increased in both dorsal (2.1-fold) and ventral (3.4-fold) BNST after estradiol implantation. E) ADCYAP1R1 transcripts are also increased in dorsal BNST (1.6-fold, N=4 per group). Bars represent mean ± SEM, (* p < 0.05).