Splice-specific deficiency of the PTSD-associated gene PAC1 leads to a paradoxical age-dependent stress behavior

Abstract

The pituitary adenylate cyclase-activating polypeptide receptor (PAC1, also known as ADCYAP1R1) is associated with post-traumatic stress disorder and modulation of stress response in general. Alternative splicing of PAC1 results in multiple gene products, which differ in their mode of signalling and tissue distribution. However, the roles of distinct splice variants in the regulation of stress behavior is poorly understood. Alternative splicing of a short exon, which is known as the "hop cassette", occurs during brain development and in response to stressful challenges. To examine the function of this variant, we generated a splice-specific zebrafish mutant lacking the hop cassette, which we designated 'hopless'. We show that hopless mutant larvae display increased anxiety-like behavior, including reduced dark exploration and impaired habituation to dark exposure. Conversely, adult hopless mutants displayed superior ability to rebound from an acute stressor, as they exhibited reduced anxiety-like responses to an ensuing novelty stress. We propose that the developmental loss of a specific PAC1 splice variant mimics prolonged mild stress exposure, which in the long term, predisposes the organism's stress response towards a resilient phenotype. Our study presents a unique genetic model demonstrating how early-life state of anxiety paradoxically correlates with reduced stress susceptibility in adulthood. PAC1 (a.k.a. Adcyap1r1) is a G-protein coupled receptor (GPCR) that serves as the high-affinity receptor for the pituitary adenylate cyclase-activating polypeptide (PACAP). PAC1 has pleiotropic functions and was demonstrated to be involved in the regulation of several homeostatic processes including metabolic rate and food consumption 1,2 , circadian rhythm 3 and, in particular, stress response 4,5. Intracerebroventricular injection of PACAP increased phosphorylated cyclic AMP response element binding protein (pCREB) and corticotropin-releasing hormone (CRH) immunoreactivity in the rat paraventricular nucleus 6. PACAP knockout mice display blunted hypothalamic CRH levels in response to restraint challenge 7. PACAP/PAC1 signaling was also associated with hypothalamo-pituitary-adrenal activity and stress-related behaviors in humans and rodents 5,8,9. Furthermore, this pathway was correlated with stress-related risky behaviors in human and rodents 10,11. Overall, these findings support positive stress regulation by PAC1; yet, some reports suggest that it may also act to suppress stress phenotypes 4,12. It has been suggested that genetic vulnerability to post-traumatic stress disorder (PTSD) may depend on PAC1 expression and single-nucleotide polymorphism (SNP) in the PAC1 gene. Ressler et al. demonstrated that a specific PAC1 genotype is strongly correlated with susceptibility to PTSD in women, probably due to perturbed expression of PAC1 resulting in impaired stress responses 13. The same PAC1 SNP was associated with PTSD in African-American females, emotional numbing in traumatized earthquake Chinese survivors, dark-e...

Genome Editing Using the CRISPR-Cas9 System to Generate a Solid-Red Germline of Nile Tilapia (Oreochromis niloticus)

“…Single guide RNA (sgRNA) design and synthesis was performed, as previously described 32 with slight modifications. Briefly, gRNA was designed using CHOP-CHOP, 33 transcribed in vitro using MEGAshortscript T7 kit (Life Technologies, Carlsbad, CA), and purified using a miRNeasy kit (Qiagen, Germantown, MD).…”

“…Microinjection to tilapia zygotes and gRNA/Cas9 heterocomplexing were performed as previously described. 26,32 Tilapia zygotes were collected immediately after natural spawning or IVF and chilled on ice to 21°C to prolong microinjection time at the single-cell stage. In each microinjection session, zygotes were injected with a mix containing a single or two to three mixed slc45a2 gRNAs (100 pmol for each crRNA and tracrRNA; 39.72 pmol for sgRNA) mixed with rCas9 (4.4 lg) to a final volume of 9 lL.…”

Effects of maternal immune activation in porcine transcript isoforms of neuropeptide and receptor genes

“…Comparison of the pig ADCYAP1R1 protein sequences identified that the different transcripts corresponded to the third intercellular loop that generate differential responses to PACAP-27 [70]. Compared to normal zebrafish (Danio renio), larvae lacking the hop exon had increased anxiety-like behavior [71,72]. AD-CYAP1 is a member of the circadian entrainment pathway and considered a potential target for therapies to ameliorate drug abuse, migraine, stress-related psychopathologies and inflammatory diseases [69,[73][74][75][76][77][78].…”

The Protective Effects of Endogenous PACAP in Oxygen-Induced Retinopathy

“…The role of endogenous PACAP has been explored with the help of PACAP gene-deficient mice. These mice show several developmental and behavioral alterations, including altered neurobehavioral, bone, and tooth development (Farkas et al 2017;Fulop et al 2019b;Jozsa et al 2018), paradoxical age-dependent stress behavior (Biran et al 2020), and altered light responses (Riedel et al 2020). The strong neuro-and general cytoprotective role of PACAP are mainly due to its anti-apoptotic, antioxidant, and anti-inflammatory effects (Soles-Tarres et al 2020;Toth et al 2020).…”