Psychiatric-disorder-related behavioral phenotypes and cortical hyperactivity in a mouse model of 3q29 deletion syndrome

3q29 microdeletion, a rare recurrent copy number variant (CNV), greatly confers an increased risk of psychiatric disorders, such as schizophrenia and autism spectrum disorder (ASD), as well as intellectual disability. However, disease-relevant cellular phenotypes of 3q29 deletion syndrome remain to be identified. To reveal the molecular and cellular etiology of 3q29 deletion syndrome, we generated a mouse model of human 3q29 deletion syndrome by chromosome engineering, which achieved construct validity. 3q29 deletion (Df/+) mice showed reduced body weight and brain volume and, more importantly, impaired social interaction and prepulse inhibition. Importantly, the schizophrenia-related impaired prepulse inhibition was reversed by administration of antipsychotics. These findings are reminiscent of the growth defects and neuropsychiatric behavioral phenotypes in patients with 3q29 deletion syndrome and exemplify that the mouse model achieves some part of face validity and predictive validity. Unbiased whole-brain imaging revealed that neuronal hyperactivation after a behavioral task was strikingly exaggerated in a restricted region of the cortex of Df/+ mice. We further elucidated the cellular phenotypes of neuronal hyperactivation and the reduction of parvalbumin expression in the cortex of Df/+ mice. Thus, the 3q29 mouse model provides invaluable insight into the disease-causative molecular and cellular pathology of psychiatric disorders

Differential gene expression profiles in neurons generated from lymphoblastoid B-cell line-derived iPS cells from monozygotic twin cases with treatment-resistant schizophrenia and discordant responses to clozapine

Schizophrenia is a chronic psychiatric disorder with complex genetic and environmental origins. While many antipsychotics have been demonstrated as effective in the treatment of schizophrenia, a substantial number of schizophrenia patients are partially or fully unresponsive to the treatment. Clozapine is the most effective antipsychotic drug for treatment-resistant schizophrenia; however, clozapine has rare but serious side-effects. Furthermore, there is inter-individual variability in the drug response to clozapine treatment. Therefore, the identification of the molecular mechanisms underlying the action of clozapine and drug response predictors is imperative. In the present study, we focused on a pair of monozygotic twin cases with treatment-resistant schizophrenia, in which one twin responded well to clozapine treatment and the other twin did not. Using induced pluripotent stem (iPS) cell-based technology, we generated neurons from iPS cells derived from these patients and subsequently performed RNA-sequencing to compare the transcriptome profiles of the mock or clozapine-treated neurons. Although, these iPS cells similarly differentiated into neurons, several genes encoding homophilic cell adhesion molecules, such as protocadherin genes, showed differential expression patterns between these two patients. These results, which contribute to the current understanding of the molecular mechanisms of clozapine action, establish a new strategy for the use of monozygotic twin studies in schizophrenia research

Impaired extinction of cued fear memory and abnormal dendritic morphology in the prelimbic and infralimbic cortices in VPAC2 receptor (VIPR2)-deficient mice.

The structurally related neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in stress regulation and learning and memory. Several bodies of research have shown the impact of the PACAP specific receptor PAC1 on fear memory, but the roles of other PACAP receptors in regulating fear stress responses remain to be elucidated. Here we aimed to investigate the effects of genetic deletion of VIPR2 encoding the VPAC2 receptor, which binds both VIP and PACAP, on fear-related memory and on dendritic morphology in the brain regions of the fear circuitry. Male VPAC2 receptor knockout (VPAC2-KO) and littermate wild-type control mice were subjected to Pavlovian fear conditioning paradigm. VPAC2-KO mice displayed normal acquisition of fear conditioning, contextual and cued fear memory, but impaired extinction of cued fear memory. Morphological analyses revealed reductions in cell body size and total branch number and length of apical and basal dendrites of prelimbic cortex neurons in VPAC2-KO mice. In addition, Sholl analysis indicated that the amount of dendritic material distal to the soma was decreased, while proximal dendritic material was increased. In the infralimbic cortex, the amount of apical dendritic material proximal to the soma was increased in VPAC2-KO mice, while other indices of morphology did not differ. Finally, there were no differences in dendritic morphology in basolateral amygdala neurons between genotypes. These findings suggest that the VPAC2 receptor plays an important role in the fear extinction processes and the regulation of the dendritic morphology in the prelimbic and infralimbic cortices

Impaired extinction of cued fear memory and abnormal dendritic morphology in the prelimbic and infralimbic cortices in VPAC2 receptor (VIPR2)-deficient mice.

The structurally related neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in stress regulation and learning and memory. Several bodies of research have shown the impact of the PACAP specific receptor PAC1 on fear memory, but the roles of other PACAP receptors in regulating fear stress responses remain to be elucidated. Here we aimed to investigate the effects of genetic deletion of VIPR2 encoding the VPAC2 receptor, which binds both VIP and PACAP, on fear-related memory and on dendritic morphology in the brain regions of the fear circuitry. Male VPAC2 receptor knockout (VPAC2-KO) and littermate wild-type control mice were subjected to Pavlovian fear conditioning paradigm. VPAC2-KO mice displayed normal acquisition of fear conditioning, contextual and cued fear memory, but impaired extinction of cued fear memory. Morphological analyses revealed reductions in cell body size and total branch number and length of apical and basal dendrites of prelimbic cortex neurons in VPAC2-KO mice. In addition, Sholl analysis indicated that the amount of dendritic material distal to the soma was decreased, while proximal dendritic material was increased. In the infralimbic cortex, the amount of apical dendritic material proximal to the soma was increased in VPAC2-KO mice, while other indices of morphology did not differ. Finally, there were no differences in dendritic morphology in basolateral amygdala neurons between genotypes. These findings suggest that the VPAC2 receptor plays an important role in the fear extinction processes and the regulation of the dendritic morphology in the prelimbic and infralimbic cortices

Increased behavioral and neuronal responses to a hallucinogenic drug in PACAP heterozygous mutant mice.

Accumulating evidence from human genetic studies implicates the pituitary adenylate cyclase-activating polypeptide (PACAP) gene as a risk factor for psychiatric disorders, including schizophrenia and stress-related diseases. Mice with homozygous disruption of the PACAP gene display profound behavioral and neurological abnormalities that are ameliorated with the atypical antipsychotic and dopamine D2 and serotonin (5-HT)2 antagonist risperidone and the 5-HT2 receptor antagonist ritanserin; however, the underlying mechanisms remain unknown. Here, we investigated if PACAP heterozygous mutant (PACAP(+/-)) mice, which appear behaviorally normal, are vulnerable to aversive stimuli. PACAP(+/-) mice were administered a 5-HT2 receptor agonist, (±)-2,5-dimethoxy-4-iodoamphetamine (DOI), a hallucinogenic drug, and their responses were compared with the littermate wild-type mice. After DOI injection, PACAP(+/-) mice showed increased head-twitch responses, while their behavior was normal after saline. DOI induced deficits in sensorimotor gating, as determined by prepulse inhibition, specifically in PACAP(+/-) mice. However, other 5-HT2 receptor-dependent responses, such as corticosterone release and hypothermia, were similarly observed in PACAP(+/-) and wild-type mice. c-Fos expression analysis, performed in various brain regions, revealed that the DOI-induced increase in the number of c-Fos-positive cells was more pronounced in 5-HT2A receptor-negative cells in the somatosensory cortex in PACAP(+/-) mice compared with wild-type mice. These results indicate that PACAP(+/-) mice exhibit specific vulnerability to DOI-induced deficits in cortical sensory function, such as exaggerated head-twitch responses and sensorimotor gating deficits. Our findings provide insight into the neural mechanisms underlying impaired behavioral responses in which 5-HT2 receptors are implicated

β-Arrestin1 and 2 differentially regulate PACAP-induced PAC1 receptor signaling and trafficking

<div><p>A pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor, PAC1R, is coupled with multiple signal transduction pathways including stimulation of adenylate cyclase, phospholipase C and extracellular-signal regulated kinase (ERK)1/2. PAC1R has been shown to exert its long-lasting and potent signals via β-arrestin1 and β-arrestin2. However, the precise roles of the two β-arrestin isoforms in PACAP-PAC1R signaling remain unclear. Here we examined the interaction between the two β-arrestin isoforms and PAC1R, β-arrestin-dependent PAC1R subcellular localization and ERK1/2 activation. Upon PACAP stimulation, although PAC1R similarly interacted with β-arrestin1 and β-arrestin2 in HEK293T cells, the complex of PAC1R and β-arrestin2 was translocated from the cell surface into cytosol, but that of β-arrestin1 remained in the cell surface regions in HeLa cells and mouse primary cultured neurons. Silencing of β-arrestin2 blocked PACAP-induced PAC1R internalization and ERK1/2 phosphorylation, but silencing of β-arrestin1 increased ERK1/2 phosphorylation. These results show that β-arrestin1 and β-arrestin2 exert differential actions on PAC1R internalization and PAC1R-dependent ERK1/2 activation, and suggest that the two β-arrestin isoforms may be involved in fine and precise tuning of the PAC1R signaling pathways.</p></div

Time-lapse cell imaging showing PAC1R and β-arrestin coupling and translocation in HeLa cells.

<p>HeLa cells were transfected with the indicated combinations of plasmid vectors. <b>(A and B)</b> Representative images of NanoBiT luminescence at 3, 15, 30 and 60 min after stimulation with 1 μM PACAP. <b>(C and D)</b> Representative time-dependent changes of line-scan images for 60 min after stimulation with 1 μM PACAP. <b>(E and F)</b> Time course of changes in luminescence intensity at the vicinity of the plasma membrane (membrane) and the cytoplasm for 60 min after stimulation with 1 μM PACAP in HeLa cells. Scale bars, 10 μm. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM (n = 3–5). *<i>p</i> < 0.05 vs. cytoplasm, repeated measure two-way ANOVA followed by Fisher-PLSD test. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196946#pone.0196946.s004" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196946#pone.0196946.s005" target="_blank">S2</a> Movies.</p

Differential effects of silencing of β-arrestin1 and β-arrestin2 on PACAP-induced ERK1/2 activation.

<p><b>(A)</b> Representative images of western blots for total and phosphorylated ERK1/2 in HEK293T cells transfected with the indicated siRNA and treated with 1 μM PACAP or saline for 3 or 25 min. <b>(B)</b> Quantification of ERK1/2 activation by normalizing phosphorylated ERK1/2 to total ERK1/2 levels analyzed by western blotting. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM of three independent experiments. *<i>p</i> < 0.05 and **<i>p</i> < 0.01, two-way ANOVA followed by Fisher-PLSD test.</p

Differential effects of β-arrestin1 and β-arrestin2 siRNA on PAC1R internalization.

<p><b>(A)</b> Knockdown of endogenous β-arrestin1 and β-arrestin2 in HEK293T cells. The upper images, representative images of western blots. Values are mean ± SEM of three independent experiments. **<i>p</i> < 0.01 and ***<i>p</i> < 0.001, two-way ANOVA followed by Fisher-PLSD test. <b>(B–E)</b> Representative images of HEK293T cells that were transfected with PAC1R-Halo and the indicated siRNA, labeled with Alexa Fluor 488 HaloTag ligand and treated with 1 μM PACAP or saline for 30 min. <b>(F)</b> Quantification of PAC1R-Halo internalization. Scale bar, 10 μm. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM of 60 cells obtained from three independent experiments. *<i>p</i> < 0.05 and **<i>p</i> < 0.01, two-way ANOVA followed by Fisher-PLSD test.</p