PACAP is Implicated in the Stress Axes

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved pleiotropic neuropeptide that functions as a neurotransmitter, neuromodulator and neurotrophic factor. Accumulating evidence implicates PACAP as an important regulator of both central and/or peripheral components of the stress axes, particularly exposure to prolonged or traumatic stress. Indeed, PACAP and its cognate receptors are widely expressed in the brain regions and peripheral tissues that mediate stress-related responses. In the sympathoadrenomedullary system, PACAP is required for sustained epinephrine secretion during metabolic stress. It is likely that PACAP regulates autonomic function and contributes to peripheral homeostasis by maintaining a balance between sympathetic and parasympathetic activity, favoring stimulation of the sympathetic system. Furthermore, PACAP is thought to act centrally on the paraventricular nucleus of the hypothalamus to regulate both the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Intriguingly, PACAP is also active in brain structures that mediate anxiety- and fear-related behaviors, and the expression of PACAP and its receptors are dynamically altered under pathologic conditions. Thus PACAP may influence both hard-wired (genetically determined) stress responses and gene-environment interactions in stress-related psychopathology. This article aims to overview the molecular mechanisms and psychiatric implications of PACAP-dependent stress responses

Inhibition of the mitochondria-shaping protein Opa1 restores sensitivity to Gefitinib in a lung adenocarcinomaresistant cell line

Drug resistance limits the efficacy of chemotherapy and targeted cancer treatments, calling for the identification of druggable targets to overcome it. Here we show that the mitochondria-shaping protein Opa1 participates in resistance against the tyrosine kinase inhibitor gefitinib in a lung adenocarcinoma cell line. Respiratory profiling revealed that oxidative metabolism was increased in this gefitinib-resistant lung cancer cell line. Accordingly, resistant cells depended on mitochondrial ATP generation, and their mitochondria were elongated with narrower cristae. In the resistant cells, levels of Opa1 were increased and its genetic or pharmacological inhibition reverted the mitochondrial morphology changes and sensitized them to gefitinib-induced cytochrome c release and apoptosis. In vivo, the size of gefitinib-resistant lung orthotopic tumors was reduced when gefitinib was combined with the specific Opa1 inhibitor MYLS22. The combo gefitinib-MYLS22 treatment increased tumor apoptosis and reduced its proliferation. Thus, the mitochondrial protein Opa1 participates in gefitinib resistance and can be targeted to overcome it

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

PACAP centrally mediates emotional stress-induced corticosterone responses in mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide widely distributed in the nervous system. Recently, PACAP was shown to be involved in restraint stress-induced corticosterone release and concomitant expression of the genes involved in hypothalamic–pituitary–adrenal (HPA) axis activation. Therefore, in this study, we have addressed the types of stressors and the levels of the HPA axis in which PACAP signaling is involved using mice lacking PACAP (PACAP−/−). Among four different types of stressors, open-field exposure, cold exposure, ether inhalation, and restraint, the corticosterone response to open-field exposure and restraint, which are categorized as emotional stressors, but not the other two, was markedly attenuated in PACAP−/− mice. Peripheral administration of corticotropin releasing factor (CRF) or adrenocorticotropic hormone induced corticosterone increase similarly in PACAP and wild-type mice. In addition, the restraint stress-induced c-Fos expression was significantly decreased in the paraventricular nucleus (PVN) and medial amygdala (MeA), but not the medial prefrontal cortex, in PACAP−/− mice. In the PVN of PACAP−/− mice, the stress-induced c-Fos expression was blunted in the CRF neurons. These results suggest that PACAP is critically involved in activation of the MeA and PVN CRF neurons to centrally regulate the HPA axis response to emotional stressors

Ultrasensitive Imaging of Ca2+ Dynamics in Pancreatic Acinar Cells of Yellow Cameleon-Nano Transgenic Mice

Yellow Cameleons are genetically encoded Ca2+ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca2+-sensor probe. To achieve ultrasensitive Ca2+ imaging for low resting Ca2+ or small Ca2+ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca2+ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca2+ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca2+ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca2+ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca2+ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from Rmax and Rmin values under in vivo condition. The mice will be useful for ultrasensitive Ca2+ imaging in vivo

代替授業実践報告からみる保育者養成課程における英語教材に関する考察

020年4～5月にかけて行われた、保育者養成課程における英語履修者に対して行ったオンライン授業（代替授業）の概要、及び代替授業後のアンケートを行った結果を考察した。また、実際に使用したオンライン教材の概要についてまとめた

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

Analysis of Oral and Gut Microbiome Composition and Its Impact in Patients with Oral Squamous Cell Carcinoma

The impact of gut and oral microbiota on the clinical outcomes of patients with oral squamous cell carcinoma (OSCC) is unknown. We compared the bacterial composition of dental plaque and feces between patients with OSCC and healthy controls (HCs). Fecal and dental plaque samples were collected from 7 HCs and 18 patients with OSCC before treatment initiation. Terminal restriction fragment-length polymorphism analysis of 16S rRNA genes was performed. Differences in bacterial diversity between the HC and OSCC groups were examined. We compared the occupancy of each bacterial species in samples taken from patients with OSCC and HCs and analyzed the correlation between PD-L1 expression in the tumor specimens and the occupancy of each bacterial species. The gut and oral microbiota of patients with OSCC were more varied than those of HCs. Porphyromonas and Prevotella were significantly more abundant in patients with OSCC than in HCs. The abundance of Clostridium subcluster XIVa in the gut microbiota of the PD-L1-positive group was significantly greater than that in the PD-L1-negative group. The oral and gut microbiomes of patients with OSCC were in a state of dysbiosis. Our results suggest the possibility of new cancer therapies targeting these disease-specific microbiomes using probiotics and synbiotics

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