Gs共役受容体GPR3の細胞内動態は小脳顆粒神経細胞内におけるPKAの局所活性に寄与する

内容の要旨 , 審査の要旨広島大学(Hiroshima University)博士(医学)Doctor of Philosophy in Medical Sciencedoctora

The role of glucocorticoid receptors in the induction and prevention of hippocampal abnormalities in an animal model of posttraumatic stress disorder

Rationale: Since the precise mechanisms of posttraumatic stress disorder (PTSD) remain unknown, effective treatment interventions have not yet been established. Numerous clinical studies have led to the hypothesis that elevated glucocorticoid levels in response to extreme stress might trigger a pathophysiological cascade which consequently leads to functional and morphological changes in the hippocampus. Objectives: To elucidate the pathophysiology of PTSD, we examined the alteration of hippocampal gene expression through the glucocorticoid receptor (GR) in the single prolonged stress (SPS) paradigm, a rat model of PTSD. Methods: We measured nuclear GRs by western blot, and the binding of GR to the promoter of Bcl-2 and Bax genes by chromatin immunoprecipitation-qPCR as well as the expression of these 2 genes by RT-PCR in the hippocampus of SPS rats. In addition, we examined the preventive effects of a GR antagonist on SPS-induced molecular, morphological, and behavioral alterations (hippocampal gene expression of Bcl-2 and Bax, hippocampal apoptosis using TUNEL staining, impaired fear memory extinction (FME) using the contextual fear conditioning paradigm). Results: Exposure to SPS increased nuclear GR expression and GR binding to Bcl-2 gene, and decreased Bcl-2 mRNA expression. Administration of GR antagonist immediately after SPS prevented activation of the glucocorticoid cascade, hippocampal apoptosis, and impairment FME in SPS rats. Conclusion: The activation of GRs in response to severe stress may trigger the pathophysiological cascade leading to impaired FME and hippocampal apoptosis. In contrast, administration of GR antagonist could be useful for preventing the development of PTSD.This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (a grant-in aid for Scientific Research, C) Grant Number JP18K07562, and Takeda Science Foundation

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed “The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research” based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap‐frozen samples should be stored in liquid nitrogen (about −180°C) until use. When intending to use genomic DNA extracted from formalin‐fixed paraffin‐embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine

The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons.

G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation

The subcellular distribution of fluorescently tagged GPR3 in CGNs.

<p>(A) We employed a GPR3-HT expression vector to analyze the localization of GPR3. After transfecting the GPR3-HT vector into the CGNs, the cells were labeled with the TMR HaloTag ligand at forty-eight hours after transfection. A representative image was shown. (B) To evaluate the localization of GPR3 at the plasma membrane, the CGNs were co-transfected with the GPR3-HT and YFP-mem expression vectors, followed by TMR HaloTag ligand labeling. YFP-mem fluorescence was shown in the green pseudo-color to easily visualize the co-localization. (C-F) To evaluate the localization of GPR3 in the endoplasmic reticulum, Golgi body, and endosomes, the GPR3-mAGFL expression vector was transfected into the CGNs. Forty-eight hours after transfection, the cells were immunostained with various organelle markers: anti-KDEL antibody (endoplasmic reticulum), WGA633 (Golgi body), anti-Rab5 antibody (early endosome), and anti-Rab7 antibody (late endosome). Co-localization of GPR3 with the organelle markers was evaluated by confocal microscopy using built-in Z-stack analysis software. Scale bar = 10 μm.</p

The local PKA activity was modulated by the expression of GPR3 in the CGNs.

<p>(A-B) The local PKA activity was compared between the soma and neurite tips in GPR3-transfected CGNs. The CGNs were transfected with Mock+control siRNA, pGPR3-HT+control siRNA, Mock+GPR3 siRNA, and pGPR3-HT+GPR3 siRNA, respectively. Forty-eight hours after transfection, the FRET/CFP images were captured using a fluorescent microscope. After capturing the images, the FRET/CFP intensity ratio was calculated and compared between the soma and neurite tips. (A) Representative FRET/CFP images from CGNs in each condition were shown (left raw). Magnifications of neurite tip were also shown (right raw) (B) The ratios of the PKA activity (tip to soma) in each condition were analyzed identically. The data represent the means ± SEM for each condition (n = 7). The asterisk (*) represents p < 0.005. (C-D) The PKA activity at the neurite tips was analyzed in CGNs transfected with Mock+control siRNA, pGPR3-HT+control siRNA, Mock+GPR3 siRNA, and pGPR3-HT+GPR3 siRNA, respectively. Forty-eight hours after transfection, the FRET/CFP images were captured using a fluorescent microscope. After capturing the images, the cells were treated with 1 mM dbcAMP for 15 min, and FRET/CFP images were captured again in the same cell to evaluated fully activated PKA. (C) Representative FRET/CFP images from the neurite tips of CGNs in each condition were shown. (D) The FRET/CFP ratio in each group were analyzed identically. The FRET/CFP ratios are expressed as the percentage of the maximum level stimulated by 1 mM dbcAMP in each cell. The data represent the means ± SEM for each condition (n = 9). The asterisk (*) represents p < 0.005.</p

Fluorescence time-lapse imaging of a single GPR3-HT-expressing CGN.

<p>(A-B) The CGNs were transfected with the pGPR3-HT expression vector and were plated onto 0.03% PEI-coated plates. Forty-five hours after transfection, GPR3-HT was labeled with the TMR HaloTag ligand for 15 min. After three sequential washes in PBS, the cells were incubated with various inhibitors for another two hours. The cells were treated with 1 μM Latrunculin B, 3 μM Nocodazole, 50 μM Blebbistatin, or 2 mM Monastrol in the culture medium, respectively. As a control, a 1:1000 dilution of DMSO was added to the culture medium. The pGPR3-HT fluorescence in the CGNs was captured every 10 minutes using a fluorescent microscope. (A) Representative images of the CGNs from each condition were shown. The arrow indicates the GPR3 fluorescent puncta at each time point. Serial images were shown every 20 min. (B) The directions that the GPR3 puncta moved along the neurite were analyzed in each condition. The directions of puncta movement were divided into three groups: the plus end movement group, the minus end movement group, and the immobile group. The values were then expressed as the percentage of the number of puncta in each direction out of the total numbers of puncta. The numbers of puncta in plus end direction were significantly decreased when Latrunculin B, Nocodazole, Blebbistatin were included in the medium. The data represent the means ± SEM for each condition (n = 16). The asterisk (*) represents p < 0.0001 and the double asterisk (**) represents p < 0.05. (C) The speed of the GPR3 puncta were calculated from the time-lapse images. The mean speed of the GPR3 puncta in plus-end and minus-end directions were separately shown (n = 25). (D-E) The fluorescent intensity of GPR3 in the membranes at the neurite tips were evaluated with or without addition of various actin and tubulin inhibitors. The CGNs were co-transfected with pGPR3-HT and pYFP-mem. Twenty-four hours after transfection, some groups were treated with inhibitors as described above. Forty-six hours after transfection, the cells were labeled with the TMR HaloTag ligand for 2 hours. The cells were then fixed and the fluorescent images of GPR3-HT and YFP-mem were captured using a fluorescent microscope from the cells in each condition. The fluorescent intensity of GPR3 in the membranes at the neurite tips were evaluated by the line profiling method (detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147466#sec002" target="_blank">Materials and methods</a>). (D) Representative images of the CGNs and the line profiling analyses from each condition were shown. (E) The fluorescent intensities of GPR3-HT in the plasma membrane and cytosol at the neurite tips were compared for each condition. The values were then expressed as the ratio of the fluorescent intensity at the plasma membrane to the cytosol at the neurite tips. The data represent the means ± SEM for each condition (n = 7). The asterisk (*) represents p < 0.0001.</p