Motohide Hori

Unraveling the ischemic brain transcriptome in a permanent middle cerebral artery occlusion mouse model by DNA microarray analysis

SUMMARY Brain ischemia, also termed cerebral ischemia, is a condition in which there is insufficient blood flow to the brain to meet metabolic demand, leading to tissue death (cerebral infarction) due to poor oxygen supply (cerebral hypoxia). Our group is interested in the protective effects of neuropeptides for alleviating brain ischemia, as well as the underlying mechanisms of their action. The present study was initiated to investigate molecular responses at the level of gene expression in ischemic brain tissue. To achieve this, we used a mouse permanent middle cerebral artery occlusion (PMCAO) model in combination with high-throughput DNA microarray analysis on an Agilent microarray platform. Briefly, the right (ipsilateral) and left (contralateral) hemispheres of PMCAO model mice were dissected at two time points, 6 and 24 hours post-ischemia. Total RNA from the ischemic (ipsilateral) hemisphere was subjected to DNA microarray analysis on a mouse whole genome 4x44K DNA chip using a dye-swap approach. Functional categorization using the gene ontology (GO, MGD/AMIGO) of numerous changed genes revealed expression pattern changes in the major categories of cellular process, biological regulation, regulation of biological process, metabolic process and response to stimulus. Reverse-transcriptase PCR (RT-PCR) analysis on randomly selected highly up- or downregulated genes validated, in general, the microarray data. Using two time points for this analysis, major and minor trends in gene expression and/or functions were observed in relation to early- and late-response genes and differentially regulated genes that were further classified into specific pathways or disease states. We also examined the expression of these genes in the contralateral hemisphere, which suggested the presence of bilateral effects and/or differential regulation. This study provides the first ischemia-related transcriptome analysis of the mouse brain, laying a strong foundation for studies designed to elucidate the mechanisms regulating ischemia and to explore the neuroprotective effects of agents such as target neuropeptides.

PACAP Attenuates NMDA-Induced Retinal Damage in Association with Modulation of the Microglia/Macrophage Status into an Acquired Deactivation Subtype

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been known as a neuroprotectant agent in several retinal injury models. However, a detailed mechanism of this effect is still not well understood. In this study, we examined the retinoprotective effects and associated underlying mechanisms of action of PACAP in the mouse N-methyl-d-aspartic acid (NMDA)-induced retinal injury model, focusing on the relationship between PACAP and retinal microglia/macrophage (MG/MΦ) status. Adult male C57BL/6 mice received an intravitreal injection of NMDA to induce retinal injury. Three days after NMDA injection, the number of MG/MΦ increased significantly in the retinas. The concomitant intravitreal injection of PACAP suppressed NMDA-induced cell loss in the ganglion cell layer (GCL) and significantly increased the number of MG/MΦ. These outcomes associated with PACAP were attenuated by cotreatment with PACAP6-38, while the beneficial effects of PACAP were not seen in interleukin-10 (IL-10) knockout mice. PACAP significantly elevated the messenger RNA levels of anti-inflammatory cytokines such as transforming growth factor beta 1 and IL-10 in the injured retina, with the immunoreactivities seen to overlap with markers of MG/MΦ. These results suggest that PACAP enhances the proliferation and/or infiltration of retinal MG/MΦ and modulates their status into an acquired deactivation subtype to favor conditions for neuroprotection.

Transcriptomics and proteomics analyses of the PACAP38 influenced ischemic brain in permanent middle cerebral artery occlusion model mice

IntroductionThe neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is considered to be a potential therapeutic agent for prevention of cerebral ischemia. Ischemia is a most common cause of death after heart attack and cancer causing major negative social and economic consequences. This study was designed to investigate the effect of PACAP38 injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with corresponding SHAM control that used 0.9% saline injection.MethodsIschemic and non-ischemic brain tissues were sampled at 6 and 24 hours post-treatment. Following behavioral analyses to confirm whether the ischemia has occurred, we investigated the genome-wide changes in gene and protein expression using DNA microarray chip (4x44K, Agilent) and two-dimensional gel electrophoresis (2-DGE) coupled with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), respectively. Western blotting and immunofluorescent staining were also used to further examine the identified protein factor.ResultsOur results revealed numerous changes in the transcriptome of ischemic hemisphere (ipsilateral) treated with PACAP38 compared to the saline-injected SHAM control hemisphere (contralateral). Previously known (such as the interleukin family) and novel (Gabra6, Crtam) genes were identified under PACAP influence. In parallel, 2-DGE analysis revealed a highly expressed protein spot in the ischemic hemisphere that was identified as dihydropyrimidinase-related protein 2 (DPYL2). The DPYL2, also known as Crmp2, is a marker for the axonal growth and nerve development. Interestingly, PACAP treatment slightly increased its abundance (by 2-DGE and immunostaining) at 6 h but not at 24 h in the ischemic hemisphere, suggesting PACAP activates neuronal defense mechanism early on.ConclusionsThis study provides a detailed inventory of PACAP influenced gene expressions and protein targets in mice ischemic brain, and suggests new targets for thereaupetic interventions.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Stimulates Proliferation of Reactive Astrocytes In Vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalamus. Recently, we have shown that the PACAP receptor (PAC1-R) is expressed in reactive astrocytes following an in vivo stub wound brain injury. However, the functional role of PACAP has not yet been clarified. In order to investigate the effect of PACAP on the proliferation of reactive astrocytes, a scratch wound paradigm was applied to astrocytic monolayers. Following injury, there was an increase in PAC1-R and glial fibrillary acidic protein (GFAP) immunoreactivity in the astrocytes surrounding the scratch line. PACAP at concentrations of 10−15 to 10−7 M was applied immediately after scratching, and the proliferating astrocytes were visualized by multiple immunofluorescence labeling. The percentage of cells that colabeled for Ki67 (a marker of proliferating cells) and GFAP increased in the 10−11- and 10−13-M PACAP-treated groups. The proliferating astrocytes induced by PACAP treatment mainly occurred in the proximal wound area where many reactive astrocytes were observed. Pretreatment with the PACAP receptor antagonist PACAP6-38 significantly suppressed the PACAP-induced effects. These results strongly suggest that PACAP plays an important role in the proliferation of reactive astrocytes following nerve injury.

Distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) in the human testis and in testicular germ cell tumors.

Pituitary adenylate cyclase‐activating peptide (PACAP) is a neuropeptide expressed in the central nervous system and peripheral organs. Previous studies revealed the role and distribution of PACAP in the rodent testis, however, its presence in the human testis and in testicular germ cell tumors is not known. We used RT‐PCR and immunohistological observations to investigate whether human testicular tissue and testicular germ cell tumors contain PACAP. The mRNAs for PACAP and its receptors were detected in total RNA extracted from human testes. PACAP immunoreactivity was observed in spermatogonia and spermatids from normal testes. In contrast, diffuse PACAP immunopositivity was observed in seminoma tumor cells, while only faint immunoreactivity was observed in embryonal carcinoma cells. Our data suggest that PACAP may play a role in human spermatogenesis and in testicular germ cell tumor development.

Two-color Dye-swap DNA Microarray approach toward confident gene expression profiling in PMCAO mouse model for ischemia-related and PACAP38-influenced genes.

Toward twin goals of identifying molecular factors in brain injured by ischemic stroke, and the effects of neuropeptide pituitary adenylate-cyclase activating polypeptide (PACAP) on the ischemic brain, we have established the permanent middle cerebral artery occlusion (PMCAO) mouse model and utilized the Agilent mouse whole genome 4 × 44 K DNA chip. PACAP38 (1 pmol) injection was given intracerebroventrically in comparison to a control saline (0.9% NaCl) injection, to screen genes responsive to PACAP38. Two sets of tissues were prepared, whole hemispheres (ischemic and non-ischemic) and infract core and penumbra regions at 6 and 24 h. In this study, we have detailed the experimental design and protocol used therein and explained the quality controls for the use of total RNA in the downstream DNA microarray experiment utilizing a two-color dye-swap approach for stringent and confident gene identification published in a series of papers by Hori and coworkers (Hori et al., 2012–2015).

PACAP38 differentially effects genes and CRMP2 protein expression in ischemic core and penumbra regions of permanent middle cerebral artery occlusion model mice brain.

Pituitary adenylate-cyclase activating polypeptide (PACAP) has neuroprotective and axonal guidance functions, but the mechanisms behind such actions remain unclear. Previously we examined effects of PACAP (PACAP38, 1 pmol) injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with control saline (0.9% NaCl) injection. Transcriptomic and proteomic approaches using ischemic (ipsilateral) brain hemisphere revealed differentially regulated genes and proteins by PACAP38 at 6 and 24 h post-treatment. However, as the ischemic hemisphere consisted of infarct core, penumbra, and non-ischemic regions, specificity of expression and localization of these identified molecular factors remained incomplete. This led us to devise a new experimental strategy wherein, ischemic core and penumbra were carefully sampled and compared to the corresponding contralateral (healthy) core and penumbra regions at 6 and 24 h post PACAP38 or saline injections. Both reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to examine targeted gene expressions and the collapsin response mediator protein 2 (CRMP2) protein profiles, respectively. Clear differences in expression of genes and CRMP2 protein abundance and degradation product/short isoform was observed between ischemic core and penumbra and also compared to the contralateral healthy tissues after PACAP38 or saline treatment. Results indicate the importance of region-specific analyses to further identify, localize and functionally analyse target molecular factors for clarifying the neuroprotective function of PACAP38.

2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima

The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves.

Unraveling the Specific Ischemic Core and Penumbra Transcriptome in the Permanent Middle Cerebral Artery Occlusion Mouse Model Brain Treated with the Neuropeptide PACAP38

Our group has been systematically investigating the effects of the neuropeptide pituitary adenylate-cyclase activating polypeptide (PACAP) on the ischemic brain. To do so, we have established and utilized the permanent middle cerebral artery occlusion (PMCAO) mouse model, in which PACAP38 (1 pmol) injection is given intracerebroventrically and compared to a control saline (0.9% sodium chloride, NaCl) injection, to unravel genome-wide gene expression changes using a high-throughput DNA microarray analysis approach. In our previous studies, we have accumulated a large volume of data (gene inventory) from the whole brain (ipsilateral and contralateral hemispheres) after both PMCAO and post-PACAP38 injection. In our latest research, we have targeted specifically infarct or ischemic core (hereafter abbreviated IC) and penumbra (hereafter abbreviated P) post-PACAP38 injections in order to re-examine the transcriptome at 6 and 24 h post injection. The current study aims to delineate the specificity of expression and localization of differentially expressed molecular factors influenced by PACAP38 in the IC and P regions. Utilizing the mouse 4 × 44 K whole genome DNA chip we show numerous changes (≧/≦ 1.5/0.75-fold) at both 6 h (654 and 456, and 522 and 449 up- and down-regulated genes for IC and P, respectively) and 24 h (2568 and 2684, and 1947 and 1592 up- and down-regulated genes for IC and P, respectively) after PACAP38 treatment. Among the gene inventories obtained here, two genes, brain-derived neurotrophic factor (Bdnf) and transthyretin (Ttr) were found to be induced by PACAP38 treatment, which we had not been able to identify previously using the whole hemisphere transcriptome analysis. Using bioinformatics analysis by pathway- or specific-disease-state focused gene classifications and Ingenuity Pathway Analysis (IPA) the differentially expressed genes are functionally classified and discussed. Among these, we specifically discuss some novel and previously identified genes, such as alpha hemoglobin stabilizing protein (Ahsp), cathelicidin antimicrobial peptide (Camp), chemokines, interferon beta 1 (Ifnb1), and interleukin 6 (Il6) in context of PACAP38-mediated neuroprotection in the ischemic brain. Taken together, the DNA microarray analysis provides not only a great resource for further study, but also reinforces the importance of region-specific analyses in genome-wide identification of target molecular factors that might play a role in the neuroprotective function of PACAP38.

Expression and Distribution of Pituitary Adenylate Cyclase-Activating Polypeptide Receptor in Reactive Astrocytes Induced by Global Brain Ischemia in Mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neuroprotectant. We previously showed that PACAP receptor (PAC1R) immunoreactivity was elevated in reactive astrocytes after stab wound injury. However, the pattern of PAC1R expression in astrocytes after brain injury is still unknown. In this study, PAC1R expression was evaluated in mouse hippocampal astrocytes after bilateral common carotid artery occlusion. PAC1R mRNA levels in the hippocampus peaked on day 7, and glial fibrillary acidic protein (GFAP) mRNA levels increased from day 3 to day 7 after ischemia. We then observed co-localization of PAC1R and GFAP by double immunostaining. GFAP-immunopositive cells showed signs of hypertrophy 3 days after the ischemia, and by day 7 had fine processes, were hypertrophied, and are known as reactive astrocytes. A low number of PAC1R-immunopositive astrocytes were detectable in the hippocampal area until 3 days after ischemia. PAC1R-positive astrocytes were widely distributed in the hippocampus between day 7 and day 14 after ischemia, and they were converging around the damaged CA1 pyramidal cell layer by day 28. These results suggest that PAC1R might be expressed in the middle to late stage of reactive astrocytes and PACAP plays an important role in the reactive astrocytes after brain injury.