Distribution and protective function of pituitary adenylate cyclase-activating polypeptide in the retina

Pituitary adenylate cyclase-activating polypeptide (PACAP), which is found in 27- or 38-amino acid forms, belongs to the VIP/glucagon/secretin family. PACAP and its three receptor subtypes are expressed in neural tissues, with PACAP known to exert a protective effect against several types of neural damage. The retina is considered to be part of the central nervous system, and retinopathy is a common cause of profound and intractable loss of vision. This review will examine the expression and morphological distribution of PACAP and its receptors in the retina, and will summarize the current state of knowledge regarding the protective effect of PACAP against different kinds of retinal damage, such as that identified in association with diabetes, ultraviolet light, hypoxia, optic nerve transection, and toxins. This article will also address PACAP-mediated protective pathways involving retinal glial cells

Expression Patterns of PACAP and PAC1R Genes and Anorexigenic Action of PACAP1 and PACAP2 in Zebrafish

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with potent suppressive effects on feeding behavior in rodents, chicken, and goldfish. Teleost fish express two PACAPs (PACAP1, encoded by the adcyap1a gene, and PACAP2, encoded by the adcyap1b gene) and two PACAP receptors (PAC1Rs; PAC1Ra, encoded by the adcyap1r1a gene, and PAC1Rb, encoded by the adcyap1r1b gene). However, the mRNA expression patterns of the two PACAPs and PAC1Rs, and the influence and relationship of the two PACAPs on feeding behavior in teleost fish remains unclear. Therefore, we first examined mRNA expression patterns of PACAP and PAC1R in tissue and brain. All PACAP and PAC1Rs mRNAs were dominantly expressed in the zebrafish brain. However, adcyap1a mRNA was also detected in the gut and testis. In the brain, adcyap1b and adcyap1r1a mRNA levels were greater than that of adcyap1a and adcyap1r1b, respectively. Moreover, adcyap1b and adcyap1r1a mRNA were dominantly expressed in telencephalon and diencephalon. The highest adcyap1a mRNA levels were detected in the brain stem and diencephalon, while the highest levels of adcyap1r1b were detected in the cerebellum. To clarify the relationship between PACAP and feeding behavior in the zebrafish, the effects of zebrafish (zf) PACAP1 or zfPACAP2 intracerebroventricular (ICV) injection were examined on food intake, and changes in PACAP mRNA levels were assessed against feeding status. Food intake was significantly decreased by ICV injection of zfPACAP1 (2 pmol/g body weight), zfPACAP2 (2 or 20 pmol/g body weight), or mammalian PACAP (2 or 20 pmol/g). Meanwhile, the PACAP injection group did not change locomotor activity. Real-time PCR showed adcyap1 mRNA levels were significantly increased at 2 and 3 h after feeding compared with the pre-feeding level, but adcyap1b, adcyap1r1a, and adcyap1r1b mRNA levels did not change after feeding. These results suggest that the expression levels and distribution of duplicated PACAP and PAC1R genes are different in zebrafish, but the anorexigenic effects of PACAP are similar to those seen in other vertebrates

Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury

<p>Abstract</p> <p>Background</p> <p>We hypothesized that gp91^phox(NOX2), a subunit of NADPH oxidase, generates superoxide anion (O₂^-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91^phoxand reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91^phoxknockout mice (gp91^phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91^phoxgeneration.</p> <p>Methods</p> <p>Unilateral TBI was induced in gp91^phox-/-and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91^phoxafter TBI were investigated using immunoblotting and staining techniques. Levels of O₂^-and peroxynitrite were determined <it>in situ </it>in the mouse brain. The activated phenotype in microglia that expressed gp91^phoxwas determined in a microglial cell line, BV-2, in the presence of IFNγ or IL-4.</p> <p>Results</p> <p>Gp91^phoxexpression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O₂^-and peroxynitrite metabolites produced were less in gp91^phox-/-mice than in Wt. In the presence of IFNγ, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91^phox.</p> <p>Conclusions</p> <p>Classical activated microglia promote ROS formation through gp91^phoxand have an important role in brain damage following TBI. Modulating gp91^phoxand gp91^phox-derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.</p

Discovery of PACAP and its receptors in the brain

Abstract Pituitary adenylate-cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which belongs to the vasoactive intestinal polypeptide (VIP)/glucagon/secretin family. PACAP shows particularly high homology (~ 68%) to VIP. Because of the high homology of the amino acid sequences of PACAP and VIP, these peptides share three class B-G-protein coupled receptors: the PAC1-Receptor (PAC1-R), the VPAC1-Receptor (VPAC1-R) and VPAC2-Receptor (VPAC2-R). These receptors have high homology to each other, and their high homology is utilized for these discoveries. This review provides mainly an overview of the history of the discovery of PACAP and its three receptors

Comprehensive Analysis of Neonatal versus Adult Unilateral Decortication in a Mouse Model Using Behavioral, Neuroanatomical, and DNA Microarray Approaches

Previously, studying the development, especially of corticospinal neurons, it was concluded that the main compensatory mechanism after unilateral brain injury in rat at the neonatal stage was due in part to non-lesioned ipsilateral corticospinal neurons that escaped selection by axonal elimination or neuronal apoptosis. However, previous results suggesting compensatory mechanism in neonate brain were not correlated with high functional recovery. Therefore, what is the difference among neonate and adult in the context of functional recovery and potential mechanism(s) therein? Here, we utilized a brain unilateral decortication mouse model and compared motor functional recovery mechanism post-neonatal brain hemisuction (NBH) with adult brain hemisuction (ABH). Three analyses were performed: (1) Quantitative behavioral analysis of forelimb movements using ladder walking test; (2) neuroanatomical retrograde tracing analysis of unlesioned side corticospinal neurons; and (3) differential global gene expressions profiling in unlesioned-side neocortex (rostral from bregma) in NBH and ABH on a 8 × 60 K mouse whole genome Agilent DNA chip. Behavioral data confirmed higher recovery ability in NBH over ABH is related to non-lesional frontal neocortex including rostral caudal forelimb area. A first inventory of differentially expressed genes genome-wide in the NBH and ABH mouse model is provided as a resource for the scientific community

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

PACAP

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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

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)