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

Abstract

Introduction: The 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.

Methods: Ischemic 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.

Results: Our 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.

Conclusions: This study provides a detailed inventory of PACAP influenced gene expressions and protein targets in mice ischemic brain, and suggests new targets for thereaupetic interventions.

Figure 1

The experimental outline and workflow. Effect of intracerebroventricular administration of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 into the ischemic mouse brain (permanent middle cerebral artery occlusion, PMCAO model) is evaluated at the molecular level in the ipsilateral (right) hemisphere. Sham control treated with saline is used for the comparison. TTC staining shows he ischemic region in the brain. The ipsilateral hemisphere is sampled and finely powdered in liquid nitrogen, followed by investigation into molecular level changes at the level of gene and protein expressions by DNA microarray and proteomics approaches, respectively.

Figure 2

The mRNA expression profiles of differentially expressed genes. Both the upregulated and downregulated genes were selected randomly. Gel images on top show the polymerase chain reaction (PCR) product bands stained with ethidium bromide; the band intensities are also presented graphically below for clarity. Lane numbers 1 to 8 indicate sham control (lanes 1, 3, 5, and 7) and permanent middle cerebral artery occlusion (PMCAO) treatment (lanes 2, 4, 6, and 8), respectively. P indicates pituitary adenylate cyclase-activating polypeptide (PACAP) treatment; C is the control (minus PACAP). GAPDH and beta-actin genes were used a positive control. Semi-quantitative RT-PCR was performed as described in Methods, and the specific 3’-UTR primers are detailed in Additional file 2: Table S2.

Figure 3

Pathway and disease states-focused gene classification of pituitary adenylate cyclase-activating polypeptide (PACAP)- influenced genes at 6 h post-ischemia. The up- and downregulated genes at 6 h hours after ischemia (ipsilateral hemisphere) were classified based on the available categories of more than 100 biological pathways or specific disease states in the SABiosciences PCR array list (QIAGEN; http://www.sabiosciences.com) for Mus musculus. The numbers in the y-axis represent the number of genes in each category, which are indicated on the x-axis.

Figure 4

Pathway and disease states-focused gene classification of pituitary adenylate cyclase-activating polypeptide (PACAP)-influenced genes at 24 h post-ischemia. The up- and downregulated genes at 24 h hours after ischemia (ipsilateral hemisphere) were classified based on the available categories of more than 100 biological pathways or specific disease states the same as mentioned in Figure 3 in the SABiosciences PCR array list (QIAGEN; http://www.sabiosciences.com) for Mus musculus. The numbers in the y-axis represent the number of genes in each category, which are indicated on the x-axis.

Figure 5

Two-dimensional gel electrophoresis of the mouse brain. (A) Total protein in the Sham (control) and PMCAO (ischemic) hemispheres were stained with Flamingo stain. (A) Separated proteins at 6 h after control and ischemia treatments, with (gels on right-hand side) or without PACAP38 (gels on left-hand side), respectively. (B) Separated proteins in the same profile as above at 24 h. Newly appearing protein/s in the ischemic hemispheres are indicated by the red dotted line circles, and the green dotted line circles represent the corresponding areas of the saline and PACAP samples. Inset: enlarged circle protein profiles. Total protein extraction, separation, staining, and image analyses procedures are detailed in Methods.

Figure 6

Western blot analysis of the CRMP2 protein cross-reacting proteins in sham control and permanent middle cerebral artery occlusion (PMCAO) with or without pituitary adenylate cyclase-activating polypeptide (PACAP) treatment. Proteins cross-reacting with the anti-CRMP2 protein antibody are visible as three constitutively present proteins (approximately 70, 65, and 63 kDa size) in all samples. The approximately 56 kDa cross-reacting protein is seen only in the ischemic hemisphere (PMCAO). Lanes 1, 2 and 5, 6 are hemispheres dissected out at 6 h after control and ischemia treatments respectively. The symbol – (minus) indicates without PACAP38, that is, 1 μL of 0.9% saline, whereas + (plus) indicates with 1 pmol (1 μL) of PACAP38 injection. Total protein extraction, separation, Western blotting and image analyses procedures are detailed in Methods. Total protein in the Sham (control) and PMCAO (ischemic) hemispheres were stained with Flamingo stain as shown in Additional file 6: Figure S4.

Figure 7

Immunofluorescent staining. Neurons express CRMP2 at 6 and 24 h after permanent middle cerebral artery occlusion (PMCAO). A,B,C: triple immunofluorescence staining for CRMP2, NeuN and 4′, 6-diamidine-2-phenylindole dihydrochloride (DAPI); the merged picture displays expression of CRMP2 by NeuN positive neuron but not DAPI-positive neurons. D,G,J,M,P: healthy; E,H,K,N,Q: penumbra; and F,I,L,O,R: core. D-R: double immunofluorescence staining for CRMP2, and DAPI; the merged picture shows mutually exclusive expression of neuron. The merged picture shows membranous localization of CRMP2 surrounding DAPI-positive nuclei. All images were captured in the ipsilateral hippocampus. Intact (double): A, B, C; 6 h saline: D, E, F; 6-h pituitary adenylate cyclase-activating polypeptide (PACAP): G, H, I; 24 h saline: J, K, L; 24 h PACAP: M, N, O; negative control: P, Q, R. Green - NeuN, Red - CRMP2, and Blue - DAPI. Scale bar: 20 μm. The sections were prepared as described in Methods; see also Additional file 8: Figure S6.