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

Hori M, Nakamachi T, Rakwal R, Shibato J, Ogawa T, Aiuchi T, Tsuruyama T, Tamaki K, Shioda S - J Neuroinflammation (2012)

Bottom Line: Our results revealed numerous changes in the transcriptome of ischemic hemisphere (ipsilateral) treated with PACAP38 compared to the saline-injected SHAM control hemisphere (contralateral).In parallel, 2-DGE analysis revealed a highly expressed protein spot in the ischemic hemisphere that was identified as dihydropyrimidinase-related protein 2 (DPYL2).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Forensic Medicine and Molecular Pathology, School of Medicine, Kyoto University, Kyoto 606-8315, Japan.

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.

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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.
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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.

Mentions: Total soluble protein profiles were first examined by one-dimensional gel electrophoresis (SDS-PAGE). Staining the separated proteins with Flamingo fluorescent stain showed no significant differences in polypeptide patterns among all the samples examined (Additional file 6: Figure S4). Therefore, we proceeded to two-dimensional analysis. Flamingo-stained two-dimensional gel protein spots clearly revealed a major and newly appeared spot with a molecular weight of approximately 60 kDa and a pI of 5.5, in the ischemic hemisphere at 6 h (lower left two-dimensional gel image, Figure 5A) over the sham saline sample; this spot was also observed in the PACAP38-treated ischemic brain (lower right two-dimensional gel, Figure 5A) over the sham PACAP sample. Interestingly, the same spot was also observed at a much stronger intensity (more than 2-fold) in the ischemic hemisphere at 24 h (lower left two dimensional gel image, Figure 5B) but not in the sham saline sample. Two additional minor spots appeared at approximately the same molecular weight range but with a slightly acidic pI (isoelectric point). Surprisingly, and in contrast to the 6 h results, the major and minor spots were not found in the PACAP38-treated brain (lower right two-dimensional gel image, Figure 5B) over the sham PACAP sample. To identify the protein in these newly appearing spots, we excised these three spots from the stained gels and after in-gel trypsin digestion analyzed the peptides by MALDI-TOF-MS, as described in Methods. The MASCOT search revealed the major abundant protein to be a dihydropyrimidinase-related protein 2 (DPYL2_MOUSE) or collapsin response mediator protein 2 (CRMP2_MOUSE). No significant data (protein IDs) could be obtained for the two protein spots with the lowest abundance appearing exclusively at 24 h post ischemia. The CRMP2 protein, as it more popularly known, is involved in axonal growth and neuronal differentiation [17]. Although there is no information to date on the effects of PACAP on the CRMP2 protein, a few studies have already shown that the CRMP2 protein is induced in the brain after focal cerebral ischemia, in old mouse brains, and in the cerebral cortex of rats [38-40].


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

Hori M, Nakamachi T, Rakwal R, Shibato J, Ogawa T, Aiuchi T, Tsuruyama T, Tamaki K, Shioda S - J Neuroinflammation (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526409&req=5

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.
Mentions: Total soluble protein profiles were first examined by one-dimensional gel electrophoresis (SDS-PAGE). Staining the separated proteins with Flamingo fluorescent stain showed no significant differences in polypeptide patterns among all the samples examined (Additional file 6: Figure S4). Therefore, we proceeded to two-dimensional analysis. Flamingo-stained two-dimensional gel protein spots clearly revealed a major and newly appeared spot with a molecular weight of approximately 60 kDa and a pI of 5.5, in the ischemic hemisphere at 6 h (lower left two-dimensional gel image, Figure 5A) over the sham saline sample; this spot was also observed in the PACAP38-treated ischemic brain (lower right two-dimensional gel, Figure 5A) over the sham PACAP sample. Interestingly, the same spot was also observed at a much stronger intensity (more than 2-fold) in the ischemic hemisphere at 24 h (lower left two dimensional gel image, Figure 5B) but not in the sham saline sample. Two additional minor spots appeared at approximately the same molecular weight range but with a slightly acidic pI (isoelectric point). Surprisingly, and in contrast to the 6 h results, the major and minor spots were not found in the PACAP38-treated brain (lower right two-dimensional gel image, Figure 5B) over the sham PACAP sample. To identify the protein in these newly appearing spots, we excised these three spots from the stained gels and after in-gel trypsin digestion analyzed the peptides by MALDI-TOF-MS, as described in Methods. The MASCOT search revealed the major abundant protein to be a dihydropyrimidinase-related protein 2 (DPYL2_MOUSE) or collapsin response mediator protein 2 (CRMP2_MOUSE). No significant data (protein IDs) could be obtained for the two protein spots with the lowest abundance appearing exclusively at 24 h post ischemia. The CRMP2 protein, as it more popularly known, is involved in axonal growth and neuronal differentiation [17]. Although there is no information to date on the effects of PACAP on the CRMP2 protein, a few studies have already shown that the CRMP2 protein is induced in the brain after focal cerebral ischemia, in old mouse brains, and in the cerebral cortex of rats [38-40].

Bottom Line: Our results revealed numerous changes in the transcriptome of ischemic hemisphere (ipsilateral) treated with PACAP38 compared to the saline-injected SHAM control hemisphere (contralateral).In parallel, 2-DGE analysis revealed a highly expressed protein spot in the ischemic hemisphere that was identified as dihydropyrimidinase-related protein 2 (DPYL2).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Forensic Medicine and Molecular Pathology, School of Medicine, Kyoto University, Kyoto 606-8315, Japan.

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.

Show MeSH
Related in: MedlinePlus