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Quantification of Protein Expression Changes in the PKC‐epsilon/Ca2+/CaMKII‐alpha Pathway after Early and Late Onset of Ischemic‐reperfusion Injury in Adult Male Rats
Author(s) -
Bauza John M.,
CarsonMarino Morgan W.,
DeMars Kelly M.,
Tuna Kubra M.,
Montgomery Darrice S.,
Sanz Brian D.,
CandelarioJalil Eduardo,
Alli Abdel A.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.551.5
Subject(s) - glial fibrillary acidic protein , protein kinase c , protein kinase a , medicine , neuroprotection , kinase , endocrinology , biology , pathology , microbiology and biotechnology , immunohistochemistry
Background According to 2018 AHA/ASA guidelines, stroke remains a leading cause of death and long‐term disability in the U.S. and worldwide. Despite prevention efforts, risk factors such as hypertension attributes to worse clinical outcomes. The therapeutic window for stroke is limited to fibrinolytic therapy. Protein Kinase C‐epsilon activity provides resistance against oxidative stress. Objective The objective of this study was to test the hypothesis that the cell‐specific Protein Kinase C‐epsilon /Ca2+/calmodulin‐dependent protein kinase II‐alpha (CaMKII‐alpha) system contributes to homeostatic dysfunction, glial hypersensitivity, and cell death mechanisms. Here, we aimed to investigate key protein expression changes in tissue after middle cerebral artery (MCA) onset in animals. Our primary and secondary endpoints assess significant expression differences for astrocyte‐specific glial fibrillary acidic protein and microglia/macrophage‐specific ionized calcium adaptor molecule 1. Methods We used a transient MCA occlusion model to quantify cell and tissue‐type‐specific expression changes by Western blot after oxygen deprivation of N2:O2 (99:1) and ischemic‐reperfusion intervals of 4, 14, 24, 48, 72‐hrs after a 90‐min occlusion. Neuroprotective potential is determined by Protein Kinase C‐epsilon expression after reperfusion onset. IHC and TTC staining is used to evaluate lesion volume and metabolic activity. All experiments were randomized and double‐blinded. Results Oxygen‐deprived cell lines and tissue were confirmed by HIF1‐alpha expression and distribution. Global expression changes reveal an inverse correlation between Protein Kinase C‐epsilon and glial immunoreactivity. Calcium‐independent Protein Kinase C‐epsilon expression levels are most significant at 24‐hrs after MCA reperfusion onset in cortical and subcortical tissue. Conclusion Our data provides new evidence of a tissue‐ and cell‐type‐specific mechanism of protein regulation in the Protein Kinase C‐epsilon /Ca2+/CaMKII‐alpha pathway after stroke. The findings suggest an inverse relationship between glial activation and Protein Kinase C‐epsilon expression. Reduced Protein Kinase C‐epsilon expression in cells and tissue may exacerbate gliosis. Significant changes to Protein Kinase C‐epsilon and CaMKII‐alpha activities in tissue often results in abnormal Ca2+ homeostasis, dysfunctional calcium‐dependent potassium conductance, and cytoskeletal deformation. Additional studies are needed to further elucidate the complex role of Ca2+ and its regulation in the context of acute and chronic neurodegenerative conditions. Support or Funding Information This project was funded by the U.S. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (Grant K01 DK099617; to A.A.A.) and the University of Florida College of Medicine. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .