Protein kinase C delta mediates cerebral reperfusion injury in vivo.
Protein kinase C (PKC) has been implicated in mediating ischemic and reperfusion damage in multiple organs. However, conflicting reports exist on the role of individual PKC isozymes in cerebral ischemic injury. Using a peptide inhibitor selective for deltaPKC, deltaV1-1, we found that deltaPKC inhibition reduced cellular injury in a rat hippocampal slice model of cerebral ischemia [oxygen-glucose deprivation (OGD)] when present both during OGD and for the first 3 hr of reperfusion. We next demonstrated peptide delivery to the brain parenchyma after in vivo delivery by detecting biotin-conjugateddeltaV1-1 and by measuring inhibition of intracellular deltaPKC translocation, an indicator of deltaPKC activity. Delivery of deltaV1-1 decreased infarct size in an in vivo rat stroke model of transient middle cerebral artery occlusion. Importantly, deltaV1-1 had no effect when delivered immediately before ischemia. However, delivery at the onset, at 1 hr, or at 6 hr of reperfusion reduced injury by 68, 47, and 58%, respectively. Previous work has implicated deltaPKC in mediating apoptotic processes. We therefore determined whether deltaPKC inhibition altered apoptotic cell death or cell survival pathways in our models. We found that deltaV1-1 reduced numbers of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells, indicating decreased apoptosis, increased levels of phospho-Akt, a kinase involved in cell survival pathways, and inhibited BAD (Bcl-2-associated death protein) protein translocation from the cell cytosol to the membrane, indicating inhibition of proapoptotic signaling. These data support a deleterious role for deltaPKC during reperfusion and suggest that deltaV1-1 delivery, even hours after commencement of reperfusion, may provide a therapeutic advantage after cerebral ischemia.
Department of Molecular Pharmacology, Stanford Stroke Center, Stanford University School of Medicine, Stanford, California 94305-5174, USA.
Rachel
R
Bright
Ami P
AP
Raval
Jeffrey M
JM
Dembner
Miguel A
MA
Pérez-Pinzón
Gary K
GK
Steinberg
Midori A
MA
Yenari
Daria
D
Mochly-Rosen
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D002545:Brain Ischemia / Q000503:physiopathology*; D004195:Disease Models, Animal; D015696:Gene Products, tat; D006624:Hippocampus; D066298:In Vitro Techniques; D020244:Infarction, Middle Cerebral Artery; D008297:Male; D018696:Neuroprotective Agents / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D051745:Protein Kinase C-delta; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins; D015427:Reperfusion Injury / Q000503:physiopathology* / Q000517:prevention & control; D015398:Signal Transduction
D015696:Gene Products, tat; D018696:Neuroprotective Agents; D011993:Recombinant Fusion Proteins; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta