ABSTRACT
A recently discovered protein phosphatase PHLPP (PH domain Leucine-rich repeat Protein Phosphatase) has been shown to dephosphorylate Akt on its hydrophobic motif (Ser473) thereby decreasing Akt kinase activity. We generated PHLPP1 knockout (KO) mice and used them to explore the ability of enhanced in vivo Akt signaling to protect the brain against ischemic insult. Brains from KO mice subjected to middle cerebral artery occlusion (MCAO) for 2 hours showed significantly greater increases in Akt activity and less neurovascular damage after reperfusion than wild-type (WT) mice. Remarkably, infarct volume in the PHLPP1 KO was significantly reduced compared with WT (12.7±2.7% versus 22.9±3.1%) and this was prevented by Akt inhibition. Astrocytes from KO mice and neurons in which PHLPP1 was downregulated showed enhanced Akt activation and diminished cell death in response to oxygen-glucose deprivation. Thus, deletion of PHLPP1 can enhance Akt activation in neurons and astrocytes, and can significantly increase cell survival and diminish infarct size after MCAO. Inhibition of PHLPP could be a therapeutic approach to minimize damage after focal ischemia.
Subject(s)
Brain Infarction/enzymology , Brain Injuries/enzymology , Gene Deletion , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Reperfusion Injury/enzymology , Animals , Astrocytes/enzymology , Astrocytes/pathology , Brain Infarction/genetics , Brain Infarction/pathology , Brain Infarction/prevention & control , Brain Injuries/genetics , Brain Injuries/pathology , Brain Injuries/prevention & control , Enzyme Activation/genetics , Glucose/metabolism , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Neurons/enzymology , Neurons/pathology , Nuclear Proteins/genetics , Oxygen/metabolism , Phosphoprotein Phosphatases/genetics , Phosphorylation/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Reperfusion Injury/genetics , Reperfusion Injury/pathology , Reperfusion Injury/prevention & control , Signal Transduction/geneticsABSTRACT
Pre-clinical development of therapy for acute ischemic stroke requires robust animal models; the rodent middle cerebral artery occlusion (MCAo) model using a nylon filament inserted into the internal carotid artery is the most popular. Drug screening requires targeted delivery of test substance in a controlled manner. To address these needs, we developed a novel method for delivering substances directly into the ischemic brain during MCAo in the awake rat. An indwelling catheter is placed in the common carotid artery ipsilateral to the occlusion at the time of the surgical placement of the occluding filament. The internal and common carotid arteries are left patent to allow superfusion anterograde. The surgeries can be completed quickly to allow rapid recovery from anesthesia; tests substances can be infused at any given time for any given duration. To simulate clinical scenarios, the occluding filament can be removed minutes or hours later (reperfusion) followed by therapeutic infusions. By delivering drug intra-arterially to the target tissue, "first pass" loss in the liver is reduced and drug effects are concentrated in the ischemic zone. To validate our method, rats were infused with Evans blue dye either intra-arterially or intravenously during a 4 h MCAo. After a 30 min reperfusion period, the dye was extracted from each hemisphere and quantitated with a spectrophotometer. Significantly more dye was measured in the ischemic hemispheres that received the dye intra-arterially.
