Rachel Bright

Protein Kinase C δ 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 δPKC, δV1-1, we found that δPKC 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-conjugatedδV1-1 and by measuring inhibition of intracellular δPKC translocation, an indicator of δPKC activity. Delivery of δV1-1 decreased infarct size in an in vivo rat stroke model of transient middle cerebral artery occlusion. Importantly, δV1-1 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 δPKC in mediating apoptotic processes. We therefore determined whether δPKC inhibition altered apoptotic cell death or cell survival pathways in our models. We found that δV1-1 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 δPKC during reperfusion and suggest that δV1-1 delivery, even hours after commencement of reperfusion, may provide a therapeutic advantage after cerebral ischemia.

The Role of Protein Kinase C in Cerebral Ischemic and Reperfusion Injury

Background and Purpose— Stroke is a leading cause of disability and death in the United States, yet limited therapeutic options exist. The need for novel neuroprotective agents has spurred efforts to understand the intracellular signaling pathways that mediate cellular response to stroke. Protein kinase C (PKC) plays a central role in mediating ischemic and reperfusion damage in multiple tissues, including the brain. However, because of conflicting reports, it remains unclear whether PKC is involved in cell survival signaling, or mediates detrimental processes. Summary of Review— This review will examine the role of PKC activity in stroke. In particular, we will focus on more recent insights into the PKC isozyme-specific responses in neuronal preconditioning and in ischemia and reperfusion-induced stress. Conclusion— Examination of PKC isozyme activities during stroke demonstrates the clinical promise of PKC isozyme-specific modulators for the treatment of cerebral ischemia.

Protein Kinase C δ (δPKC)-Annexin V Interaction A REQUIRED STEP IN δPKC TRANSLOCATION AND FUNCTION

Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia, and participates in a variety of signal transduction pathways such as apoptosis, cell proliferation, and tumor suppression. Though much is known about PKC downstream signaling events, the mechanisms of regulation of PKC activation and subsequent translocation have not been elucidated. Protein-protein interactions regulate and determine the specificity of many cellular signaling events. Such a specific protein-protein interaction is described here between δPKC and annexin V. We demonstrate, at physiologically relevant conditions, that a transient interaction between annexin V and δPKC occurs in cells after δPKC stimulation, but before δPKC translocates to the particulate fraction. Evidence of δPKC-annexin V binding is provided also by FRET and by in vitro binding studies. Dissociation of the δPKC-annexin V complex requires ATP and microtubule integrity. Furthermore, depletion of endogenous annexin V, but not annexin IV, with siRNA inhibits δPKC translocation following PKC stimulation. A rationally designed eight amino acid peptide, corresponding to the interaction site for δPKC on annexin V, inhibits δPKC translocation and δPKC-mediated function as evidenced by its protective effect in a model of myocardial infarction. Our data indicate that translocation of δPKC is not simply a diffusion-driven process, but is instead a multi-step event regulated by protein-protein interactions. We show that following cell activation, δPKC-annexin V binding is a transient and an essential step in the function of δPKC, thus identifying a new role for annexin V in PKC signaling and a new step in PKC activation.

δPKC mediates microcerebrovascular dysfunction in acute ischemia and in chronic hypertensive stress in vivo

Maintaining cerebrovascular function is a priority for reducing damage following acute ischemic events such as stroke, and under chronic stress in diseases such as hypertension. Ischemic episodes lead to endothelial cell damage, deleterious inflammatory responses, and altered neuronal and astrocyte regulation of vascular function. These, in turn, can lead to impaired cerebral blood flow and compromised blood-brain barrier function, promoting microvascular collapse, edema, hemorrhagic transformation, and worsened neurological recovery. Multiple studies demonstrate that protein kinase C (PKC), a widely expressed serine/threonine kinase, is involved in mediating arterial tone and microvascular function. However, there is no clear understanding about the role of individual PKC isozymes. We show that intraperitoneal injection of deltaV1-1-TAT(47-57) (0.2 mg/kg in 1 mL), an isozyme-specific peptide inhibitor of deltaPKC, improved microvascular pathology, increased the number of patent microvessels by 92% compared to control-treated animals, and increased cerebral blood flow by 26% following acute focal ischemia induced by middle cerebral artery occlusion in normotensive rats. In addition, acute delivery of deltaV1-1-TAT(47-57) in hypertensive Dahl rats increased cerebral blood flow by 12%, and sustained delivery deltaV1-1-TAT(47-57) (5 uL/h, 1 mM), reduced infarct size by 25% following an acute stroke induced by MCA occlusion for 90 min. Together, these findings demonstrate that deltaPKC is an important therapeutic target for protection of microvascular structure and function under both acute and chronic conditions of cerebrovascular stress.

Cell-specific role for ε- and βI-protein kinase C isozymes in protecting cortical neurons and astrocytes from ischemia-like injury

Activation of epsilon protein kinase C (epsilonPKC) has been shown to protect cardiac myocytes against ischemia and reperfusion injury. However, the role of PKC in ischemic brain injury is less well defined. Western blot analysis of murine neurons and astrocytes in primary culture demonstrated epsilon- and betaIPKC expression in both cell types. Activation of epsilonPKC increased in neuronal cultures in response to the ischemia-like insult of oxygen-glucose deprivation (OGD). Isozyme-specific peptide activators or inhibitors of PKC were applied at various times before, during and after the OGD period. Neuron-astrocyte mixed cultures pretreated with a selective epsilonPKC activator peptide showed a significant reduction in neuronal injury after OGD and reperfusion, compared to cultures pretreated with control peptide. The epsilonPKC activator peptide counteracted the increased damage induced by pretreatment with the epsilonPKC-selective inhibitor peptide in relatively pure neuronal cultures subjected to OGD. Neither epsilonPKC activator nor inhibitor peptides affected injury of neurons when applied after OGD onset. In contrast, the betaIPKC-selective inhibitor peptide increased injury in astrocyte cultures exposed to OGD at all application times tested. Our data demonstrate protection of neurons by selective activation of epsilonPKC but enhanced astrocyte cell death with selective inhibition of betaIPKC. Thus PKC isozymes exhibit cell type-specific effects on ischemia-like injury.

δPKC-annexin V interaction; a required step in δPKC translocation and function

Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia, and participates in a variety of signal transduction pathways such as apoptosis, cell proliferation, and tumor suppression. Though much is known about PKC downstream signaling events, the mechanisms of regulation of PKC activation and subsequent translocation have not been elucidated. Protein-protein interactions regulate and determine the specificity of many cellular signaling events. Such a specific protein-protein interaction is described here between δPKC and annexin V. We demonstrate, at physiologically relevant conditions, that a transient interaction between annexin V and δPKC occurs in cells after δPKC stimulation, but before δPKC translocates to the particulate fraction. Evidence of δPKC-annexin V binding is provided also by FRET and by in vitro binding studies. Dissociation of the δPKC-annexin V complex requires ATP and microtubule integrity. Furthermore, depletion of endogenous annexin V, but not annexin IV, with siRNA inhibits δPKC translocation following PKC stimulation. A rationally designed eight amino acid peptide, corresponding to the interaction site for δPKC on annexin V, inhibits δPKC translocation and δPKC-mediated function as evidenced by its protective effect in a model of myocardial infarction. Our data indicate that translocation of δPKC is not simply a diffusion-driven process, but is instead a multi-step event regulated by protein-protein interactions. We show that following cell activation, δPKC-annexin V binding is a transient and an essential step in the function of δPKC, thus identifying a new role for annexin V in PKC signaling and a new step in PKC activation.

EPKC confers acute tolerance to cerebral ischemic reperfusion injury

In response to mild ischemic stress, the brain elicits endogenous survival mechanisms to protect cells against a subsequent lethal ischemic stress, referred to as ischemic tolerance. The molecular signals that mediate this protection are thought to involve the expression and activation of multiple kinases, including protein kinase C (PKC). Here we demonstrate that epsilonPKC mediates cerebral ischemic tolerance in vivo. Systemic delivery of psiepsilonRACK, an epsilonPKC-selective peptide activator, confers neuroprotection against a subsequent cerebral ischemic event when delivered immediately prior to stroke. In addition, activation of epsilonPKC by psiepsilonRACK treatment decreases vascular tone in vivo, as demonstrated by a reduction in microvascular cerebral blood flow. Here we demonstrate the role of acute and transient epsilonPKC in early cerebral tolerance in vivo and suggest that extra-parenchymal mechanisms, such as vasoconstriction, may contribute to the conferred protection.