Ischemic Preconditioning Targets the Respiration of Synaptic Mitochondria via Protein Kinase Cε

Abstract

In the brain, ischemic preconditioning (IPC) diminishes mitochondrial dysfunction after ischemia and confers neuroprotection. Activation of protein kinase C (PKC) has been proposed to be a key neuroprotective pathway during IPC. We tested the hypothesis that IPC increases the levels of PKC in synaptosomes from rat hippocampus, resulting in improved synaptic mitochondrial respiration. Preconditioning significantly increased the level of hippocampal synaptosomal PKC to 152% of sham-operated animals at 2 d of reperfusion, the time of peak neuroprotection. We tested the effect of PKC activation on hippocampal synaptic mitochondrial respiration 2 d after preconditioning. Treatment with the specific PKC activating peptide, tat-RACK (tat--receptor for activated C kinase), increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV to 157, 153, and 131% of control (tat peptide alone). In parallel, we found that PKC activation in synaptosomes from preconditioned animals resulted in altered levels of phosphorylated mitochondrial respiratory chain proteins: increased serine and tyrosine phosphorylation of 18 kDa subunit of complex I, decreased serine phosphorylation of FeS protein in complex III, increased threonine phosphorylation of COX IV (cytochrome oxidase IV), increased mitochondrial membrane potential, and decreased H 2 O 2 production. In brief, ischemic preconditioning promoted significant increases in the level of synaptosomal PKC. Activation of PKC increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins. We propose that, at 48 h of reperfusion after ischemic preconditioning, PKC is poised at synaptic mitochondria to respond to ischemia either by direct phosphorylation or activation of the PKC signaling pathway.

Novel Mitochondrial Targets for Neuroprotection

“…These increases in the rate of respiration were correlated with increased levels of serine and tyrosine phosphorylation of 18 kDa subunit of complex I, threonine phosphorylation of COX IV, increased mitochondrial membrane potential, and decreased H 2 O 2 production. In addition, induction of in-vitro ischemia decreased mitochondrial cytochrome c release (Dave et al, 2008). These results suggest that after IPC, ePKC is readily available for activation in synaptosomal mitochondria on ischemia/reperfusion, in such a manner to increase mitochondrial respiration, reduce ROS production and cytochrome c release, hallmarks of reperfusion injury.…”

“…In another study, ePKC levels were found to increase in the hippocampal synaptosomal fraction 48 hours after IPC (Dave et al, 2008). Treatment with a specific ePKC activating peptide 48 hours after IPC, increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV (Dave et al, 2008). These increases in the rate of respiration were correlated with increased levels of serine and tyrosine phosphorylation of 18 kDa subunit of complex I, threonine phosphorylation of COX IV, increased mitochondrial membrane potential, and decreased H 2 O 2 production.…”

“…After IPC, selective inhibition of ePKC activation prevented Kir6.2 phosphorylation, a specific subunit of the mt K + ATP channel, consistent with Kir6.2 as a phosphorylation target of ePKC or its downstream effectors, and inhibition of this channel inhibited IPC-induced neuroprotection. In another study, ePKC levels were found to increase in the hippocampal synaptosomal fraction 48 hours after IPC (Dave et al, 2008). Treatment with a specific ePKC activating peptide 48 hours after IPC, increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV (Dave et al, 2008).…”

The neuroprotective mechanism of brain ischemic preconditioning

“…Ischemic preconditioning promoted significant increases in the levels of synaptosomal PKCε in rat hippocampus. Activation of PKCε increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins [40] . Delivery of a PKCε inhibitory peptide abated NMDA-induced preconditioning in cell culture and isolated hippocampal slice models [41] .…”

Protein Kinase C Epsilon Regulates Mitochondrial Pools of Nampt and NAD Following Resveratrol and Ischemic Preconditioning in the Rat Cortex

“…5,6 Ischemic preconditioning (IPC), a paradigm where a brief ischemic insult protects the brain against a subsequent lethal ischemic injury, or treatment with the polyphenol resveratrol, has been shown to protect mitochondria and enhance levels of NAD in the brain. [7][8][9][10] The mechanism by which resveratrol and IPC regulate NAD levels has not been clearly defined. However, two key signaling pathways that may be involved are protein kinase C epsilon (PKCe) and AMP-activated protein kinase (AMPK), enzymes that regulate mitochondrial ischemic neuroprotection after IPC and resveratrol treatment.…”