Mechanisms of Mitochondrial Regulation and Ischemic Neuroprotection by the PKCε Pathway

Mitochondrial dysfunction following cerebral ischemia is a major contributor to tissue injury and neurodegeneration. Therefore, investigating the pathways that lead to mitochondrial protection following cerebral ischemic injury represents a major strategy in the development of therapeutic approaches. The PKCepsilon enzyme has been shown to translocate to the mitochondria where it is involved in providing global mitochondrial protection against ischemic insults. The goal of this study was to better understand the downstream pathways that drive PKCepsilon-mediated mitochondrial neuroprotection which may facilitate therapeutic approaches to cerebral ischemic injury. Initial experiments performed in vitro revealed that PKCepsilon functionally cooperates with the transcriptional regulator AMPK, another master regulator of mitochondrial function. AMPK and PKCepsilon were shown to reciprocally regulate each other’s activity and require each other to provide ischemic neuroprotection. Further investigations both in vitro and in vivo showed that the PKCepsilon-AMPK pathway is involved in enhancing mitochondrial-localized Nampt. Furthermore, PKCepsilon was shown to be crucial for the upregulation or translocation of mitochondrial pools of Nampt following AMPK activation, IPC or resveratrol treatment. PKCepsilon regulation of Nampt was both sufficient and necessary to increase the mitochondrial NAD+/NADH ratio, a redox signal involved in promoting sirtuin activity. The PKCepsilon-Nampt-NAD+ pathway enhanced SIRT5 desuccinylase activity, which was shown to regulate mitochondrial respiration and was required for PKCepislon-mediated ischemic neuroprotection against apoptosis and necrosis. Collectively, the results of this study identify a novel pathway by which PKCepsilon regulates wide-scale mitochondrial function through activation of SIRT5.</p

Metabolomics Based Identification of SIRT5 and Protein Kinase C Epsilon Regulated Pathways in Brain

The role of Sirtuins in brain function is emerging, yet little is known about SIRT5 in this domain. Our previous work demonstrates that protein kinase C epsilon (PKCε)-induced protection from focal ischemia is lost in SIRT5−/− mice. Thus, metabolic regulation by SIRT5 contributes significantly to ischemic tolerance. The aim of this study was to identify the SIRT5-regulated metabolic pathways in the brain and determine which of those pathways are linked to PKCε. Our results show SIRT5 is primarily expressed in neurons and endothelial cells in the brain, with mitochondrial and extra-mitochondrial localization. Pathway and enrichment analysis of non-targeted primary metabolite profiles from Sirt5−/− cortex revealed alterations in several pathways including purine metabolism (urea, adenosine, adenine, xanthine), nitrogen metabolism (glutamic acid, glycine), and malate-aspartate shuttle (malic acid, glutamic acid). Additionally, perturbations in β-oxidation and carnitine transferase (pentadecanoic acid, heptadecanoic acid) and glutamate transport and glutamine synthetase (urea, xylitol, adenine, adenosine, glycine, glutamic acid) were predicted. Metabolite changes in SIRT5−/− coincided with alterations in expression of amino acid (SLC7A5, SLC7A7) and glutamate (EAAT2) transport proteins as well as key enzymes in purine (PRPS1, PPAT), fatty acid (ACADS, HADHB), glutamine-glutamate (GAD1, GLUD1), and malate-aspartate shuttle (MDH1) metabolic pathways. Moreover, PKCε activation induced alternations in purine metabolites (urea, glutamine) that overlapped with putative SIRT5 pathways in WT but not in SIRT5−/− mice. Finally, we found that purine metabolism is a common metabolic pathway regulated by SIRT5, PKCε and ischemic preconditioning. These results implicate Sirt5 in the regulation of pathways central to brain metabolism, with links to ischemic tolerance

Ischemic Preconditioning-Mediated Signaling Pathways Leading to Tolerance Against Cerebral Ischemia

Cerebral ischemia, most notably in the form of stroke, is the leading cause of morbidity and mortality resulting in long-term disability in the USA. Approximately 800,000 strokes occur each year in the USA, and 87 % of all strokes in the world are caused by embolism, thrombosis, or systemic hemorrhage/hypoperfusion, all of which are a form of cerebral ischemia (Roger et al. 2011). The medical cost for the treatment of stroke in the USA was estimated to be $25 billion in 2007 (Roger et al. 2011). Due to this great burden, a fundamental understanding of cerebral ischemia and the inciting cellular dysfunction is imperative for the development of new therapies to combat this growing epidemic

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

Preserving mitochondrial pools of nicotinamide adenine dinucleotide (NAD) or nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in NAD production, maintains mitochondrial function and confers neuroprotection after ischemic stress. However, the mechanisms involved in regulating mitochondrial-localized Nampt or NAD have not been defined. In this study, we investigated the roles of protein kinase C epsilon (PKCɛ) and AMP-activated protein kinase (AMPK) in regulating mitochondrial pools of Nampt and NAD after resveratrol or ischemic preconditioning (IPC) in the cortex and in primary neuronal-glial cortical cultures. Using the specific PKCɛ agonist ψɛRACK, we found that PKCɛ induced robust activation of AMPK in vitro and in vivo and that AMPK was required for PKCɛ-mediated ischemic neuroprotection. In purified mitochondrial fractions, PKCɛ enhanced Nampt levels in an AMPK-dependent manner and was required for increased mitochondrial Nampt after IPC or resveratrol treatment. Analysis of intrinsic NAD autofluorescence using two-photon microscopy revealed that PKCɛ modulated NAD in the mitochondrial fraction. Further assessments of mitochondrial NAD concentrations showed that PKCɛ has a key role in regulating the mitochondrial NAD(+)/nicotinamide adenine dinucleotide reduced (NADH) ratio after IPC and resveratrol treatment in an AMPK- and Nampt-dependent manner. These findings indicate that PKCɛ is critical to increase or maintain mitochondrial Nampt and NAD after pathways of ischemic neuroprotection in the brain

Metabolomics Based Identification of SIRT5 and Protein Kinase C Epsilon Regulated Pathways in Brain

The role of Sirtuins in brain function is emerging, yet little is known about SIRT5 in this domain. Our previous work demonstrates that protein kinase C epsilon (PKCε)-induced protection from focal ischemia is lost in SIRT5 −/− mice. Thus, metabolic regulation by SIRT5 contributes significantly to ischemic tolerance. The aim of this study was to identify the SIRT5-regulated metabolic pathways in the brain and determine which of those pathways are linked to PKCε. Our results show SIRT5 is primarily expressed in neurons and endothelial cells in the brain, with mitochondrial and extra-mitochondrial localization. Pathway and enrichment analysis of non-targeted primary metabolite profiles from Sirt5 −/− cortex revealed alterations in several pathways including purine metabolism (urea, adenosine, adenine, xanthine), nitrogen metabolism (glutamic acid, glycine), and malate-aspartate shuttle (malic acid, glutamic acid). Additionally, perturbations in β-oxidation and carnitine transferase (pentadecanoic acid, heptadecanoic acid) and glutamate transport and glutamine synthetase (urea, xylitol, adenine, adenosine, glycine, glutamic acid) were predicted. Metabolite changes in SIRT5 −/− coincided with alterations in expression of amino acid (SLC7A5, SLC7A7) and glutamate (EAAT2) transport proteins as well as key enzymes in purine (PRPS1, PPAT), fatty acid (ACADS, HADHB), glutamine-glutamate (GAD1, GLUD1), and malate-aspartate shuttle (MDH1) metabolic pathways. Moreover, PKCε activation induced alternations in purine metabolites (urea, glutamine) that overlapped with putative SIRT5 pathways in WT but not in SIRT5 −/− mice. Finally, we found that purine metabolism is a common metabolic pathway regulated by SIRT5, PKCε and ischemic preconditioning. These results implicate Sirt5 in the regulation of pathways central to brain metabolism, with links to ischemic tolerance

Abstract TP267: Protein Kinase C Epsilon-activation Mediates Ischemic Neuroprotection by Activating an Activity-regulated Cytoskeleton-associated Protein-dependent Mechanism

Introduction: Cerebral ischemia can trigger cell death in the CA1 region of the hippocampus, an area important for memory. Protein kinase C epsilon (PKCε) activation prior to ischemia protects the CA1 from injury by modulating neurotransmission. The underlying mechanism needs further study. Hypothesis: PKCε-induced neuroprotection increases latency until anoxic depolarization (AD) through an activity-regulated cytoskeleton-associated protein (arc)-dependent mechanism of regulating AMPAR currents. Results: In vivo activation of PKCε by the PKCε-activator ψε-Receptor of Activated C Kinase (ψεRACK) increased BDNF 5.99 +/- 0.11 fold and TrkB phosphorylation levels 2.94 +/- 0.32 fold (enhancers of arc protein levels) (n = 4, p < 0.005, t-test). Arc mRNA and protein was increased 143.97 +/-7.68 % and 1.91 +/- 0.22 fold (n = 9, p < 0.005, t-test). Inhibition of arc using antisense oligodeoxynucleotides (arc AS ODNs) in cultured slices blocked PKCε-mediated neuroprotection against lethal oxygen and glucose deprivation (OGD) from 35.91 +/- 5.97 to 74.93 +/- 4.24 % cell death (n = 6, p < 0.005, ANOVA, Bonferroni). ΨεRACK decreased AMPAR-mediated mEPSC amplitude to 12.75 +/- 0.35 pA from 14.80 +/- 0.39 pA (n = 20, p < 0.01, ANOVA, Bonferroni). This effect was arc-dependent. Additionally, ψεRACK treatment increased latency until AD from 29.27 +/- 3.6 min 50.77 +/- 5.08 min (n = 13, p < 0.01, ANOVA, Bonferroni). This increase was arc-dependent, and required AMPAR internalization. Inhibiting internalization reduced AD latency from 54.5 +/- 8.40 min to 22.3 + 5.17 min (n = 6, p <0.005, t-test). Conclusion: Arc expression is necessary for neuroprotection afforded by PKCε-activation, modulates excitatory synaptic strength, and increases latency until AD. Methods: Western blot: Proteins (40 μg) were separated on a 12% SDS-PAGE gel. Immunofluorescence : 30 μm sections were incubated with 1:500 NeuN and 1:50 arc in PBS with 0.8% triton. Cultured slices preparation : sections from P9-11 rats were plated on inserts and cultured for 14 days. PI measurements of cell death : Slices were incubated in medium with 2 μg/mL PI. mEPSC measurements : Whole-cell voltage clamp was performed. AD: OGD, perfusate was switched to glucose free media, and bubbled with a 95% N 2 and 5% CO 2 gas

Protein kinase C epsilon delays latency until anoxic depolarization through arc expression and GluR2 internalization

Global cerebral ischemia is a debilitating injury that damages the CA1 region of the hippocampus, an area important for learning and memory. Protein kinase C epsilon (PKCɛ) activation is a critical component of many neuroprotective treatments. The ability of PKCɛ activation to regulate AMPA receptors (AMPARs) remains unexplored despite the role of AMPARs in excitotoxicity after brain ischemia. We determined that PKCɛ activation increased expression of a protein linked to learning and memory, activity-regulated cytoskeleton-associated protein (arc). Also, arc is necessary for neuroprotection and confers protection through decreasing AMPAR currents via GluR2 internalization. In vivo, activation of PKCɛ increased arc expression through a BDNF/TrkB pathway, and decreased GluR2 mRNA levels. In hippocampal cultured slices, PKCɛ activation decreased AMPAR current amplitudes in an arc- and GluR2-dependent manner. Additionally, PKCɛ activation triggered an arc- and GluR2 internalization-dependent delay in latency until anoxic depolarization. Inhibiting arc also blocked PKCɛ-mediated neuroprotection against lethal oxygen and glucose deprivation. These data characterize a novel PKCɛ-dependent mechanism that for the first time defines a role for arc and AMPAR internalization in conferring neuroprotection

Signaling pathways leading to ischemic mitochondrial neuroprotection

There is extensive evidence that ischemic/reperfusion mediated mitochondrial dysfunction is a major contributor to ischemic damage. However data also indicates that mild ischemic stress induces mitochondrial dependent activation of ischemic preconditioning. Ischemic preconditioning is a neuroprotective mechanism which is activated upon a brief sub-injurious ischemic exposure and is sufficient to provide protection against a subsequent lethal ischemic insult. Current research demonstrates that mitochondria are not only the inducers of but are also an important target of ischemic preconditioning mediated protection. Numerous proteins and signaling pathways are activated by ischemic preconditioning which protect the mitochondria against ischemic damage. In this review we examine some of the proteins activated by ischemic precondition which counteracts the deleterious effects of ischemia/reperfusion thereby maintaining normal mitochondrial activity and lead to ischemic tolerance