Redox modification of proteins as essential mediators of CNS autophagy and mitophagy.

Production of cellular reactive oxygen species (ROS) is typically associated with protein and DNA damage, toxicity, and death. However, ROS are also essential regulators of signaling and work in concert with redox-sensitive proteins to regulate cell homeostasis during stress. In this review, we focus on the redox regulation of mitophagy, a process that contributes to energetic tone as well as mitochondrial form and function. Mitophagy has been increasingly implicated in diseases including Parkinson's, Amyotrophic Lateral Sclerosis, and cancer. Although these disease states employ different genetic mutations, they share the common factors of redox dysregulation and autophagic signaling. This review highlights key redox sensitive signaling molecules which can enhance neuronal survival by promoting temporally and spatially controlled autophagic signaling and mitophagy.

Alteration of isocitrate dehydrogenase following acute ischemic injury as a means to improve cellular energetic status in neuroadaptation.

The isocitrate dehydrogenase (IDH) enzymes were initially identified as essential components of the Krebs cycle. IDH mutations were thought to be incompatible with cell survival. However, 90% of glioblastomas were recently shown to be associated with somatic mutations in these enzymes, indicating a possible role for IDH in promoting cellular survival in hypoxic environments. Our proteomic analysis of rats given 10 minutes of middle cerebral artery occlusion to induce transient ischemia demonstrates a significant decrease in IDH expression. We have recapitulated this decrease in an in vitro model using primary cortical neurons exposed to acute oxygen and glucose deprivation. Given the role of IDHs in energy metabolism and antioxidant production, we hypothesize that the IDHs may serve as first-line, rapid-response enzymes that regulate survival in environments of energetic or oxidative stress. In order to identify the specific events that regulate IDH enzymes, HT-22 neural cells were subjected to either a selective energetic challenge or a pure oxidative stress. In response to the non-lethal energetic challenge induced by substituting galactose for glucose, we observed increased IDH1, 2, and 3 expression and cessation of cellular proliferation. No change in expression of any IDH isoform was observed when neural cells were subjected to subtoxic oxidative stress via glutathione depletion. Taken together, these data imply that IDH expression rapidly responds to changes in energetic status, but not to oxidative stress. These data also suggest that IDH enzymes respond not only to allosteric modulation, but can also change patterns of expression in response to moderate stress in an effort to maximize ATP production and survival.

Perioperative plasma F(2)-Isoprostane levels correlate with markers of impaired ventilation in infants with single-ventricle physiology undergoing stage 2 surgical palliation on the cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) produces inflammation and oxidative stress, which contribute to postoperative complications after cardiac surgery. F(2)-Isoprostanes (F(2)-IsoPs) are products of lipid oxidative injury and represent the most accurate markers of oxidative stress. In adults undergoing cardiac surgery, CPB is associated with elevated IsoPs. The relationship between F(2)-IsoPs and perioperative end-organ function in infants with single-ventricle physiology, however, has not been well studied. This study prospectively enrolled 20 infants (ages 3-12 months) with univentricular physiology undergoing elective stage 2 palliation (bidirectional cavopulmonary anastomosis). Blood samples were collected before the surgical incision (T0), 30 min after initiation of CPB (T1), immediately after separation from CPB (T2), and 24 h postoperatively (T3). Plasma F(2)-IsoP levels were measured at each time point and correlated with indices of pulmonary function and other relevant clinical variables. Plasma F(2)-IsoPs increased significantly during surgery, with highest levels seen immediately after separation from CPB (p < 0.001). After separation from CPB, increased F(2)-IsoP was associated with lower arterial pH (ρ = -0.564; p = 0.012), higher partial pressure of carbon dioxide (PaCO(2); ρ = 0.633; p = 0.004), and decreased lung compliance (ρ = -0.783; p ≤ 0.001). After CPB, F(2)-IsoPs did not correlate with duration of CPB, arterial lactate, or immediate postoperative outcomes. In infants with single-ventricle physiology, CPB produces oxidative stress, as quantified by elevated F(2)-IsoP levels. Increased F(2)-IsoP levels correlated with impaired ventilation in the postoperative period. The extent to which F(2)-IsoPs and other bioactive products of lipid oxidative injury might predict or contribute to organ-specific stress warrants further investigation.

The fatty acid oxidation product 15-A3t-isoprostane is a potent inhibitor of NFκB transcription and macrophage transformation.

Fatty acids such as eicosapentaenoic acid (EPA) have been shown to be beneficial for neurological function and human health. It is widely thought that oxidation products of EPA are responsible for biological activity, although the specific EPA peroxidation product(s) which exert these responses have not yet been identified. In this work we provide the first evidence that the synthesized representative cyclopentenone IsoP, 15-A(3t)-IsoP, serves as a potent inhibitor of lipopolysaccharide-stimulated macrophage activation. The anti-inflammatory activities of 15-A(3t)-IsoP were observed in response not only to lipopolysaccharide, but also to tumor necrosis factor alpha and IL-1b stimulation. Subsequently, this response blocked the ability of these compounds to stimulate nuclear factor kappa b (NFκB) activation and production of proinflammatory cytokines. The bioactivity of 15-A(3t)-IsoP was shown to be dependent upon an unsaturated carbonyl residue which transiently adducts to free thiols. Site directed mutagenesis of the redox sensitive C179 site of the Ikappa kinase beta subunit, blocked the biological activity of 15-A(3t)-IsoP and NFκB activation. The vasoprotective potential of 15-A(3t)-IsoP was underscored by the ability of this compound to block oxidized lipid accumulation, a critical step in foam cell transformation and atherosclerotic plaque formation. Taken together, these are the first data identifying the biological activity of a specific product of EPA peroxidation, which is formed in abundance in vivo. The clear mechanism linking 15-A(3t)-IsoP to redox control of NFκB transcription, and the compound's ability to block foam cell transformation suggest that 15-A(3t)-IsoP provides a unique and potent tool to provide vaso- and cytoprotection under conditions of oxidative stress.

Assessing neuronal bioenergetic status.

Drug discovery and therapeutic development for disorders of the central nervous system (CNS) represents one of the largest unmet markets in modern medicine. We have increasingly recognized that the lack of stringent assessment of mitochondrial function during the discovery process has resulted in drug recalls, black box warnings, and an urgent need to understand the metabolic liability of small molecules in neural systems. Given that the brain is the most energetically demanding organ, even modest perturbations in neuronal energetic pathways have been shown to impact growth, signaling, connectivity, and the restorative capacity of the CNS. In this work, we describe several tools to assess metabolic activity of primary neuronal cultures and neural cell lines using an acute model of injury induced by oxygen glucose deprivation. Methods include the measurement of total ATP and NADH, enzymatic assessment of lactate production by anaerobic respiration, as well as viability assays. We also present a modified screening method for assessing aerobic respiration of immortalized cell lines using galactose challenge.

C-terminus of heat shock cognate 70 interacting protein increases following stroke and impairs survival against acute oxidative stress.

The decision to remove or refold oxidized, denatured, or misfolded proteins by heat shock protein 70 and its binding partners is critical to determine cell fate under pathophysiological conditions. Overexpression of the ubiquitin ligase C-terminus of HSC70 interacting protein (CHIP) can compensate for failure of other ubiquitin ligases and enhance protein turnover and survival under chronic neurological stress. The ability of CHIP to alter cell fate after acute neurological injury has not been assessed. Using postmortem human tissue samples, we provide the first evidence that cortical CHIP expression is increased after ischemic stroke. Oxygen glucose deprivation in vitro led to rapid protein oxidation, antioxidant depletion, proteasome dysfunction, and a significant increase in CHIP expression. To determine if CHIP upregulation enhances neural survival, we overexpressed CHIP in vitro and evaluated cell fate 24 h after acute oxidative stress. Surprisingly, CHIP overexpressing cells fared worse against oxidative injury, accumulated more ubiquitinated and oxidized proteins, and experienced decreased proteasome activity. Conversely, using small interfering RNA to decrease CHIP expression in primary neuronal cultures improved survival after oxidative stress, suggesting that increases in CHIP observed after stroke like injuries are likely correlated with diminished survival and may negatively impact the neuroprotective potential of heat shock protein 70.

Essential role of the redox-sensitive kinase p66shc in determining energetic and oxidative status and cell fate in neuronal preconditioning.

Ischemic preconditioning is a phenomenon in which low-level stressful stimuli upregulate endogenous defensive programs, resulting in subsequent resistance to otherwise lethal injuries. We previously observed that signal transduction systems typically associated with neurodegeneration such as caspase activation are requisite events for the expression of tolerance and induction of HSP70. In this work, we sought to determine the extent and duration of oxidative and energetic dysfunction as well as the role of effector kinases on metabolic function in preconditioned cells. Using an in vitro neuronal culture model, we observed a robust increase in Raf and p66(Shc) activation within 1 h of preconditioning. Total ATP content decreased by 25% 3 h after preconditioning but returned to baseline by 24 h. Use of a free radical spin trap or p66(shc) inhibitor increased ATP content whereas a Raf inhibitor had no effect. Phosphorylated p66(shc) rapidly relocalized to the mitochondria and in the absence of activated p66(shc), autophagic processing increased. The constitutively expressed chaperone HSC70 relocalized to autophagosomes. Preconditioned cells experience significant total oxidative stress measured by F(2)-isoprostanes and neuronal stress evaluated by F(4)-neuroprostane measurement. Neuroprostane levels were enhanced in the presence of Shc inhibitors. Finally, we found that inhibiting either p66(shc) or Raf blocked neuroprotection afforded by preconditioning as well as upregulation of HSP70, suggesting both kinases are critical for preconditioning but function in fundamentally different ways. This is the first work to demonstrate the essential role of p66(shc) in mediating requisite mitochondrial and energetic compensation after preconditioning and suggests a mechanism by which protein and organelle damage mediated by ROS can increase HSP70.

New approaches to neuroprotection in infant heart surgery.

Advances in surgical techniques and perioperative management have led to dramatic improvements in outcomes for children with complex congenital heart disease (CHD). As the number of survivors continues to grow, clinicians are becoming increasingly aware that adverse neurodevelopmental outcomes after surgical repair of CHD represent a significant cause of morbidity, with widespread neuropsychologic deficits in as many as 50% of these children by the time they reach school age. Modifications of intraoperative management have yet to measurably impact long-term neurologic outcomes. However, exciting advances in our understanding of the underlying mechanisms of cellular injury and of the events that mediate endogenous cellular protection have provided a variety of new potential targets for the assessment, prevention, and treatment of neurologic injury in patients with CHD. In this review, we will discuss the unique challenges to developing neuroprotective strategies in children with CHD and consider how multisystem approaches to neuroprotection, such as ischemic preconditioning, will be the focus of ongoing efforts to develop new diagnostic tools and therapies. Although significant challenges remain, tremendous opportunity exists for the development of diagnostic and therapeutic interventions that can serve to limit neurologic injury and ultimately improve outcomes for infants and children with CHD.

Electrophilic cyclopentenone neuroprostanes are anti-inflammatory mediators formed from the peroxidation of the omega-3 polyunsaturated fatty acid docosahexaenoic acid.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflammatory properties are poorly understood. DHA is highly susceptible to peroxidation, which yields an array of potentially bioactive lipid species. One class of compounds are cyclopentenone neuroprostanes (A(4)/J(4)-NPs), which are highly reactive and similar in structure to anti-inflammatory cyclopentenone prostaglandins. Here we show that a synthetic A(4)/J(4)-NP, 14-A(4)-NP (A(4)-NP), potently suppresses lipopolysaccharideinduced expression of inducible nitric-oxide synthase and cyclooxygenase-2 in macrophages. Furthermore, A(4)-NP blocks lipopolysaccharide-induced NF-kappaB activation via inhibition of Ikappa kinase-mediated phosphorylation of IkappaBalpha. Mutation on Ikappa kinase beta cysteine 179 markedly diminishes the effect of A(4)-NP, suggesting that A(4)-NP acts via thiol modification at this residue. Accordingly, the effects of A(4)-NP are independent of peroxisome proliferator-activated receptor-gamma and are dependent on an intact reactive cyclopentenone ring. Interestingly, free radical-mediated oxidation of DHA greatly enhances its anti-inflammatory potency, an effect that closely parallels the formation of A(4)/J(4)-NPs. Furthermore, chemical reduction or conjugation to glutathione, both of which eliminate the bioactivity of A(4)-NP, also abrogate the anti-inflammatory effects of oxidized DHA. Thus, we have demonstrated that A(4)/J(4)-NPs, formed via the oxidation of DHA, are potent inhibitors of NF-kappaB signaling and may contribute to the anti-inflammatory actions of DHA. These findings have implications for understanding the anti-inflammatory properties of omega-3 fatty acids, and elucidate novel interactions between lipid peroxidation products and inflammation.

Electrophilic cyclopentenone isoprostanes in neurodegeneration.

Although oxidative stress has been implicated in the pathogenesis of numerous neurodegenerative conditions, the precise mechanisms by which reactive oxygen species (ROS) induce neuronal death are still being explored. The generation of reactive lipid peroxidation products is thought to contribute to ROS neurotoxicity. Isoprostanes (IsoPs), prostaglandin-like molecules formed in vivo via the ROS-mediated oxidation of arachidonic acid, have been previously demonstrated to be formed in increased amounts in the brains of patients with various neurodegenerative diseases. Recently, we have identified a new class of IsoPs, known as A(2)- and J(2)-IsoPs or cyclopentenone IsoPs, which are highly reactive electrophiles and form adducts with thiol-containing molecules, including cysteine residues in proteins and glutathione. Cyclopentenone IsoPs are favored products of the IsoP pathway in the brain and are formed abundantly after oxidant injury. These compounds also potently induce neuronal apoptosis by a mechanism which involves glutathione depletion, ROS generation, and activation of several redox-sensitive pathways that overlap with those involved in other forms of oxidative neurodegeneration. Cyclopentenone IsoPs also enhance neurodegeneration caused by other insults at biologically relevant concentrations. These data are reviewed, whereas new data demonstrating the neurotoxicity of J-ring IsoPs and a discussion of the possible role of cyclopentenone IsoPs as contributors to neurodegeneration are presented.

Slit modulates cerebrovascular inflammation and mediates neuroprotection against global cerebral ischemia.

Cerebrovascular inflammation contributes to secondary brain injury following ischemia. Recent in vitro studies of cell migration and molecular guidance mechanisms have indicated that the Slit family of secreted proteins can exert repellant effects on leukocyte recruitment in response to chemoattractants. Utilizing intravital microscopy, we addressed the role of Slit in modulating leukocyte dynamics in the mouse cortical venular microcirculation in vivo following TNFalpha application or global cerebral ischemia. We also studied whether Slit affected neuronal survival in the mouse global ischemia model as well as in mixed neuronal-glial cultures subjected to oxygen-glucose deprivation. We found that systemically administered Slit significantly attenuated cerebral microvessel leukocyte-endothelial adherence occurring 4 h after TNFalpha and 24 h after global cerebral ischemia. Administration of RoboN, the soluble receptor for Slit, exacerbated the acute chemotactic response to TNFalpha. These findings are indicative of a tonic repellant effect of endogenous Slit in brain under acute proinflammatory conditions. Three days of continuous systemic administration of Slit following global ischemia significantly attenuated the delayed neuronal death of hippocampal CA1 pyramidal cells. Moreover, Slit abrogated neuronal death in mixed neuronal-glial cultures exposed to oxygen-glucose deprivation. The ability of Slit to reduce the recruitment of immune cells to ischemic brain and to provide cytoprotective effects suggests that this protein may serve as a novel anti-inflammatory and neuroprotective target for stroke therapy.

Cyclopentenone isoprostanes are novel bioactive products of lipid oxidation which enhance neurodegeneration.

Oxidative stress and subsequent lipid peroxidation are involved in the pathogenesis of numerous neurodegenerative conditions, including stroke. Cyclopentenone isoprostanes (IsoPs) are novel electrophilic lipid peroxidation products formed under conditions of oxidative stress via the isoprostane pathway. These cyclopentenone IsoPs are isomeric to highly bioactive cyclopentenone prostaglandins, yet it has not been determined if these products are biologically active or are formed in the brain. Here we demonstrate that the major cyclopentenone IsoP isomer 15-A2t-IsoP potently induces apoptosis in neuronal cultures at submicromolar concentrations. We present a model in which 15-A2t-IsoP induced neuronal apoptosis involves initial depletion of glutathione and enhanced production of reactive oxygen species, followed by 12-lipoxygenase activation and phosphorylation of extracellular signal-regulated kinase 1/2 and the redox sensitive adaptor protein p66shc, which results in caspase-3 cleavage. 15-A2t-IsoP application also dramatically potentiates oxidative glutamate toxicity at concentrations as low as 100 nm, demonstrating the functional importance of these molecules in neurodegeneration. Finally, we employ novel mass spectrometric methods to show that cyclopentenone IsoPs are formed abundantly in brain tissue under conditions of oxidative stress. Together these findings suggest that cyclopentenone IsoPs may contribute to neuronal death caused by oxidative insults, and that their activity should perhaps be addressed when designing neuroprotective therapies.

Caspase 3 activation is essential for neuroprotection in preconditioning.

Sublethal insults can induce tolerance to subsequent stressors in neurons. As cell death activators such as ROS generation and decreased ATP can initiate tolerance, we tested whether other cellular elements normally associated with neuronal injury could add to this process. In an in vivo model of ischemic tolerance, we were surprised to observe widespread caspase 3 cleavage, without cell death, in preconditioned tissue. To dissect the preconditioning pathways activating caspases, and the mechanisms by which these proteases are held in check, we developed an in vitro model of excitotoxic tolerance. In this model, antioxidants and caspase inhibitors blocked ischemia-induced protection against N-methyl-d-aspartate toxicity. Moreover, agents that blocked preconditioning also attenuated induction of HSP 70; transient overexpression of a constitutive form of this protein prevented HSP 70 up-regulation and blocked tolerance. We outline a neuroprotective pathway where events normally associated with apoptotic cell death are critical for cell survival.