Luke I. Szweda

The glial glutamate transporter, GLT‐1, is oxidatively modified by 4‐hydroxy‐2‐nonenal in the Alzheimer's disease brain: the role of Aβ1–42

Glutamate transporters are involved in the maintenance of synaptic glutamate concentrations. Because of its potential neurotoxicity, clearance of glutamate from the synaptic cleft may be critical for neuronal survival. Inhibition of glutamate uptake from the synapse has been implicated in several neurodegenerative disorders. In particular, glutamate uptake is inhibited in Alzheimers disease (AD); however, the mechanism of decreased transporter activity is unknown. Oxidative damage in brain is implicated in models of neurodegeneration, as well as in AD. Glutamate transporters are inhibited by oxidative damage from reactive oxygen species and lipid peroxidation products such as 4‐hydroxy‐2‐nonenal (HNE). Therefore, we have investigated a possible connection between the oxidative damage and the decreased glutamate uptake known to occur in AD brain. Western blots of immunoprecipitated HNE‐immunoreactive proteins from the inferior parietal lobule of AD and control brains suggest that HNE is conjugated to GLT‐1 to a greater extent in the AD brain. A similar analysis of beta amyloid (Aβ)‐treated synaptosomes shows for the first time that Aβ1–42 also increases HNE conjugation to the glutamate transporter. Together, our data provide a possible link between the oxidative damage and neurodegeneration in AD, and supports the role of excitotoxicity in the pathogenesis of this disorder. Furthermore, our data suggests that Aβ may be a possible causative agent in this cascade.

Modulation of Mitochondrial Function by Hydrogen Peroxide

During normal cellular metabolism, mitochondrial electron transport results in the formation of superoxide anion (O⨪2) and subsequently hydrogen peroxide (H2O2). Because H2O2 increases in concentration under certain physiologic and pathophysiologic conditions and can oxidatively modify cellular components, it is critical to understand the response of mitochondria to H2O2. In the present study, treatment of isolated rat heart mitochondria with H2O2 resulted in a decline and subsequent recovery of state 3 NADH-linked respiration. Alterations in NADH levels induced by H2O2 closely paralleled changes in the rate of state 3 respiration. Assessment of electron transport chain complexes and Krebs cycle enzymes revealed that α-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and aconitase were susceptible to H2O2 inactivation. Of particular importance, KGDH and SDH activity returned to control levels, concurrent with the recovery of state 3 respiration. Inactivation is not because of direct interaction of H2O2 with KGDH and SDH. In addition, removal of H2O2alone is not sufficient for reactivation. Enzyme activity does not recover unless mitochondria remain intact. The sensitivity of KGDH and SDH to H2O2-mediated inactivation and the reversible nature of inactivation suggest a potential role for H2O2 in the regulation of KGDH and SDH.

INHIBITION OF THE MULTICATALYTIC PROTEINASE (PROTEASOME) BY 4-HYDROXY-2-NONENAL CROSS-LINKED PROTEIN

© 1997 Federation of European Biochemical Societies.

The Oxygen-Rich Postnatal Environment Induces Cardiomyocyte Cell-Cycle Arrest through DNA Damage Response

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

Caloric restriction of rhesus monkeys lowers oxidative damage in skeletal muscle

In laboratory rodents, caloric restriction (CR) retards several age‐dependent physiological and biochemical changes in skeletal muscle, including increased steady‐state levels of oxidative damage to lipids, DNA, and proteins. We used immunogold electron microscopic (EM) techniques with antibodies raised against 4‐hydroxy‐2‐nonenal (HNE) ‐modified proteins, dinitrophenol, and nitrotyrosine to quantify and localize the age‐dependent accrual of oxidative damage in rhesus monkey vastus lateralis skeletal muscle. Using immunogold EM analysis of muscle from rhesus monkeys ranging in age from 2 to 34 years old, a fourfold maximal increase in levels of HNE‐modified proteins was observed. Likewise, carbonyl levels increased ~ twofold with aging. Comparing 17‐ to 23‐year‐old normally fed to age‐matched monkeys subjected to CR for 10 years, levels of HNE‐modified proteins, carbonyls, and nitrotyrosine in skeletal muscle from the CR group were significantly less than control group values. Oxidative damage largely localized to myofibrils, with lesser labeling in other subcellular compartments. Accumulation of lipid peroxidation‐derived aldehydes, such as malondialdehyde and 4‐hydroxy‐2‐alkenals, and protein carbonyls were measured biochemically and confirmed the morphological data. Our study is the first to quantify morphologically and localize the agedependent accrual of oxidative damage in mammalian skeletal muscle and to demonstrate that oxidative damage in primates is lowered by CR.—Zainal, T. A., Oberley, T. D., Allison, D. B., Szweda, L. I., Weindruch, R. Caloric restriction of rhesus monkeys lowers oxidative damage in skeletal muscle. FASEB J. 14, 1825–1836 (2000)

Aldose reductase functions as a detoxification system for lipid peroxidation products in vasculitis

Giant cell arteritis (GCA) is a systemic vasculitis preferentially affecting large and medium-sized arteries. Inflammatory infiltrates in the arterial wall induce luminal occlusion with subsequent ischemia and degradation of the elastic membranes, allowing aneurysm formation. To identify pathways relevant to the disease process, differential display-PCR was used. The enzyme aldose reductase (AR), which is implicated in the regulation of tissue osmolarity, was found to be upregulated in the arteritic lesions. Upregulated AR expression was limited to areas of tissue destruction in inflamed arteries, where it was detected in T cells, macrophages, and smooth muscle cells. The production of AR was highly correlated with the presence of 4-hydroxynonenal (HNE), a toxic aldehyde and downstream product of lipid peroxidation. In vitro exposure of mononuclear cells to HNE was sufficient to induce AR production. The in vivo relationship of AR and HNE was explored by treating human GCA temporal artery-severe combined immunodeficiency (SCID) mouse chimeras with the AR inhibitors Sorbinil and Zopolrestat. Inhibition of AR increased HNE adducts twofold and the number of apoptotic cells in the arterial wall threefold. These data demonstrate that AR has a tissue-protective function by preventing damage from lipid peroxidation. We propose that AR is an oxidative defense mechanism able to neutralize the toxic effects of lipid peroxidation and has a role in limiting the arterial wall injury mediated by reactive oxygen species.

Reperfusion-Induced Translocation of δPKC to Cardiac Mitochondria Prevents Pyruvate Dehydrogenase Reactivation

Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive &dgr;-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the &dgr;PKC activator H2O2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of &dgr;PKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of &dgr;PKC is associated with phosphorylation of the &agr;E1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that &dgr;PKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of &dgr;PKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.

Mitochondrial protein oxidation and degradation in response to oxidative stress and aging.

Mitochondria are a major source of intracellular reactive oxygen species (ROS), the production of which increases with age. These organelles are also targets of oxidative damage. The deleterious effects of ROS may be responsible for impairment of mitochondrial function observed during various pathophysiological states associated with oxidative stress and aging. An important factor for protein maintenance in the presence of oxidative stress is enzymatic reversal of oxidative modifications and/or protein degradation. Failure of these protein maintenance systems is likely a critical component of the aging process. Mitochondrial matrix proteins are sensitive to oxidative inactivation and oxidized proteins are known to accumulate during aging. The ATP-stimulated mitochondrial Lon protease is a highly conserved protease found in prokaryotes and the mitochondrial compartment of eukaryotes and is believed to play an important role in the degradation of oxidized mitochondrial matrix proteins. Age-dependent declines in the activity and regulation of this proteolytic system may underlie accumulation of oxidatively modified and dysfunctional protein and loss in mitochondrial viability.

Reversible redox-dependent modulation of mitochondrial aconitase and proteolytic activity during in vivo cardiac ischemia/reperfusion

Prooxidents can induce reversible inhibition or irreversible inactivation and degradation of the mitochondrial enzyme aconitase. Cardiac ischemia/reperfusion is associated with an increase in mitochondrial free radical production. In the current study, the effects of reperfusion-induced production of prooxidants on mitochondrial aconitase and proteolytic activity were determined to assess whether alterations represented a regulated response to changes in redox status or oxidative damage. Evidence is provided that ATP-dependent proteolytic activity increased during early reperfusion followed by a time-dependent reduction in activity to control levels. These alterations in proteolytic activity paralleled an increase and subsequent decrease in the level of oxidatively modified protein. In vitro data supports a role for prooxidants in the activation of ATP-dependent proteolytic activity. Despite inhibition during early periods of reperfusion, aconitase was not degraded under the conditions of these experiments. Aconitase activity exhibited a decline in activity followed by reactivation during cardiac reperfusion. Loss and regain in activity involved reversible sulfhydryl modification. Aconitase was found to associate with the iron binding protein frataxin exclusively during reperfusion. In vitro, frataxin has been shown to protect aconitase from [4Fe-4S]2+ cluster disassembly, irreversible inactivation, and, potentially, degradation. Thus, the response of mitochondrial aconitase and ATP-dependent proteolytic activity to reperfusion-induced prooxidant production appears to be a regulated event that would be expected to reduce irreparable damage to the mitochondria.

Proteolysis, free radicals, and aging,

Aging is accompanied by declines in cellular proteolytic capacity. Proteolytic processing is an important step in numerous cellular processes required for normal metabolic function. These include regulation of protein turnover, degradation of altered forms of protein, signal transduction, protein sorting/trafficking, receptor-mediated endo- and exocytosis, stress/immune responses, and activation of gene transcription. Thus, loss of cellular proteolytic function is likely to contribute to the enhanced fragility of cells from senescent relative to young and adult organisms. Free radicals have been implicated as contributing factors to observed age-dependent declines in proteolytic capacity. The current review offers an overview of the evidence linking free radical events to functional alterations in the lysosomal system and the proteasome, two major pathways by which proteins are degraded within cells. Implications for future investigations in the field are discussed in light of these findings.