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Free Radicals: Key to Brain Aging and Heme Oxygenase as a Cellular Response to Oxidative Stress

¹ Department of Chemistry, Center of Membrane Sciences, and Sanders-Brown Center on Aging, University of Kentucky, Lexington.
² Section of Biochemistry and Molecular Biology, Department of Chemistry, Faculty of Medicine, University of Catania, Italy.

Address correspondence to Professor D. Allan Butterfield, Department of Chemistry, Center of Membrane Sciences and Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40506-0055. E-mail: dabcns@uky.edu

	Abstract

Top Abstract Lipid Peroxidation Protein Oxidation The Heat Shock Pathway... HO for Neuroprotective... Conclusion References

 
Aging is one of the unique features in all organisms. The impaired functional capacity of many systems characterizes aging. When such impairments occur in the brain, the susceptibility to neurodegenerative diseases amplifies considerably. The free radical theory of aging posits that the functional impairments in brains are due to the attack on critical cellular components by free radicals, reactive oxygen species, and reactive nitrogen species produced during normal metabolism. In this review, we examine this concept based on the parameters of oxidative stress in correlation to aging. The parameters for lipid peroxidation are phospholipid composition, reactive aldehydes, and isoprostanes. The parameters for protein oxidation are protein carbonyl levels, protein 3-nitrotyrosine levels, electron paramagnetic resonance, and oxidative stress-sensitive enzyme activities. We conclude that free radicals are, at least partially, responsible for the functional impairment in aged brains. The aging brain, under oxidative stress, responds by induction of various protective genes, among which is heme oxygenase. The products of the reaction catalyzed by heme oxygenase, carbon monoxide, iron, and biliverdin (later to bilirubin) each have profound effects on neurons. Although there may be other factors contributing to brain aging, free radicals are involved in the damaging processes associated with brain aging, and cellular stress response genes are induced under free radical oxidative stress. Therefore, this review supports the proposition that free radicals are, indeed, a key to brain aging.

The free radical theory of aging is based on the reactive free radical reactions and their ubiquitous prominent presence in living systems. Since all aerobic organisms utilize O₂ for energy, generation of free radicals by enzymatic or nonenzymatic reaction as well as by "leakage" from mitochondria is inevitable. When oxygen is partially reduced, less-toxic species with longer half-lives are formed, and these species can attack cell components distant from where those free radicals were produced. These less-toxic species and free radicals are collectively called reactive oxygen species (ROS) and reactive nitrogen species (RNS). Reactive oxygen species refer to molecules that contain oxygen with higher reactivity than the ground-state O₂. Reactive oxygen species include, among others, superoxide (O₂·), hydroxyl radical (HO·), hydrogen peroxide (H₂O₂), and hypochlorous acid (HOCl). Reactive nitrogen species refer to nitric oxide (NO) and molecules derived from NO, such as peroxynitrite (-OONO), nitroyl (·NO), and nitrogen dioxide (NO₂). Reactive oxygen species and RNS have the capacity to modify proteins, lipids, and nucleic acids to develop or enhance age-related manifestations.

Fortunately, living organisms evolved several mechanisms to protect themselves from the attack of ROS and RNS. These antioxidant defense mechanisms include: (a) scavenging ROS/RNS and their precursors; (b) binding catalytic metal ions needed for ROS formation; and (c) generating and up-regulating endogenous antioxidant defenses. Two major groups of antioxidant defense systems are low molecular weight antioxidant compounds, e.g., vitamins C and E, lipoic acid, and ubiquinones, and antioxidant enzymes, e.g., superoxide dismutases (SOD), superoxide reductases (SOR), catalase, and glutathione peroxidases (GPx), among others (4). When the production of ROS and RNS exceed the capacity of the antioxidant defenses systems, the new state is termed oxidative stress.

The brain is more susceptible to oxidative stress for the following reasons: (a) high content of peroxidizable unsaturated fatty acids; (b) high oxygen consumption per unit weight; (c) high content of lipid peroxidation key ingredients (iron and ascorbate); and (d) the scarcity of antioxidant defenses systems (5). It is widely held that free radical-induced oxidative stress increases in brain aging. We review this concept based on the parameters of oxidative stress in the brain. This review provides a critical evaluation on whether free radicals are the key to brain aging.

	LIPID PEROXIDATION

Top Abstract Lipid Peroxidation Protein Oxidation The Heat Shock Pathway... HO for Neuroprotective... Conclusion References

 
Background
The mechanism of lipid peroxidation can be divided into 3 stages: initiation, propagation, and termination. In initiation, since OH· is a highly reactive ROS (6), it attacks the hydrogens from nearly any C–H bond to form H₂O. The electron-rich hydrogens at the CH₂ group in the system -CH=CH-CH₂-CH=CH- of polyunsaturated fatty acids (PUFA) are even more vulnerable to the OH· attack due to the electron-donating double bonds (7). The OH·, along with other radicals (8), generate racemic peroxyl radicals that attack the hydrogens from other PUFAs and begin a chain reaction (7). These chain reactions are collectively called propagation. The peroxyl radicals then undergo the Esterbauer dioxetane mechanism to form multiple aldehydes with varying lengths of carbons, such as 4-hydroxyl-2-nonenal (HNE) (9), and terminate the chain reaction, thus the termination stage. The end products of lipid peroxidation are usually more stable than the free radicals, serving as excellent markers for free radical reactions. Although these aldehydes have lower activity than free radicals, their longer half-life potentially allows them to be more neurotoxic.

Phospholipid Composition
One way to assess lipid peroxidation is to use the compositional alterations of brain phospholipids. PUFAs, such as arachidonic (AA, 20:4n-6), docosatetraenoic acid (DCS, 22:4n-6), and decosohexaenic acid (DCH, 22:6n-6), are abundant in brains and highly susceptible to free radical attack by the mechanism mentioned above. When PUFAs act as substrates of lipid peroxidation and become oxidized, their lipid peroxidation end-product fatty acids, e.g., oleic acid (18:1n-9), godonic acid (20:1n-9), and arachidonic acid, process reduced number of double bonds as well as different chemical and physical properties. Therefore, the quantity of lipid peroxidation can be estimated by measuring the depletion of PUFAs or the ratio change between PUFAs and saturated/monounsaturated fatty acids. These measurements can be performed by simple extraction (10) and separation of the fatty acids by thin layer chromatography (TLC) (11,12), gas chromatography (GC) (13), or high-performance liquid chromatography (HPLC) (14) followed by ultraviolet (UV) spectrophotometry, gas chromatography, or mass spectrometry for identification of the fatty acids.

Brain aging is accompanied by changes in the overall membrane lipid composition (15,16). The most important changes involve a decreased level in PUFA (20:4n-6, 22:4n-6, 22:6n-3) and an increase in monounsaturated fatty acids (18:1n-9 and 20:1n-9) in cerebral cortex and cerebellum of aged rats (17). Moreover, arachidonic acid, along with n-6 and n-3 fatty acids, were also reported to decrease in the brains of aged rats with cognitive deficit (18). This study suggested that low levels of AA might be related to the cognitive deficit in rats, as membrane AA concentration were shown to be correlated with the long-term potentiation (LTP). Long-term potentiation is a form of synaptic plasticity characterized by persistent synaptic efficacious increases, which follow tetanic stimulation from an afferent pathway to one of the hippocampal subfields (19). The ability to sustain LTP is usually used as a parameter of cognitive function. The changes in phospholipid composition provide evidence that free radical-mediated lipid peroxidation occurs in brains, and such changes might also be responsible for the cognitive deficit observed in aged animals and humans.

Reactive Aldehydes
HNE is an end product of AA or linoic acid (mainly, but not exclusively) peroxidation (9). Because of its extensive half-life, HNE is able to diffuse to sites that are distant from that of its formation and cause further damage (20). HNE causes neurotoxicity and apoptosis in many ways. This major product of lipid peroxidation can inhibit proliferation and cell cycle progression by the down-regulation of D1, D2, and A cyclin expression (21). It can also react with some bases of DNA and lipid amino groups by Michael addition (22,23) to alter the proper function of these biological molecules. The most damaging effect of HNE comes from its ability to form covalent adducts of histidine, lysine, and cysteine residues in proteins to modify their activity (20). HNE deactivates aldose reductase and glutathione peroxidase, which detoxify HNE (24) and catalyze the reduction of H₂O₂ by glutathione (25), respectively. HNE reduces Na⁺/K⁺–ATPase activity (26). Reduction of these critical ion pumps causes neurotoxicity in rat hippocampal neurons by disrupting the Ca²⁺ homeostasis (27) and impairing glucose transport (28).

Acrolein (2-propen-1-al) is the most reactive of the ,ß-unsaturated aldehydes formed by lipid peroxidation of fatty acid sources that are not fully characterized as yet (29). Sharing the chemical properties of HNE, acrolein induces toxicity to primary hippocampal culture (30). This toxicity can result from the distortion of the transmembrane and cytoskeletal proteins structures (31) as well as the inhibition of the glucose and glutamate uptake (32).

Since acrolein and HNE adducts are more stable than most free radicals, acrolein- and HNE adducts, in particular acrolein-modified and HNE-modified proteins, provide an effective marker for free radical-mediated lipid peroxidation. It was also suggested that the protein-bound acrolein is a potential marker of oxidative stress in aging (29). Studies have shown that the amount of HNE-modified protein staining increased logarithmically with age in human oculomotor neurons but not melanized neurons (33). Additionally, the HNE-modified proteins, along with neurofibrillary tangles, are also observed in the senile plaques in aged dogs (34). Both HNE and acrolein are elevated in the AD brain (30,35,36).

Malondialdehyde (MDA) is the most abundant toxic aldehyde formed when AA acts as the PUFA source for the lipid peroxidation (37–39). Lysine or arginine can catalytically hydrolyze MDA to formic acid or acetaldehydes (40). Similar to HNE, MDA can react with amino acids in proteins to for MDA adducts. Lysine, tryptophan, and histidine react with MDA to form N-ß-lysyl-amino-acrolein (ß-LAA) (41,42), whereas arginine reacts with MDA to form N-(2-pyrimidyl)-l-ornithine (NPO) (43,44). ß-LAA can then further react with another MDA molecule to form N-lysyl-4-methyl-2,6-dihydropyridine-3,5-dicarbaldehyde (NLMDD) (40,45). This latter molecule contains an aldehyde side-chain on the dihydropyridine ring that is responsible for reactions with lysine, to form a reversible cross-link (42). Although there is no evidence that such a reaction occurs physiologically, it is possible that HNE, arolein, and so forth can also react, in place of the second MDA, with NLMDD to form a reversible cross-link reaction. (46). MDA is also a highly reactive genotoxic compound that induces DNA damage by reacting with nucleic acids to form adducts, thus disrupting base pairing (47). One of these adducts, deoxyguanosine-MDA (dG-MDA), was shown to induce cell cycle arrests in human cell cultures (48).

MDA reacts with thiobarbituric acid (TBA) to produce fluorescent thiobarbituric acid-reacting substances (TBARS), commonly used to measure MDA levels (49,50). However, TBA also reacts with other carbonyl-containing compounds. Therefore, TBARS are generally considered a nonspecific measurement of MDA. Such a problem can be corrected through HPLC coupling with postcolumn TBA derivatization. This column can specifically identify TBA–MDA complexes, thereby providing a relevant assay in biological systems (51–53). The cellular distribution of MDA can also be assessed immunohistochemically (46).

In aged human brain, it was shown that the MDA was increased in the inferior temporal corticies (54) and in the cytoplasm of neurons and astrocytes (55) compared with young controls. Through electron microscopic immunohistochemistry, MDA was shown to form linear deposits. These deposits are cap-like when associating with lipofuscin in neurons and vacuole encircling when found in the glia in the CA4 region of the human hippocampus (55). In a human aging canine model, which naturally develops extensive diffused deposits of human-type amyloid ß-peptide (Aß), MDA was increased in the prefrontal cortex of aged brains (56). Although there is no significant increase of MDA-modified protein observed in aged rat brains (57), it was shown that the basal MDA level was significantly elevated (19%) in the hippocampi of old rats (58). Additionally, 5 dG-MDA adducts were identified as the indigenous DNA (iDNA) adducts, which is considered a biomarker of the aging brain (59) due to the increase of iDNA with age (60–63). The above studies suggest that the presence of free radical-mediated MDA production could induce further damage of the proteins or DNAs in the brain. In the Mooradian and colleagues study (57), the increased MDA-modified proteins were not observed. This could be due to the fact that MDA-modified protein reacts with lipid peroxidation products to cross-link with each other. Therefore, MDA-modified proteins could not be detected immunochemically.

Isoprostanes
Isoprostanes are another group of compounds that result from lipid peroxidation. Two groups of isoprostanes were commonly used to index lipid peroxidation: isoprostane (IP) and neuroprostanes (NP). IP is a prostaglandin F₂-like compound formed by the nonenzymatic peroxidation of AA (64). In the initiation stage when O₂· attacks AA, 3 arachidonyl radicals are formed to yield 4 prostaglandin H₂-like bicyclic endoperoxide intermediate regioisomers. These regioisomers are then reduced to F-ring regioisomers to form F₂-IP in different series, according to the carbon number on the hydroxyl side of the chain. However, during the reduction, rearrangement could occur to create E-, D-, throboxin-, A-, and J-ring IP as well as highly active acyclic -ketoaldehydes, namely isoketals (65–67). All of these isomers are termed as IP (68). F₂-IP and E₂- IP are involved in mediation of receptor action, such as vasoconstriction, due to their inherent chemical reactivity. A₂-IPs and J₂-IP, as well as isoketals, are involved in adduct formation due to their reactivity (65,69–71). F₂-IPs are an ideal index for lipid peroxidation because of their stability and specificity (72,73). F₂-IPs are initially found in esterified form. They are then released in free form by a phospholipase(s) because most AAs are esterified in phospholipids (74). Similar to assessments of PUFAs level, the assessments of free and esterified F₂-IPs can be accomplished immunochemically (75) or by TLC, HPLC (73), or GC (76) coupled with MS (77).

Neuroprostanes are a set of IPs uniquely formed from the free radical-mediated peroxidation of DCH in brains (78,79). Although DCH is highly enriched in neurons, those neurons are not able to elongate and desaturate essential fatty acids to form DCH. DCH is synthesized and secreted by astrocytes and then captured by neurons (80). Similar to the mechanisms of IP formation, O₂· attacks DCH in the initiation stage of NP formation. However, 8 peroxyl radicals are formed, rather than 4 in AA, since DCH is 2 carbons longer than AA and able to form different regioisomers. These peroxyl radicals then undergo a similar process as in the AA reaction to develop F₄-IP or NP (79). The rearrangement during the reduction step can also result in reactive -ketoaldehydes, named neuroketals (81). Both neuroketals and isoketals demonstrate the inhibition of proteasome function (82). Due to the similar chemical properties of F₄-IPs, NP can be detected by the same methodologies described above.

Although elevated free and esterified F₂-IPs in plasma were reported in aged animals (72), interestingly, the study of isoprostane levels in the brain found that F₂-NP and NP showed no significant differences among the brains of young, adult, and old rats (83). This finding is rather controversial to the other findings of lipid peroxidation during aging (15–18,29,33,34,54–56,58,60–62). However, such concealed differences of the IP and NP levels in aged brains could be due to the reduced glutathione (GSH) level generally in aged brains (84,85). Since GSH is an important effector for the reduction of endoperoxide intermediates to F-ring IPs (86), reduced GSH levels may not effectively reduce all the peroxyl radicals to F₂-IPs. Since GSH levels are age dependent (87) and abnormal in AD brains (88,89), the increased levels of F₂-isoprostanes were observed in the cerebrospinal fluid (CSF) of AD patients (90), as well as in the brains of AD models, e.g., mice with apolipoprotein E (ApoE) deficiency (91) and AD amyloidosis (92). Therefore, lack of differences observed in the IPs and NPs level in aged brains could be due to the reduced GSH level.

Lipid Peroxidation Summary
In short, the results above suggest that free radicals are important in lipid peroxidation in the brain. Although others might argue that lipid hydroperoxides can also be formed enzymatically by lipoxygenases (93), most lipoxygenases require as substrates free PUFAs, which are only present in trace amounts in healthy tissue (94). Free PUFAs are generated by the activation of membrane-bound phospholipase A₂ (95,96). Oxidative stress-associated activation and enhanced function of cytosolic phospholipase A₂ have been proposed to be critical factors in the accumulation of PUFAs (97). These alterations of cytosolic phospholipase A₂ are due to the increase in H₂O₂-induced intracellular Ca²⁺ concentration (98). Therefore, it seems that free radicals play a key role in lipid peroxidation, whether that be in enzymatically or nonenzymatically driven reactions.

	PROTEIN OXIDATION

Top Abstract Lipid Peroxidation Protein Oxidation The Heat Shock Pathway... HO for Neuroprotective... Conclusion References

 
Introduction
Protein oxidation is an exothermic event where peptides react with free radicals. This reaction is mainly, though not exclusively, mediated by the OH; which is mostly produced by the Fenton reaction through the decomposition of H₂O₂ in the presence of redox metals (Cu⁺ and Fe²⁺) (20). Free radicals can attack peptides at two locations: back bone and side chain. In backbone modification, a free radical attacks the hydrogen on the -carbon to form a carbon-centered radical. In the presence of oxygen, this radical then further converts into a peroxyl radical (20), which can attack other hydrogens of the same or differing peptides to propagate the free radical oxidation in a similar fashion as the initial and propagation stage of free radical-mediated lipid peroxidation. Such oxidation can lead to protein cross-linking and/or peptide bond cleavage. Four types of cleavage are reported: (a) -amidation, which yields RCOCOOH and NH₃; (b) diamide, which yields RCOOH, CO₂, and NH₃; (c) glutamate oxidation, which yields CH₃COCOOH, HOOC-COOH, and NH₃; and (d) proline oxidation, which yields H₂NCH₂(CH₂)₂COOH and CO₂ (99). In side-chain modification, the free radicals attack the amino acid side chains of a peptide. Although most amino acid side chains are vulnerable to oxidative modification, only some of these modifications are fully characterized: (a) histidine modified to aspartic acid, aspargine, or oxo-histidine; (b) tyrosine modified to 3,4-dihydroxyphenylalanine and Tyr–Tyr cross-links; (c) phenylalanine modified to 2-, 3-, 4-hydroxyphenylalanine; (d) tryptophan modified to N-formylkynureine, kynurenine, 2-, 4-, 5-, and 6-hydroxyvaline; (e) valine modified to 3-hydroxyvaline; (f) leucine modified to 3- and 4-hydroxyleucine; (g) lysine modified to 2-aminoadipic semialdehyde; (h) arginine modified to glutamic semialdehyde; (i) proline modified to glutamic semialdehyde, pyroglutamic acid, 2-pyroglutamic acid, 2-pyrrolidone, and 4-hydroxyproline; (j) glutamic acid modified to 4-hydroxyglutamic acid, and pyruvate -ketoglutaric acid; (k) threonine modified to 2-amino-3-ketobutyric acid; (l) methionine modified to methionine sulfoxide and methionine sulfone; and (m) cysteine modified to disulfides such as Cys-S-S-Cys and Cys-S-S-R (100). These oxidative modifications generally cause the loss of catalytic or structural function in the affected protein and contribute serious deleterious effects on cellular and organ functions (99).

Protein Carbonyl Levels
Carbonyl level is probably the most commonly used method of assessing the oxidative modification of proteins (20). Besides introducing carbonyls to proteins directly as previously described, free radicals can introduce carbonyls to protein through covalent reaction with lipid peroxidation end products such as HNE, acrolein, and MDA (20). Due to the resonance effect between the double bonds, the -carbon of these reactive aldehydes are highly electrophilic and able to covalently bind to the electron-rich site of Cys, His, and Lys by Michael addition (Figure 1). Therefore, the increased carbonyl content is introduced to the proteins, as these aldehydes process at least one carbonyl group. Carbonyl content of proteins are generally indexed by its 2,4-dinitrophenylhydrazone (DNP-) adduct formed by reacting the proteins with 2,4-dinitrophenylhydrazine (DNPH) (20). The DNP adduct can be detected by UV-Vis, fluorescence spectroscopy, or immunochemically. It should be noticed that the quantity of the protein carbonyl level depends not only on its formation, but also the degradation of the oxidized protein. Therefore, we can only conclude that increased carbonyl levels are due to increased free radical attack if the protein degradation system is normal.

View larger version (12K): [in this window] [in a new window]   Figure 1. The carbonyl group was introduced to protein lysine (P-Lys), histidine (P-His), or cysteine (P-Cys) residues by Michael addition (233)

Protein 3-Nitrotyrosine (3-NT) Level
3-Nitrotyrosines result from the nitration of the ortho position of the aromatic ring on tyrosine residues of proteins (20,109). Such reaction is a form of protein oxidation by definition (27). 3-Nitrotyrosines can make the aromatic moiety more hydrophilic, thereby altering the secondary and tertiary structures of a protein. It can also sterically or electronically hinder the phosphorylation or dephosphorylation on the para-OH group of Tyr, thus interfering with the cell signaling (110). Protein 3-NT levels can be detected immunochemically or by HPLC coupled with MS/MS to avoid the electrochemical potential complications of 3-NT (111).

Although protein 3-NT levels of brain homogenate were found to decrease in aged Wistar rats (104), it was increased in the hippocampus and the cerebral cortex of aged rats (112), the CSF of aged human (113), and the subcortical white matter of aged monkeys (114). Moreover, the most prominent labeling of 3-NT was discovered in Prukije cell layers and molecular layers of the cerebellar cortex, as well as in the surroundings of neuropil in the cerebellar nuclei of aged rats (115). These findings suggest that increases in protein 3-NT levels are in a regionally specific fashion in aging. Such specificity could be attributed to the level of nitric oxide synthase, which produces NO to activate tyrosine nitration, in cells (113); in contrast, 3-NT occurs in a wide-spread manner in the AD brain (116,117). Recently, proteomics analysis identified that specific proteins were increased in 3-NT in the AD brain (118).

Electron Paramagnetic Resonance
Free radical-mediated protein oxidation can also be indirectly assessed by EPR spin labels. A 2,2,6,6-tetramethyl-4-maleimidopiperdin-oxyl (MAL-6) spin label (119–125) can covalently bond to SH groups on Cys. The ratio of the EPR signal intensity of the low-field weakly immobilized resonance line (W) to that of the strongly immobilized line (S) (the W/S ratio) is a parameter that reflects motion of the spin label bound to proteins (119–125). The amplitude of the low-field weakly immobilized line indicates the relatively fast MAL-6 motion, as this protein-specific spin label binds to SH groups in which the MAL-6 motion is only weakly immobilized. In contrast, the amplitude of the low-field strongly immobilized line indicates the relatively slow MAL-6 motion as this spin label binds to SH groups in which the MAL-6 motion is strongly immobilized. Therefore, the W/S ratio is lowered when protein cross-linkings increase, protein–protein interactions increase, and segmental motion of spin-labeled proteins decrease. Since all of these protein alterations occur when free radicals induce protein oxidation, a change in the W/S ratio indexes the protein conformation change due to free radical modification (119–125).

It was found that the W/S ratio is extremely precise when assessing the oxidative conformational changes of proteins (119–125). It was also found that the W/S ratio decreases when OH· induce oxidation to rodents' synaptosomes in vivo and in vitro (123,124). Such a W/S ratio decrease is also observed in the cortical synaptosomes and erythrocyte membranes of hyperoxia-exposed rodents (125) and the synaptosomes of senescence-accelerated mice (SAM) (108). This suggests that free radical-induced protein oxidations, and thus conformational alterations, are significant events that occur during brain aging.

Oxidative Stress-Sensitive Enzyme Activity
Oxidative deactivation of enzymes is another index of free radical oxidative damage to proteins. As described above, the free radical-induced oxidative modification of proteins usually results in structural and functional change in these proteins (119–125). Decreased activities of glutamine synthetase (GS), creatine kinase (CK), and tyrosine hydroxylase (126–129) by free radical-induced modification were observed. Since the activities of these enzymes, particularly CK and GS, can be easily determined, their activities serve as efficient indices for oxidative modification of proteins.

Studies shows that the activities of CK and GS were decreased in aged brown Norway rats brains (102), in aged gerbil brains (130), and in aged human frontal and occipital lobes (101). Since CK and GS activity is very sensitive to free radical-induced oxidative modification, the declined activity of CK and GS in aged animals demonstrates direct evidence that free radical-induced oxidative modification of proteins are essential during the aging process. That diminution of CK activity occurs by free radical-induced reactions was confirmed by the observation that vitamin E protected against the loss of CK activity by Aß (131). It should be noticed that the activities of many enzymes are also altered during aging. These proteins include: (a) lipid metabolic enzymes phospholipase D, phosphatase phosphohydrolase, diacylglycerol lipase, phosphatidylserine synthase, and phosphatidylserine decarboxylase (17); (b) metabolic enzyme, L -ketoglutarate dehydrogenase complex (132), pyruvate kinase, NAD-isocitrate dehydrogenase, NAD-malate dehydrogenase (133), enolase (134), and lactate dehydrogenase (135–138); (c) mitochondrial enzymes, citrate synthetase, and electron transport complex I-IV (139); (d) ionic homeostatic and signaling proteins: Na⁺, K⁺-ATPase (140), and acetylcholine esterase (141); and (e) antioxidant enzymes, superoxide dismutase, catalase, glutathione peroxidase, -glutamylcysteine synthetase, glutathione reductase, glutathione-S-transferase, and -glutamyl transpeptidase (142–144). Although the alteration of these enzymes are most likely due to oxidative modification of the proteins, no direct correlation has been established between the oxidative modification of the proteins and their activity decline until recently, In the AD brain, we found direct correlation of loss of CK and GS activities (125) with specific protein oxidation using proteomics (145,146). This suggests that oxidative modification of enzymes leads to their dysfunction. Further studies of this notion are required.

Protein Oxidation Summary
The studies mentioned show that free radical modification of proteins may be responsible for the gradual alteration of physiological function and accumulation of altered enzymes in aged brain. In a recent review (99), evidence supporting this view is classified into 7 major areas: (a) in vitro free radical alteration of catalytic activities, thermal stability, and sensitivity to photolytic degradation is similar to what is observed in aging; (b) inducing free radicals in young animals in vivo can change the enzymes to aged-like forms; (c) increasing the life span of animals by factors or physiological conditions can lead to decreases of protein carbonyl levels, and vice versa; (d) increased levels of oxidized protein in the frontal pole and occipital pole concur with the age-related loss of cognitive function; (e) proteins from aged animals are more sensitive to oxidative damage, compared with the proteins from young animals; (f) protein oxidation levels are correlated to increased surface hydrophobicity of protein in aged animals; and (g) protein carbonyl levels increase exponentially with age in different animal species and tissues. The review further proposed that free radical-induced protein oxidation might be the cause of changes in aging. In short, free radicals and free radical-mediated protein modification is essential to the brain aging process.

Recently, proteomics studies enabled the identification of specific proteins that undergo oxidative modification in AD patients (118,145,146). Applying the proteomics technology to brain aging potentially can increase understanding of the mechanisms of brain aging associated with protein oxidation. However, such studies conceivably could encounter problems such as the difficulty of removing lipids from transmembrane proteins, and unknown mass spectrum of proteins. Another potential major problem is that a large amount of oxidized proteins may be present in the brain due to nonspecific protein oxidation in normal aging brains. However, proteomic technology is advancing rapidly. Large-scale proteomics will eventually develop and, therefore, the complete oxidative proteome will be obtained in normal aging brains. This database, with the help of bioinformatics, will provide very valuable information and a foundation about protein oxidation in aging and in many age-related neurodegenerative diseases and dementia (147).

	THE HEAT SHOCK PATHWAY OF CELL STRESS TOLERANCE

Top Abstract Lipid Peroxidation Protein Oxidation The Heat Shock Pathway... HO for Neuroprotective... Conclusion References

 
Background
It is well known that living cells are continually challenged by conditions that cause acute or chronic stress. To adapt to environmental changes and survive different types of injuries, eukaryotic cells have evolved networks of different responses that detect and control diverse forms of stress. One of these responses, known as the heat shock response, has attracted a great deal of attention as a universal fundamental mechanism necessary for cell survival under a wide variety of toxic conditions. In mammalian cells, heat shock protein (HSP) synthesis is induced not only after hyperthermia but also following alterations in the intracellular redox environment, exposure to heavy metals, amino acid analogs, or cytotoxic drugs. While prolonged exposure to conditions of extreme stress is harmful and can lead to cell death, induction of HSP synthesis can result in stress tolerance and cytoprotection against stress-induced molecular damage. Furthermore, transient exposure to elevated temperatures has a cross-protective effect against sustained, normally lethal exposures to other pathogenic stimuli. Hence, the heat shock response contributes to establish a cytoprotective state in a variety of metabolic disturbances and injuries, including stroke, epilepsy, cell and tissue trauma, neurodegenerative disease, and aging (148,149).

These considerations have provided new perspectives in medicine and pharmacology, as molecules activating this defense mechanism appear as possible candidates for novel cytoprotective strategies (150–156). In mammalian cells, the induction of the heat shock response requires the activation and translocation to the nucleus of one or more heat shock transcription factors that control the expression of a specific set of genes encoding cytoprotective heat shock proteins. Some of the known HSPs include ubiquitin, HSP10, HSP27, HSP32 (or HO-1), HSP47, HSP60, HSC70, HSP70 (or HSP72), HSP90, and HSP100/105. Most of the proteins are named according to their molecular weight.

HSP70
The 70 kDa family of stress proteins is one of the most extensively studied. Included in this family are HSC70 (heat shock cognate, the constitutive form), HSP70 (the inducible form, also referred to as HSP72), and GRP75 (a constitutively expressed glucose-regulated protein found in the endoplasmic reticulum). After a variety of central nervous system (CNS) insults, HSP70 is synthesized at high levels and is present in the cytosol, nucleus, and endoplasmic reticulum). Denaturated proteins are thought to serve as stimulus for induction. These denaturated proteins activate heat shock factors (HSFs) within the cytosol by dissociating other HSPs that are normally bound to HSF (157). Free HSF is phosphorylated and forms trimers, which enter the nucleus and bind to heat shock elements (HSE) within the promoters of different heat shock genes leading to transcription and synthesis of HSPs. After heat shock, for instance, the synthesis of HSP70 increases to a point to where it becomes the most abundant single protein in a cell. Once synthesized, HSP70 binds to denaturated proteins in an ATP-dependent manner. The N-terminal end contains an ATP-binding domain, whereas the C-terminal region contains a substrate-binding domain. Heat shock proteins serve as chaperones that bind to other proteins and regulate their conformation, regulate the protein movement across membranes or through organelles, or regulate the availability of a receptor or activity of an enzyme.

In the nervous system, HSPs are induced in a variety of pathological conditions, including cerebral ischemia, neurodegenerative disorders, epilepsy, and trauma (157). Expression of the gene-encoding HSPs has been found in various cell populations within the nervous system, including neurons, glia, and endothelial cells (158). HSPs consist of both stress-inducible and constitutive family members. Whether stress proteins are neuroprotective has been the subject of much debate, as it has been speculated that these proteins might be merely an ephiphenomenon unrelated to cell survival. Only recently, however, with the availability of transgenic animals and gene transfer, has it become possible to overexpress the gene encoding HSP70 to test directly the hypothesis that stress proteins protect cells from injury, and it has been demonstrated that overproduction of HSP70 leads to protection in several different models of nervous system injury (159,160). Following focal cerebral ischemia, mRNA-encoding HSP70 is synthesized in most ischemic cells except in an area of very low blood flow because of limited ATP levels. HSP70 proteins are produced mainly in endothelial cells, in the core of infarcts in the cells that are most resistant to ischemia, in glial cells at the edges of infarcts, and in neurons outside the areas of infarction. It has been suggested that this neuronal expression of HSP70 outside an infarct can be used to define the ischemic penumbras (161). A number of in vitro studies show that both heat shock and HSP overproduction protect CNS cells against both necrosis and apoptosis. Mild heat shock protects neurons against glutamate-mediated toxicity and protects astrocytes against injury produced by lethal acidosis (162). Transfection of cultured astrocytes with HSP70 protects them from ischemia or glucose deprivation (163). HSP70 has been demonstrated to inhibit caspase-3 activation caused by ceramide and also affect JUN kinase and p38-kinase activation (164). In addition, HSP70 binds to and modulates the function of BAG-1, the bcl-2 binding protein (165), thus modulating some type of apoptosis-related cell death.

A large body of evidence now suggests a correlation between mechanisms of oxidative and/or nitrosative stress and HSP induction. Current opinion also holds the possibility that the heat shock response can exert its protective effects through inhibition of nuclear factor kappa B (NFkB)-signaling pathway (149). We have demonstrated in astroglial cell cultures that cytokine-induced nitrosative stress is associated with an increased synthesis of HSP70 stress proteins. An increase in HSP70 protein expression was also found after treatment of cells with the NO-generating compound sodium nitroprusside (SNP), thus suggesting a role for NO in inducing HSP70 proteins (167). The molecular mechanisms regulating the NO-induced activation of the heat shock signal seems to involve cellular oxidant/antioxidant balance, mainly represented by the glutathione status and the antioxidant enzymes (165–167).

Ubiquitin
Ubiquitin is one of the smallest HSPs and is expressed throughout the brain in response to ischemia. It is involved in the targeting and chaperoning of proteins degraded in proteasomes, which include NFkB, cyclins, HSFs, hypoxia-inducible factor, some apoptosis-related proteins, tumor necrosis factor (TNF), and erythropoietin receptors (168).

HSP27
HSP27 is synthesized mainly in astrocytes in response to ischemic situations or to kainic acid administration. It chaperones cytoskeletal proteins, such as intermediate filaments, actin, or glial fibrillary acidic protein following stress in astrocytes. It also protects against Fas-Apo-1, staurosporine, TNF, and etoposside-induced apoptotic cell death as well as H₂O₂-induced necrosis (169).

HSP47
HSP47 is synthesized mainly in microglia following cerebral ischemia and subarachnoid hemorrhage (170).

HSP60, Glucose-Regulated Protein 75 (GRP75), and HSP10 Chaperone Proteins
HSP60, glucose-regulated protein 75 (GRP75), and HSP10 chaperone proteins are within mitochondria. GRP75 and GRP78, also called oxygen-regulated proteins (ORPs), are produced by low levels of oxygen and glucose. These protect brain cells against ischemia and seizures in vivo, after viral-induced over-expression (171).

HSP32
HSP32, or heme oxygenase, is the rate-limiting enzyme in the production of bilirubin. There are 3 isoforms of heme oxygenase: HO-1 or inducible isoform, HO-2 or constitutive isoform, and the recently discovered HO-3 (172–174).

	HO FOR NEUROPROTECTIVE STRATEGIES IN AGE-RELATED NEURODEGENERATIVE DISEASE PATHOLOGY

Top Abstract Lipid Peroxidation Protein Oxidation The Heat Shock Pathway... HO for Neuroprotective... Conclusion References

 
Background
In the last decade, the heme oxygenase (HO) system has been strongly highlighted for its potential significance in maintaining cellular homeostasis. It is found in the endoplasmic reticulum in a complex with NADPH cytochrome c P450 reductase. It catalyzes the degradation of heme in a multistep, energy-requiring system. The reaction catalyzed by HO is the -specific oxidative cleavage of the heme molecule to form equimolar amounts of biliverdin and carbon monoxide (CO). Iron is reduced to its ferrous state through the action of NADPH cytochrome c P450 reductase. Carbon monoxide is released by elimination of the -methylene bridge of the porphyrin ring. Further degradation of biliverdin to bilirubin occurs through the action of a cytosolic enzyme, biliverdin reductase. Biliverdin complexes with iron until its final release.

Heme Oxygenase Isoforms
HO is present in various tissues with the highest activity in the brain, liver, spleen, and testes. There are 3 isoforms of heme oxygenase: HO-1 or inducible isoform (175), HO-2 or constitutive isoform (176), and HO-3, cloned only in rats to date (177). They are all products of different genes and, unlike HO-3, which is a poor heme catalyst, both HO-1 and HO-2 catalyze the same reaction (i.e., degradation of heme) but differ in many respects and are regulated under separate mechanisms. The most relevant similarity between HO-1 and HO-2 consists in a common 24 amino acid (aa) domain (differing in just one residue) called the "HO signature," which renders both proteins extremely active in their ability to catabolize heme (178). These isoforms have different localization, similar substrate, and cofactor requirements, while presenting a different molecular weight. They also display different antigenicity, electrophoretic mobility, and inducibility as well as susceptibility to degradation. The proteins for HO-1 and HO-2 are immunologically distinct and, in humans, the two genes are located on different chromosomes, i.e., 22q12 for HO-1 and 16q13.3 for HO-2, respectively (179).

Various tissues have different amounts of HO-1 and HO-2. The brain and testes have a predominance of HO-2, whereas HO-1 predominates in the spleen. In the lung not subjected to oxidative stress, more than 70% of HO activity is accounted for by HO-2, whereas in the testes, the pattern of HO isoenzyme expression differs according to the cell type, although HO-1 expression predominates after heat shock. This also occurs in brain tissue, where HO isoforms appear to be distributed in a cell-specific manner and HO-1 distribution is widely apparent after heat shock or oxidative stress. Although previous reports from our group and other groups have not found detectable levels of HO-1 protein in the normal brain (180,181), we have recently demonstrated that HO-1 mRNA expression is physiologically detectable in the brain and shows a characteristic regional distribution, with a high level of expression in the hippocampus and the cerebellum (182,183). This evidence may suggest the possible existence of a cellular reserve of HO-1 transcript quickly available for protein synthesis and a posttranscriptional regulation of its expression.

HO isoenzymes are also seen to colocalize with different enzymes depending on the cell type. In the kidney, HO-1 colocalizes with erythropoietin, whereas in smooth muscle cells, HO-1 colocalizes with nitric oxide synthase. In neurons, HO-2 colocalizes with NOS, whereas the endothelium exhibits the same isoform to colocalize with NOS III. The cellular specificity of this pattern of colocalization lends further support to the concept that CO may serve a function similar to that of NO. Furthermore, the brain expression pattern shown by HO-2 protein and HO-1 mRNA overlaps with distribution of guanylate cyclase (184), the main CO functional target.

HO-3, the third isoform of heme oxygenase, shares a high homology with HO-2, both at the nucleotide (88%) and protein (81%) levels. Both HO-2 and HO-3, but not HO-1, are endowed with 2 Cys-Pro residues considered the core of the heme-responsive motif (HRM), a domain critical for heme binding but not for its catalysis (185,186). Although the biological properties of this isoenzyme still remain to be elucidated, the presence of 2 HRM motifs in its amino acidic sequence might suggest a role in cellular heme regulation (187). Studying the HO-3 mRNA sequence (GenBank accession n.: AF058787) shows that a 5' portion corresponds to the sequence of a L-1 retrotransposon, a member of a family of retrotransposons recently involved in evolutionary mechanisms (188,189). Based on the close similarity to a paralogous gene (HO-2) and the preliminary data from our group demonstrating no introns in the HO-3 gene (182), it is possible that HO-3 could have originated from the retrotransposition of the HO-2 gene. In addition, this genetic mutation in the rat may represent a specie-specific event since no other sequence in the public databases matches the rat HO-3.

Regulation of HO Genes
The HO-2 gene consists of 5 exons and 4 introns. HO-2 has a molecular weight of 34 kDa and exhibits 40% homology in an amino acid sequence with HO-1. It is generally considered a constitutive isoenzyme, however, in situ hybridization studies have shown increases in HO-2 mRNA synthesis associated with increased HO-2 protein and enzyme activity in neonatal rat brain after treatment with corticosterone (190). The organization of the HO-2 gene needs to be fully elucidated, although a consensus sequence of the glucocorticoid response element (GRE) has been demonstrated in the promoter region of the HO-2 gene (191). In addition, endothelial cells treated with the NOS inhibitor L-NAME and HO inhibitor zinc mesophorphyrin exhibited a significant up-regulation of HO-2 mRNA.

The regulation of the HO-1 gene as well as its promoter has received more elucidation that constitutive HO-2. The HO-1 gene is induced by a variety of factors, including metallophorphyrins and hemin, as well as ultraviolet A (UVA) irradiation, hydrogen peroxide, prooxidant states, or inflammation (192,193). This characteristic inducibility of the HO-1 gene strictly relies on its configuration: the 6.8-kilobase gene is organized into 4 introns and 5 exons. A promoter sequence is located approximately 28 base pairs upstream from the transcriptional site of initiation. In addition, different transcriptional enhancer elements, such as the heat shock element and metal regulatory element, reside in the flanking 5' region. Also, inducer-responsive sequences have been identified in the proximal enhancer located upstream from the promoter and, more distally, in 2 enhancers located 4 kb and 10 kb upstream from the initiation site (194).

The transcriptional activation of HO-1 in response to hyperoxia requires the cooperation between the promoter and an enhancer element located 4 kb upstream from the transcription site (195). This response is also mediated by increased binding activity of AP-1. The HO-1 promoter contains an antioxidant responsive element with a consensus sequence (GCnnnGTA) similar to that of other antioxidant enzymes (196,197). Heme treatment results in increased binding activity of NFkB and AP-2 transcription factors at the proximal part of the promoter, but also of AP-1. The promoter region also contains 2 metal-responsive elements, similar to those found in the metallothionein-1 gene, which respond to heavy metals (cadmium and zinc) only after recruitment of another fragment located upstream, between –3.5 and 12 kbp (CdRE). In addition, a 163-bp fragment containing 2 binding sites for HSF-1, which mediates the HO-1 transcription, are located 9.5 kb upstream of the initiation site (197). The distal enhancer regions are important in regulating HO-1 in inflammation, since it has been demonstrated that these enhancer regions are responsive to endotoxin. In the promoter region also resides a 56 bp fragment that responds to the STAT-3 acute-phase response factor involved in the down-regulation of the HO-1 gene induced by 1 glucocorticoid (198).

Heme Oxygenase in Brain Function and Dysfunction
In the brain, the HO system has been reported to be very active and its modulation seems to play a crucial role in the pathogenesis of neurodegenerative disorders. The heme oxygenase pathway, in fact, has been shown to act as a fundamental defensive mechanism for neurons exposed to an oxidant challenge (199–201). Induction of HO occurs together with the induction of other HSPs in the brain during various experimental conditions including ischemia (202). Injection of blood or hemoglobin results in increased expression of the gene encoding HO-1, which has been shown to occur mainly in microglia throughout the brain (202). This suggests that microglia take up extracellular heme protein following cell lysis or hemorrhage. Once in the microglia, heme induces the transcription of HO-1. In human brains following traumatic brain injury, accumulation of HO-1 plus microglia/macrophages at the hemorrhagic lesion were detected as early as 6 hours post trauma and was still pronounced after 6 months (203).

Since the expression of HSPs is closely related to that of the amyloid precursor protein (APP), HSPs have been studied in the brain of patients with AD. Significant increases in the levels of HO-1 have been observed in AD brains in association with neurofibrillary tangles (204,205), and also HO-1 mRNA was found to be increased in AD neocortex and cerebral vessels (206). An HO-1 increase was not only in association with neurofibrillary tangles, but also colocalized with senile plaques and glial fibrillary acidic protein-positive astrocytes in AD brains (206). It is conceivable that the dramatic increase in HO-1 in AD may be a direct response to increased free heme associated with neurodegeneration and an attempt to convert the highly damaging heme into the antioxidants biliverdin and bilirubin (207).

Up-regulation of HO-1 in the substantia nigra of Parkinson's disease patients has been demonstrated. In these patients, nigral neurons containing cytoplasmic Lewy bodies exhibited in their proximity maximum HO-1 immunoreactivity (208). New evidences showed a specific up-regulation of HO-1 in the nigral dopaminergic neurons by oxidative stress (153,209).

It has been recently demonstrated that hemin, an inducer of HO-1, inhibited effectively experimental autoimmune encephalomyelitis (EAE), an animal model of the human disease multiple sclerosis (MS) (210). In contrast, tin mesoporphyrin, an inhibitor of HO-1, markedly exacerbated EAE. These results suggest that endogenous HO-1 plays an important protective role in EAE and MS.

All of these findings have opened up new perspectives in medicine and pharmacology, as molecules activating this defense mechanism appear to be possible candidates for novel therapeutic neuroprotective strategies (210) (see below).

Carbon Monoxide Effects on Brain Function in Health and Disease
Increasing evidence implicates CO as an emerging chemical messenger molecule that can influence physiological and pathological processes in both the central and peripheral nervous systems. This gaseous molecule is now considered a putative neurotransmitter, owing to its capability to diffuse freely from one cell to another, thereby influencing intracellular signal transduction mechanisms. However, unlike a conventional neurotransmitter, carbon monoxide is not stored in synaptic vesicles and is not released by membrane depolarization and exocitosis. It seems likely that CO is involved in the mechanism of cell injury (211,212). This is evidenced by the fact that CO binds to the heme moiety in guanylate cyclase to activate cGMP (178,184). It has been found that CO is responsible for maintaining endogenous levels of cGMP. This effect is blocked by potent HO inhibitors but not by NO inhibitors (213,214).

Based on the endogenous distribution of HO in the CNS, it has been suggested that CO can influence neurotransmission like NO (184). CO appears to be involved as a retrograde messenger in LTP and also is involved in mediating glutamate action at metabotropic receptors (213). This is evident from the observation that metabotropic receptor activation in the brain regulates the conductance of specific ion channels via a cGMP-dependent mechanism that is blocked by HO inhibitors (214). Experimental evidence suggests that CO plays a similar role as NO in the signal transduction mechanism for the regulation of cell function and cell-to-cell communication (212).

HO resembles NOS in that the electrons for CO synthesis are donated by cytochrome P450 reductase, which is 60% homologous at the amino acid level to the carboxyl terminal half of NOS (215). CO, like NO, binds to iron in the heme moiety of guanylate cyclase. However there are some differences in function between CO and NO. Thus, NO mainly mediates glutamate effects at NMDA receptors, while CO is primarily responsible for glutamate action at metabotropic receptors.

Taken together, it appears that CO and NO play an important role in the regulation of CNS function; thus, impairment of CO and NO metabolism results in abnormal brain function (207,212). A number of reports suggest a possible role of CO in regulating the transmission of nitrogen compounds. Endogenous CO has been suggested to control constitutive NOS activity. Moreover, CO may interfere with NO binding to guanylate cyclase. This is in addition to the important role of HO in regulating NO generation, owing to its function in the control of heme intracellular levels, as part of normal protein turnover (216). This hypothesis is sustained by recent findings showing that HO inhibition increases NO production in mouse macrophages exposed to endotoxin (217).

CO may also act as a signaling effector molecule by interacting with targets different from guanylate cyclase. Notably, it has been recently demonstrated that K_Ca channels are activated by CO in a GMPc-independent manner (218), and also that CO-induced vascular relaxation results from the inhibition of the synthesis of the vasoconstrictor endothelin-1 (218). However, little is known about how CO is sensed on a biological ground. Interestingly, the photosynthetic bacterium Rhodospirillum rubrum has the ability to respond to CO through the heme protein CooA, which, upon exposure to CO, acquires DNA-binding transcriptional activity for the CO dehydrogenase gene. This property hereby encodes for CO dehydrogenase, which is the key enzyme involved in the oxidative conversion of CO to CO₂. Remarkably, heart cytochrome c oxidase possesses CO oxygenase activity, thus metabolizing CO to CO₂ (219). Whether this occurs also in brain mitochondria remains to be elucidated.

Bilirubin: An Endogenous Antioxidant Derived From Heme Oxygenase
Supraphysiological levels (>300 µM) of nonconjugated bilirubin, as in the case of neonatal jaundice, are associated with severe brain damage. This is a plausible reason whereby bilirubin has generally been recognized as a cytotoxic waste product. However, only in recent years, its emerging role as a powerful antioxidant has received wide interest. The specific role of endogenously derived bilirubin as a potent antioxidant has been demonstrated in hippocampal and cortical neurons, in which accumulation of this metabolite protected against H₂O₂-induced cytotoxicity (220,221). Moreover, nanomolar concentrations of bilirubin resulted in a significant protection against hydrogen peroxide-induced toxicity in cultured neurons as well as in glial cells following experimental subarachnoid hemorrhage. In addition, neuronal damage following middle cerebral artery occlusion was substantially worsened in HO-2 knock-out mice (222). Bilirubin can become particularly important as a cytoprotective agent for tissues with relatively weak endogenous antioxidant defences, such as the central nervous system and the myocardium. Interestingly, increased levels of bilirubin have been found in the CSF of AD patients, which may reflect the increase of degraded bilirubin metabolites in the AD brain derived from the scavenging reaction against chronic oxidative stress (223). Similarly, a decreased risk for coronary artery disease is associated with mildly elevated serum bilirubin, with a protective effect comparable to that of high-density lipoprotein cholesterol (221). The most likely explanation for the potent neuroprotective effect of bilirubin is that a redox cycle exists between bilirubin and biliverdin, the major oxidation product of bilirubin. In mediating the antioxidant actions, bilirubin would be transformed in biliverdin, then rapidly converted back to bilirubin by biliverdine reductase, which in the brain is present in large functional excess. Remarkably, the rapid activation of HO-2 by protein kinase C (PKC) phosphorylation parallels the disposition of nNOS. Both are constitutive enzymes localized to neurons, and nNOS is activated by calcium entry into cells binding to calmodulin on nNOS. Similarly, PKC phosphorylation of HO-2 and the transient increase in intracellular bilirubin would provide a way for a rapid response to calcium entry, a major activator of PKC. Whether the antioxidant bilirubin possess antinitrosative properties still remains an open question, although this possibility could partially account for the effect of NO and peroxynitrite in up-regulating HO-1 expression (193).

	CONCLUSION

Top Abstract Lipid Peroxidation Protein Oxidation The Heat Shock Pathway... HO for Neuroprotective... Conclusion References

 
The studies discussed here provide evidence for free radical/ROS/RNS involvement in brain aging that is direct as well as correlative. Recent literature provide increasing evidence of accumulation of the oxidatively modified proteins, DNAs, and lipids in the aged brain. Numerous marker compounds of lipid peroxidation were detected, such as MDA, HNE, and acrolein. These products react with DNA and proteins to produce further damage in aged brains (20,233). It was reported that 10,000 oxidative interactions occur between DNA and endogenously generated free radicals per human cell per day (224). Additionally, at least 1 of every 3 proteins in the cells of older animals is dysfunctional as enzyme or structural protein due to oxidative modification (99). Although much evidence shows the important role of free radicals in brain aging, some argue otherwise. Some contend that damage produced by endogenously generated oxygen free radicals by mitochondria resulting in distinctive biochemical profiles only occurs under exceptional conditions (225). Others claim that elongated life span from caloric restriction is not accredited to reducing free radical production, but by metabolic-shift activation of the SIR2 protein, which slices chromatin by deacetylating the histones in targeted regions of the genome, including the rDNA (226). It was also reported that inactive SIR2 mutants shorten the life span while over-expression of SIR2 extends it (227). However, these experiments did not eliminate the likely involvement of free radicals during the aging process. As matter of fact, many studies showed a connection between other theories of aging and free radicals. The "wear and tear" theory stated that the cells and tissues in the system, such as neuroendocrine system (228), were damaged by toxin accumulation from diet and environment, such as consumption of excessive fat, alcohol, UV rays from the sun, or metal ions from the environment, and so forth. However, this toxin accumulation, such as UV and estrogens, could affect ROS production and indicate the involvement of free radicals in aging (229). Also, the programmed death theory stated that the unique genetic code and genetic inheritance within DNA decide the longevity of the individual. DNA can be compared to a biological clock set to go off at a particular time (230). Telomeres are the tails of DNA that shorten every time cells divide. Once the telomeres become too short, cell division slows and finally ceases, causing the cell to die (231). It was reported that the telomere repairing enzyme telomerase is activated by free iron, and iron is responsible for the production of O₂· by Fenton reaction and activating xanthine oxidase (232). This suggests that the loss of telomerase activity is possibly due to insufficient free iron in cells caused by free radicals.

HO provides free iron as well as CO and the antioxidant bilirubin (via biliverdin). The HSP response to oxidative stress in aging and age-related neurodegenerative diseases is primal, powerful, and pervasive (149). Therapeutic strategies aimed at inducing HO-1 may be a promising approach to the oxidative stress characteristic of brain aging and age-related neurodegenerative disorders (154,159,194,196,201).

All of the studies described do not imply that free radicals are the only direct cause of brain aging. However, while other factors may be involved in the cascade of damaging effects in the brain, the significant key role of free radicals in this process cannot be underestimated. Therefore, this review supports the proposition that free radicals are, indeed, the key to brain aging.

	Acknowledgments

 
This work was supported in part by grants from the National Institutes of Health to D. A. Butterfield (AG-10836, AG-05119) and by a Wellcome Trust Grant to V. Calabrese.

Received August 18, 2003

Accepted August 19, 2003

	References

Top Abstract Lipid Peroxidation Protein Oxidation The Heat Shock Pathway... HO for Neuroprotective... Conclusion References

 

1. Ashok BT, Ali R. The aging paradox: free radical theory of aging. Exp Gerontol.. 1999;34:293-303.[Medline]
2. Harman D. The aging process: major risk factor for disease and death. Proc Natl Acad Sci U S A.. 1991;88:5360-5363.[Abstract/Free Full Text]
3. Sohal RS, Mockett RJ, Orr WC. Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radic Biol Med.. 2002;33:575-586.[Medline]
4. Gilgun-Sherki Y, Melamed E, Offen D. Oxidative stress induced-neurodegenerative diseases: the need for antioxidants that penetrate the blood brain barrier. Neuropharmacology.. 2001;40:959-975.[Medline]
5. Floyd RA. Antioxidants, oxidative stress, and degenerative neurological disorders. Proc Soc Exp Biol Med.. 1999;222:236-245.[Abstract/Free Full Text]
6. Halliwell B, Gutteridge JM. Biologically relevant metal ion-dependent hydroxyl radical generation. An update. FEBS Lett.. 1992;307:108-112.[Medline]
7. Frankel EN. Chemistry of free radical and singlet oxidation of lipids. Prog Lipid Res.. 1984;23:197-221.[Medline]
8. Spiteller G. Peroxidation of linoleic acid and its relation to aging and age dependent diseases. Mech Ageing Dev.. 2001;122:617-657.[Medline]
9. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med.. 1991;11:81-128.[Medline]
10. Delsal JL. Nouveau procede d'extraction des lipids du serum per le methylal. Applications aux, icrodpsages du chlesterol total, des phosphoaminolipides et des proteines. Bull Soc Chim Biol.. 1944;26:99-105.
11. Vitiello F, Zanetta JP. Thin-layer chromatography of phospholipids. J Chromatogr.. 1978;166:637-640.[Medline]
12. Hedegaard E, Jensen B. Nano-scale densitometric quantitation of phospholipids. J Chromatogr.. 1981;225:450-454.[Medline]
13. Lanza E, Zyren J, Slover HT. Fatty acid analysis on short glass capillary columns. J Agric Food Chem.. 1980;28:1182-1186.[Medline]
14. Murphy EJ, Stephens R, Jurkowitz-Alexander M, Horrocks LA. Acidic hydrolysis of plasmalogens followed by high-performance liquid chromatography. Lipids.. 1993;28:565-568.[Medline]
15. Schroeder F, Goetz I, Roberts E. Sex and age alter plasma membranes of cultured fibroblasts. Eur J Biochem.. 1984;142:183-191.[Medline]
16. Lopez GH. Ilincheta de Boschero MG, Castagnet PI, Giusto NM. Age-associated changes in the content and fatty acid composition of brain glycerophospholipids. Comp Biochem Physiol B Biochem Mol Biol.. 1995;112:331-343.[Medline]
17. Giusto NM, Salvador GA, Castagnet PI, Pasquare SJ. Ilincheta de Boschero MG. Age-associated changes in central nervous system glycerolipid composition and metabolism. Neurochem Res.. 2002;27:1513-1523.[Medline]
18. Ulmann L, Mimouni V, Roux S, Porsolt R, Poisson JP. Brain and hippocampus fatty acid composition in phospholipid classes of aged-relative cognitive deficit rats. Prostagl Leukot Essent Fatty Acids.. 2001;64:189-195.[Medline]
19. Lynch MA. Analysis of the mechanisms underlying the age-related impairment in long-term potentiation in the rat. Rev Neurosci.. 1998;9:169-201.[Medline]
20. Butterfield DA, Stadtman ER. Protein oxidation processes in aging brain. Adv Cell Aging Gerontol.. 1997;2:161-191.
21. Pizzimenti S, Barrera G, Dianzani MU, Brusselbach S. Inhibition of D1, D2, and A-cyclin expression in HL-60 cells by the lipid peroxydation product 4-hydroxynonenal. Free Radic Biol Med.. 1999;26:1578-1586.[Medline]
22. Uchida K, Stadtman ER. Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. Proc Natl Acad Sci U S A.. 1992;89:4544-4548.[Abstract/Free Full Text]
23. Guichardant M, Taibi-Tronche P, Fay LB, Lagarde M. Covalent modifications of aminophospholipids by 4-hydroxynonenal. Free Radic Biol Med.. 1998;25:1049-1056.[Medline]
24. Del Corso A, Dal Monte M, Vilardo PG, et al. Site-specific inactivation of aldose reductase by 4-hydroxynonenal. Arch Biochem Biophys.. 1998;350:245-248.[Medline]
25. Boschi-Muller S, Azza S, Sanglier-Cianferani S, Talfournier F, Van Dorsselear A, Branlant G. A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli. J Biol Chem.. 2000;275:35908-35913.[Abstract/Free Full Text]
26. Siems WG, Hapner SJ, van Kuijk FJ. 4-hydroxynonenal inhibits Na(+)-K(+)-ATPase. Free Radic Biol Med.. 1996;20:215-223.[Medline]
27. Butterfield DA, Lauderback CM. Lipid peroxidation and protein oxidation in Alzheimer's disease brain: potential causes and consequences involving amyloid beta-peptide-associated free radical oxidative stress. Free Radic Biol Med.. 2002;32:1050-1060.[Medline]
28. Mark RJ, Lovell MA, Markesbery WR, Uchida K, Mattson MP. A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid beta-peptide. J Neurochem.. 1997;68:255-264.[Medline]
29. Uchida K, Kanematsu M, Sakai K, et al. Protein-bound acrolein: potential markers for oxidative stress. Proc Natl Acad Sci U S A.. 1998;95:4882-4887.[Abstract/Free Full Text]
30. Lovell MA, Xie C, Markesbery WR. Acrolein is increased in Alzheimer's disease brain and is toxic to primary hippocampal cultures. Neurobiol Aging.. 2001;22:187-194.[Medline]
31. Pocernich CB, Cardin AL, Racine CL, Lauderback CM, Butterfield DA. Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: relevance to brain lipid peroxidation in neurodegenerative disease. Neurochem Int.. 2001;39:141-149.[Medline]
32. Lovell MA, Xie C, Markesbery WR. Acrolein, a product of lipid peroxidation, inhibits glucose and glutamate uptake in primary neuronal cultures. Free Radic Biol Med.. 2000;29:714-720.[Medline]
33. Yoritaka A, Hattori N, Uchida K, Tanaka M, Stadtman ER, Mizuno Y. Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proc Natl Acad Sci U S A.. 1996;93:2696-2701.[Abstract/Free Full Text]
34. Papaioannou N, Tooten PC, van Ederen AM, et al. Immunohistochemical investigation of the brain of aged dogs. I. Detection of neurofibrillary tangles and of 4-hydroxynonenal protein, an oxidative damage product, in senile plaques. Amyloid.. 2001;8:11-21.[Medline]
35. Markesbery WR, Lovell MA. Four-hydroxynonenal, a product of lipid peroxidation, is increased in the brain in Alzheimer's disease. Neurobiol Aging.. 1998;19:33-36.[Medline]
36. Sayre LM, Zelasko DA, Harris PL, Perry G, Salomon RG, Smith MA. 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer's disease. J Neurochem.. 1997;68:2092-2097.[Medline]
37. Michiels C, Remacle J. Cytotoxicity of linoleic acid peroxide, malondialdehyde and 4-hydroxynonenal towards human fibroblasts. Toxicology.. 1991;66:225-234.[Medline]
38. Esterbauer H. Cytotoxicity and genotoxicity of lipid-oxidation products. Am J Clin Nutr.. 1993;57:779S-785S discussion 785S-786S.
39. Basu AK, Marnett LJ. Unequivocal demonstration that malondialdehyde is a mutagen. Carcinogenesis.. 1983;4:331-333.[Abstract/Free Full Text]
40. Nair V, Offerman RJ, Turner GA, Pryor AN. Fluorescent 1,4-dihydropyridines—the malondialdehyde connection. Tetrahedron,. 1988;44:2793-2803.
41. Nair V, Vietti DE, Cooper CS. Degenerative chemistry of malondialdehyde. Structure, stereochemistry, and kinetics of formation of enaminals from reaction with amino acids. J Am Chem Soc.. 1981;103:3030-3036.
42. Slatter DA, Murray M, Bailey AJ. Formation of a dihydropyridine derivative as a potential cross-link derived from malondialdehyde in physiological systems. FEBS Lett.. 1998;421:180-184.[Medline]
43. King TP. Selective chemical modification of arginyl residues. Biochemistry.. 1966;5:3454-3459.[Medline]
44. Slatter DA, Paul RG, Murray M, Bailey AJ. Reactions of lipid-derived malondialdehyde with collagen. J Biol Chem.. 1999;274:19661-19669.[Abstract/Free Full Text]
45. Xu D, Thiele GM, Beckenhauer JL, Klassen LW, Sorrell MF, Tuma DJ. Detection of circulating antibodies to malondialdehyde-acetaldehyde adducts in ethanol-fed rats. Gastroenterology.. 1998;115:686-692.[Medline]
46. Slatter DA, Bolton CH, Bailey AJ. The importance of lipid-derived malondialdehyde in diabetes mellitus. Diabetologia.. 2000;43:550-557.[Medline]
47. Agarwal S, Draper HH. Isolation of a malondialdehyde-deoxyguanosine adduct from rat liver DNA. Free Radic Biol Med.. 1992;13:695-699.[Medline]
48. Ji C, Rouzer CA, Marnett LJ, Pietenpol JA. Induction of cell cycle arrest by the endogenous product of lipid peroxidation, malondialdehyde. Carcinogenesis.. 1998;19:1275-1283.[Abstract/Free Full Text]
49. Yagi K. Lipid peroxides and related radicals in clinical medicine. Adv Exp Med Biol.. 1994;366:1-15.[Medline]
50. Yagi K. Assay for serum lipid peroxide level and its clinical significance. In: K Yagi, ed. Lipid Peroxides in Biology and Medicine. New York: Academic; 1982:223–241.
51. Fukunaga K, Yoshida M, Nakazono N. A simple, rapid, highly sensitive and reproducible quantification method for plasma malondialdehyde by high-performance liquid chromatography. Biomed Chromatogr.. 1998;12:300-303.[Medline]
52. Wong SH, Knight JA, Hopfer SM, Zaharia O, Leach CN, Jr, Sunderman FW, Jr. Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct. Clin Chem.. 1987;33:214-220.[Abstract/Free Full Text]
53. Young IS, Trimble ER. Measurement of malondialdehyde in plasma by high performance liquid chromatography with fluorimetric detection. Ann Clin Biochem.. 1991;28(Pt 5):504-508.
54. Palinski W, Yla-Herttuala S, Rosenfeld ME, et al. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis.. 1990;10:325-335.[Abstract/Free Full Text]
55. Dei R, Takeda A, Niwa H, et al. Lipid peroxidation and advanced glycation end products in the brain in normal aging and in Alzheimer's disease. Acta Neuropathol (Berl).. 2002;104:113-122.[Medline]
56. Head E, Liu J, Hagen TM, et al. Oxidative damage increases with age in a canine model of human brain aging. J Neurochem.. 2002;82:375-381.[Medline]
57. Mooradian AD, Lung CC, Shah G, Mahmoud S, Pinnas JL. Age-related changes in tissue content of malondialdehyde-modified proteins. Life Sci.. 1994;55:1561-1566.[Medline]
58. Cini M, Moretti A. Studies on lipid peroxidation and protein oxidation in the aging brain. Neurobiol Aging.. 1995;16:53-57.[Medline]
59. Cai Q, Tian L, Wei H. Age-dependent increase of indigenous DNA adducts in rat brain is associated with a lipid peroxidation product. Exp Gerontol.. 1996;31:373-385.[Medline]
60. Randerath K, Li D, Moorthy B, Randerath E. I-compounds—endogenous DNA markers of nutritional status, ageing, tumour promotion and carcinogenesis. IARC Sci Publ.. 1993;124:157-165.
61. Randerath K, Li DH, Randerath E. Age-related DNA modifications (I-compounds): modulation by physiological and pathological processes. Mutat Res.. 1990;238:245-253.[Medline]
62. Randerath K, Liehr JG, Gladek A, Randerath E. Age-dependent covalent DNA alterations (I-compounds) in rodent tissues: species, tissue and sex specificities. Mutat Res.. 1989;219:121-133.[Medline]
63. Gupta KP, van Golen KL, Randerath E, Randerath K. Age-dependent covalent DNA alterations (I-compounds) in rat liver mitochondrial DNA. Mutat Res.. 1990;237:17-27.[Medline]
64. Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ, II. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci U S A.. 1990;87:9383-9387.[Abstract/Free Full Text]
65. Chen Y, Morrow JD, Roberts LJ, II. Formation of reactive cyclopentenone compounds in vivo as products of the isoprostane pathway. J Biol Chem.. 1999;274:10863-10868.[Abstract/Free Full Text]
66. Morrow JD, Minton TA, Mukundan CR, et al. Free radical-induced generation of isoprostanes in vivo. Evidence for the formation of D-ring and E-ring isoprostanes. J Biol Chem.. 1994;269:4317-4326.[Abstract/Free Full Text]
67. Morrow JD, Awad JA, Wu A, Zackert WE, Daniel VC, Roberts LJ, II. Nonenzymatic free radical-catalyzed generation of thromboxane-like compounds (isothromboxanes) in vivo. J Biol Chem.. 1996;271:23185-23190.[Abstract/Free Full Text]
68. Taber DF, Herr RJ, Gleave DM. Diastereoselective synthesis of an isoprostane: (+/–)-8-epi-PGF(2)(alpha) ethyl ester. J Org Chem.. 1997;62:194-198.[Medline]
69. Morrow JD, Roberts LJ. The isoprostanes: unique bioactive products of lipid peroxidation. Prog Lipid Res.. 1997;36:1-21.[Medline]
70. Brame CJ, Salomon RG, Morrow JD, Roberts LJ, II. Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts. J Biol Chem.. 1999;274:13139-13146.[Abstract/Free Full Text]
71. Roberts LJ, II, Morrow JD. The generation and actions of isoprostanes. Biochim Biophys Acta.. 1997;1345:121-135.[Medline]
72. Roberts LJ, II, Reckelhoff JF. Measurement of F(2)-isoprostanes unveils profound oxidative stress in aged rats. Biochem Biophys Res Commun.. 2001;287:254-256.[Medline]
73. Roberts LJ, Morrow JD. Measurement of F(2)-isoprostanes as an index of oxidative stress in vivo. Free Radic Biol Med.. 2000;28:505-513.[Medline]
74. Morrow JD, Awad JA, Kato T, et al. Formation of novel non-cyclooxygenase-derived prostanoids (F2-isoprostanes) in carbon tetrachloride hepatotoxicity. An animal model of lipid peroxidation. J Clin Invest.. 1992;90:2502-2507.
75. Granstrom E, Kindahl H. Radioimmunoassay of prostaglandins and thromboxanes. Adv Prostaglandin Thromboxane Res.. 1978;5:119-210.[Medline]
76. Proudfoot J, Barden A, Mori TA, Burke V, Croft KD, Beilin LJ, Puddey IB. Measurement of urinary F(2)-isoprostanes as markers of in vivo lipid peroxidation-A comparison of enzyme immunoassay with gas chromatography/mass spectrometry. Anal Biochem.. 1999;272:209-215.[Medline]
77. Morrow JD, Roberts LJ, II. Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress. Methods Enzymol.. 1999;300:3-12.[Medline]
78. Reich EE, Zackert WE, Brame CJ, et al. Formation of novel D-ring and E-ring isoprostane-like compounds (D4/E4-neuroprostanes) in vivo from docosahexaenoic acid. Biochemistry.. 2000;39:2376-2383.[Medline]
79. Roberts LJ, II, Montine TJ, Markesbery WR, et al. Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid. J Biol Chem.. 1998;273:13605-13612.[Abstract/Free Full Text]
80. Moore SA, Yoder E, Murphy S, Dutton GR, Spector AA. Astrocytes, not neurons, produce docosahexaenoic acid (22:6 omega-3) and arachidonic acid (20:4 omega-6). J Neurochem.. 1991;56:518-524.[Medline]
81. Bernoud-Hubac N, Davies SS, Boutaud O, Montine TJ, Roberts LJ, II. Formation of highly reactive gamma-ketoaldehydes (neuroketals) as products of the neuroprostane pathway. J Biol Chem.. 2001;276:30964-30970.[Abstract/Free Full Text]
82. Davies SS, Amarnath V, Montine KS, et al. Effects of reactive gamma-ketoaldehydes formed by the isoprostane pathway (isoketals) and cyclooxygenase pathway (levuglandins) on proteasome function. FASEB J.. 2002;16:715-717.[Free Full Text]
83. Youssef JA, Birnbaum LS, Swift LL, Morrow JD, Badr MZ. Age-independent, gray matter-localized, brain-enhanced oxidative stress in male fischer 344 rats: brain levels of F(2)-isoprostanes and F(4)-neuroprostanes. Free Radic Biol Med.. 2003;34:1631-1635.[Medline]
84. Liu J, Mori A. Age-associated changes in superoxide dismutase activity, thiobarbituric acid reactivity and reduced glutathione level in the brain and liver in senescence accelerated mice (SAM): a comparison with ddY mice. Mech Ageing Dev.. 1993;71:23-30.[Medline]
85. Hussain S, Slikker W, Jr, Ali SF. Age-related changes in antioxidant enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione in different regions of mouse brain. Int J Dev Neurosci.. 1995;13:811-817.[Medline]
86. Morrow JD, Roberts LJ, Daniel VC, et al. Comparison of formation of D2/E2-isoprostanes and F2-isoprostanes in vitro and in vivo—effects of oxygen tension and glutathione. Arch Biochem Biophys.. 1998;353:160-171.[Medline]
87. Benzi G, Pastoris O, Marzatico F, Villa RF. Age-related effect induced by oxidative stress on the cerebral glutathione system. Neurochem Res.. 1989;14:473-481.[Medline]
88. Balazs L, Leon M. Evidence of an oxidative challenge in the Alzheimer's brain. Neurochem Res.. 1994;19:1131-1137.[Medline]
89. Perry TL, Yong VW, Bergeron C, Hansen S, Jones K. Amino acids, glutathione, and glutathione transferase activity in the brains of patients with Alzheimer's disease. Ann Neurol.. 1987;21:331-336.[Medline]
90. Montine TJ, Markesbery WR, Morrow JD, Roberts LJ, II. Cerebrospinal fluid F2-isoprostane levels are increased in Alzheimer's disease. Ann Neurol.. 1998;44:410-413.[Medline]
91. Pratico D, Rokach J, Tangirala RK. Brains of aged apolipoprotein E-deficient mice have increased levels of F2-isoprostanes, in vivo markers of lipid peroxidation. J Neurochem.. 1999;73:736-741.[Medline]
92. Pratico D, Uryu K, Leight S, Trojanoswki JQ, Lee VM. Increased lipid peroxidation precedes amyloid plaque formation in an animal model of Alzheimer amyloidosis. J Neurosci.. 2001;21:4183-4187.[Abstract/Free Full Text]
93. Yamamoto S. Mammalian lipoxygenases: molecular structures and functions. Biochim Biophys Acta.. 1992;1128:117-131.[Medline]
94. Spiteller G. Lipid peroxidation in aging and age-dependent diseases. Exp Gerontol.. 2001;36:1425-1457.[Medline]
95. Hazen SL, Ford DA, Gross RW. Activation of a membrane-associated phospholipase A2 during rabbit myocardial ischemia which is highly selective for plasmalogen substrate. J Biol Chem.. 1991;266:5629-5633.[Abstract/Free Full Text]
96. Parthasarathy S, Steinbrecher UP, Barnett J, Witztum JL, Steinberg D. Essential role of phospholipase A2 activity in endothelial cell-induced modification of low density lipoprotein. Proc Natl Acad Sci U S A.. 1985;82:3000-3004.[Abstract/Free Full Text]
97. Sapirstein A, Spech RA, Witzgall R, Bonventre JV. Cytosolic phospholipase A2 (PLA2), but not secretory PLA2, potentiates hydrogen peroxide cytotoxicity in kidney epithelial cells. J Biol Chem.. 1996;271:21505-21513.[Abstract/Free Full Text]
98. Golconda MS, Ueda N, Shah SV. Evidence suggesting that iron and calcium are interrelated in oxidant-induced DNA damage. Kidney Int.. 1993;44:1228-1234.[Medline]
99. Levine RL, Stadtman ER. Oxidative modification of proteins during aging. Exp Gerontol.. 2001;36:1495-1502.[Medline]
100. Stadtman ER. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic Biol Med.. 1990;9:315-325.[Medline]
101. Smith CD, Carney JM, Starke-Reed PE, et al. Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc Natl Acad Sci U S A.. 1991;88:10540-10543.[Abstract/Free Full Text]
102. Aksenova MV, Aksenov MY, Carney JM, Butterfield DA. Protein oxidation and enzyme activity decline in old brown Norway rats are reduced by dietary restriction. Mech Ageing Dev.. 1998;100:157-168.[Medline]
103. Dubey A, Forster MJ, Sohal RS. Effect of the spin-trapping compound N-tert-butyl-alpha-phenylnitrone on protein oxidation and life span. Arch Biochem Biophys.. 1995;324:249-254.[Medline]
104. Cakatay U, Telci A, Kayali R, Tekeli F, Akcay T, Sivas A. Relation of oxidative protein damage and nitrotyrosine levels in the aging rat brain. Exp Gerontol.. 2001;36:221-229.[Medline]
105. Nicolle MM, Gonzalez J, Sugaya K, et al. Signatures of hippocampal oxidative stress in aged spatial learning-impaired rodents. Neuroscience.. 2001;107:415-431.[Medline]
106. Agarwal S, Sohal RS. Aging and proteolysis of oxidized proteins. Arch Biochem Biophys.. 1994;309:24-28.[Medline]
107. Butterfield DA, Howard BJ, Yatin S, Allen KL, Carney JM. Free radical oxidation of brain proteins in accelerated senescence and its modulation by N-tert-butyl-alpha-phenylnitrone. Proc Natl Acad Sci U S A.. 1997;94:674-678.[Abstract/Free Full Text]
108. Farr SA, Poon HF, Dogrukol-Ak D, et al. The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice. J Neurochem.. 2003;84:1173-1183.[Medline]
109. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol.. 1996;271:C1424-C1437.
110. Butterfield DA, Kanski J. Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins. Mech Ageing Dev.. 2001;122:945-962.[Medline]
111. Althaus JS, Schmidt KR, Fountain ST, Tseng MT, Carroll RT, Galatsis P, Hall ED. LC-MS/MS detection of peroxynitrite-derived 3-nitrotyrosine in rat microvessels. Free Radic Biol Med.. 2000;29:1085-1095.[Medline]
112. Shin CM, Chung YH, Kim MJ, Lee EY, Kim EG, Cha CI. Age-related changes in the distribution of nitrotyrosine in the cerebral cortex and hippocampus of rats. Brain Res.. 2002;931:194-199.[Medline]
113. Tohgi H, Abe T, Yamazaki K, Murata T, Ishizaki E, Isobe C. Alterations of 3-nitrotyrosine concentration in the cerebrospinal fluid during aging and in patients with Alzheimer's disease. Neurosci Lett.. 1999;269:52-54.[Medline]
114. Sloane JA, Hollander W, Moss MB, Rosene DL, Abraham CR. Increased microglial activation and protein nitration in white matter of the aging monkey. Neurobiol Aging.. 1999;20:395-405.[Medline]
115. Chung YH, Shin CM, Joo KM, Kim MJ, Cha CI. Immunohistochemical study on the distribution of nitrotyrosine and neuronal nitric oxide synthase in aged rat cerebellum. Brain Res.. 2002;951:316-321.[Medline]
116. Hensley K, Maidt ML, Yu Z, Sang H, Markesbery WR, Floyd RA. Electrochemical analysis of protein nitrotyrosine and dityrosine in the Alzheimer brain indicates region-specific accumulation. J Neurosci.. 1998;18:8126-8132.[Abstract/Free Full Text]
117. Smith MA, Richey Harris PL, Sayre LM, Beckman JS, Perry G. Widespread peroxynitrite-mediated damage in Alzheimer's disease. J Neurosci.. 1997;17:2653-2657.[Abstract/Free Full Text]
118. Castegna A, Thongboonkerd V, Klein JB, Lynn B, Markesbery WR, Butterfield DA. Proteomic identification of nitrated proteins in Alzheimer's disease brain. J Neurochem.. 2003;85:1994-1401.
119. Koppal T, Drake J, Yatin S, et al. Peroxynitrite-induced alterations in synaptosomal membrane proteins: insight into oxidative stress in Alzheimer's disease. J Neurochem.. 1999;72:310-317.[Medline]
120. Hall NC, Carney JM, Cheng MS, Butterfield DA. Ischemia/reperfusion-induced changes in membrane proteins and lipids of gerbil cortical synaptosomes. Neuroscience.. 1995;64:81-89.[Medline]
121. Hall NC, Carney JM, Plante OJ, Cheng M, Butterfield DA. Effect of 2-cyclohexene-1-one-induced glutathione diminution on ischemia/reperfusion-induced alterations in the physical state of brain synaptosomal membrane proteins and lipids. Neuroscience.. 1997;77:283-290.[Medline]
122. Subramaniam R, Koppal T, Green M, et al. The free radical antioxidant vitamin E protects cortical synaptosomal membranes from amyloid beta-peptide(25-35) toxicity but not from hydroxynonenal toxicity: relevance to the free radical hypothesis of Alzheimer's disease. Neurochem Res.. 1998;23:1403-1410.[Medline]
123. Pocernich CB, La Fontaine M, Butterfield DA. In-vivo glutathione elevation protects against hydroxyl free radical-induced protein oxidation in rat brain. Neurochem Int.. 2000;36:185-191.[Medline]
124. Hensley K, Carney J, Hall N, Shaw W, Butterfield DA. Electron paramagnetic resonance investigations of free radical-induced alterations in neocortical synaptosomal membrane protein infrastructure. Free Radic Biol Med.. 1994;17:321-331.[Medline]
125. Hensley K, Howard BJ, Carney JM, Butterfield DA. Membrane protein alterations in rodent erythrocytes and synaptosomes due to aging and hyperoxia. Biochim Biophys Acta.. 1995;1270:203-206.[Medline]
126. Aksenov MY, Aksenova MV, Carney JM, Butterfield DA. Oxidative modification of glutamine synthetase by amyloid beta peptide. Free Radic Res.. 1997;27:267-281.[Medline]
127. Hensley K, Hall N, Subramaniam R, et al. Brain regional correspondence between Alzheimer's disease histopathology and biomarkers of protein oxidation. J Neurochem.. 1995;65:2146-2156.[Medline]
128. Howard BJ, Yatin S, Hensley K, Allen KL, Kelly JP, Carney J, Butterfield DA. Prevention of hyperoxia-induced alterations in synaptosomal membrane-associated proteins by N-tert-butyl-alpha-phenylnitrone and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol). J Neurochem.. 1996;67:2045-2050.[Medline]
129. Floyd RA. Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J.. 1990;4:2587-2597.[Abstract]
130. Carney JM, Starke-Reed PE, Oliver CN, et al. Reversal of age-related increase in brain protein oxidation, decrease in enzyme activity, and loss in temporal and spatial memory by chronic administration of the spin-trapping compound N-tert-butyl-alpha-phenylnitrone. Proc Natl Acad Sci U S A.. 1991;88:3633-3636.[Abstract/Free Full Text]
131. Yatin SM, Aksenov M, Butterfield DA. The antioxidant vitamin E modulates amyloid beta-peptide-induced creatine kinase activity inhibition and increased protein oxidation: implications for the free radical hypothesis of Alzheimer's disease. Neurochem Res.. 1999;24:427-435.[Medline]
132. Gibson GE, Park LC, Zhang H, Sorbi S, Calingasan NY. Oxidative stress and a key metabolic enzyme in Alzheimer brains, cultured cells, and an animal model of chronic oxidative deficits. Ann N Y Acad Sci.. 1999;893:79-94.[Medline]
133. Vitorica J, Andres A, Satrustegui J, Machado A. Age-related quantitative changes in enzyme activities of rat brain. Neurochem Res.. 1981;6:127-136.[Medline]
134. Deloulme JC, Lucas M, Gaber C, Bouillon P, Keller A, Eclancher F, Sensenbrenner M. Expression of the neuron-specific enolase gene by rat oligodendroglial cells during their differentiation. J Neurochem.. 1996;66:936-945.[Medline]
135. Ferrante F, Amenta F. Enzyme histochemistry of the choroid plexus in old rats. Mech Ageing Dev.. 1987;41:65-72.[Medline]
136. Agrawal A, Shukla R, Tripathi LM, Pandey VC, Srimal RC. Permeability function related to cerebral microvessel enzymes during ageing in rats. Int J Dev Neurosci.. 1996;14:87-91.[Medline]
137. Hrachovina V, Mourek J. [Lactate dehydrogenase and malate dehydrogenase activity in the glial and neuronal fractions of the brain tissue in rats of various ages]. Sb Lek.. 1990;92:39-44.[Medline]
138. Mizuno Y, Ohta K. Regional distributions of thiobarbituric acid-reactive products, activities of enzymes regulating the metabolism of oxygen free radicals, and some of the related enzymes in adult and aged rat brains. J Neurochem.. 1986;46:1344-1352.[Medline]
139. Sandhu SK, Kaur G. Mitochondrial electron transport chain complexes in aging rat brain and lymphocytes. Biogerontology.. 2003;4:19-29.[Medline]
140. Kocak H, Oner P, Oztas B. Comparison of the activities of Na(+), K(+)-ATPase in brains of rats at different ages. Gerontology.. 2002;48:279-281.[Medline]
141. Gorini A, Canosi U, Devecchi E, Geroldi D, Villa RF. ATPases enzyme activities during ageing in different types of somatic and synaptic plasma membranes from rat frontal cerebral cortex. Prog Neuropsychopharmacol Biol Psychiatry.. 2002;26:81-90.[Medline]
142. Vohra BP, Sharma SP, Kansal VK. Age-dependent variations in mitochondrial and cytosolic antioxidant enzymes and lipid peroxidation in different regions of central nervous system of guinea pigs. Indian J Biochem Biophys.. 2001;38:321-326.[Medline]
143. Sandhu SK, Kaur G. Alterations in oxidative stress scavenger system in aging rat brain and lymphocytes. Biogerontology.. 2002;3:161-173.[Medline]
144. Tsay HJ, Wang P, Wang SL, Ku HH. Age-associated changes of superoxide dismutase and catalase activities in the rat brain. J Biomed Sci.. 2000;7:466-474.[Medline]
145. Castegna A, Aksenov M, Aksenova M, et al. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. Free Radic Biol Med.. 2002;33:562-571.[Medline]
146. Castegna A, Aksenov M, Thongboonkerd V, et al. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71. J Neurochem.. 2002;82:1524-1532.[Medline]
147. Butterfield DA, Boyd-Kimball DL, Castegna A. Proteomics in Alzheimer's disease: insights into mechanisms of neurodegenation. J Neurochem. 203;86:1313–1327.
148. Ropeleski MJ, Tang J, Walsh-Reitz MM, Musch MW, Chang EB. Interleukin-11-induced heat shock protein 25 confers intestinal epithelial-specific cytoprotection from oxidant stress. Gastroenterology.. 2003;124:1358-1368.[Medline]
149. Ozawa T, Tanaka M, Ikebe S, Ohno K, Kondo T, Mizuno Y. Quantitative determination of deleted mitochondrial DNA relative to normal DNA in parkinsonian striatum by a kinetic PCR analysis. Biochem Biophys Res Commun.. 1990;172:483-489.[Medline]
150. Strauss E. Longevity research. Single signal unites treatments that prolong life. Science.. 2003;300:881-883.
151. Calabrese V, Scapagnini G, Ravagna A, Giuffrida Stella AM, Butterfield DA. Molecular chaperones and their roles in neural cell differentiation. Dev Neurosci.. 2002;24:1-13.[Medline]
152. Calabrese V, Renis M, Calderone A, Russo A, Reale S, Barcellona ML, Rizza V. Stress proteins and SH-groups in oxidant-induced cellular injury after chronic ethanol administration in rat. Free Radic Biol Med.. 1998;24:1159-1167.[Medline]
153. Calabrese V, Scapagnini G, Catalano C, et al. Induction of heat shock protein synthesis in human skin fibroblasts in response to oxidative stress: regulation by a natural antioxidant from rosemary extract. Int J Tissue React.. 2001;23:51-58.[Medline]
154. Wheeler DS, Dunsmore KE, Wong HR. Intracellular delivery of HSP70 using HIV-1 Tat protein transduction domain. Biochem Biophys Res Commun.. 2003;301:54-59.[Medline]
155. Meldrum KK, Burnett AL, Meng X, Misseri R, Shaw MB, Gearhart JP, Meldrum DR. Liposomal delivery of heat shock protein 72 into renal tubular cells blocks nuclear factor-kappaB activation, tumor necrosis factor-alpha production, and subsequent ischemia-induced apoptosis. Circ Res.. 2003;92:293-299.[Abstract/Free Full Text]
156. Goodman R, Blank M. Insights into electromagnetic interaction mechanisms. J Cell Physiol.. 2002;192:16-22.[Medline]
157. Calabrese V, Scapagnini G, Giuffrida Stella AM, Bates TE, Clark JB. Mitochondrial involvement in brain function and dysfunction: relevance to aging, neurodegenerative disorders and longevity. Neurochem Res.. 2001;26:739-764.[Medline]
158. Kelly S, Yenari MA. Neuroprotection: heat shock proteins. Curr Med Res Opin.. 2002;18:(Suppl 2): S55-S60.
159. Kelly S, Zhang ZJ, Zhao H, et al. Gene transfer of HSP72 protects cornu ammonis 1 region of the hippocampus neurons from global ischemia: influence of Bcl-2. Ann Neurol.. 2002;52:160-167.[Medline]
160. Yenari MA. Heat shock proteins and neuroprotection. Adv Exp Med Biol.. 2002;513:281-299.[Medline]
161. Hata R, Gass P, Mies G, Wiessner C, Hossmann KA. Attenuated c-fos mRNA induction after middle cerebral artery occlusion in CREB knockout mice does not modulate focal ischemic injury. J Cereb Blood Flow Metab.. 1998;18:1325-1335.[Medline]
162. Narasimhan P, Swanson RA, Sagar SM, Sharp FR. Astrocyte survival and HSP70 heat shock protein induction following heat shock and acidosis. Glia.. 1996;17:147-159.[Medline]
163. Fink SL, Chang LK, Ho DY, Sapolsky RM. Defective herpes simplex virus vectors expressing the rat brain stress-inducible heat shock protein 72 protect cultured neurons from severe heat shock. J Neurochem.. 1997;68:961-969.[Medline]
164. Mosser DD, Caron AW, Bourget L, Denis-Larose C, Massie B. Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol Cell Biol.. 1997;17:5317-5327.[Abstract]
165. McLaughlin B, Hartnett KA, Erhardt JA, et al. Caspase 3 activation is essential for neuroprotection in preconditioning. Proc Natl Acad Sci U S A.. 2003;100:715-720.[Abstract/Free Full Text]
166. Calabrese V, Copani A, Testa D, et al. Nitric oxide synthase induction in astroglial cell cultures: effect on heat shock protein 70 synthesis and oxidant/antioxidant balance. J Neurosci Res.. 2000;60:613-622.[Medline]
167. Calabrese V, Testa G, Ravagna A, Bates TE, Stella AM. HSP70 induction in the brain following ethanol administration in the rat: regulation by glutathione redox state. Biochem Biophys Res Commun.. 2000;269:397-400.[Medline]
168. Mayer RJ. From neurodegeneration to neurohomeostasis: the role of ubiquitin. Drug News Perspect.. 2003;16:103-108.[Medline]
169. Valentim LM, Rodnight R, Geyer AB, et al. Changes in heat shock protein 27 phosphorylation and immunocontent in response to preconditioning to oxygen and glucose deprivation in organotypic hippocampal cultures. Neuroscience.. 2003;118:379-386.[Medline]
170. Turner CP, Panter SS, Sharp FR. Anti-oxidants prevent focal rat brain injury as assessed by induction of heat shock proteins (HSP70, HO-1/HSP32, HSP47) following subarachnoid injections of lysed blood. Brain Res Mol Brain Res.. 1999;65:87-102.[Medline]
171. Izaki K, Kinouchi H, Watanabe K, et al. Induction of mitochondrial heat shock protein 60 and 10 mRNAs following transient focal cerebral ischemia in the rat. Brain Res Mol Brain Res.. 2001;88:14-25.[Medline]
172. Sahlas DJ, Liberman A, Schipper HM. Role of heme oxygenase-1 in the biogenesis of corpora amylacea. Biogerontology.. 2002;3:223-231.[Medline]
173. Goldbaum O, Richter-Landsberg C. Stress proteins in oligodendrocytes: differential effects of heat shock and oxidative stress. J Neurochem.. 2001;78:1233-1242.[Medline]
174. Schipper HM. Heme oxygenase-1: role in brain aging and neurodegeneration. Exp Gerontol.. 2000;35:821-830.[Medline]
175. Shibahara S, Muller R, Taguchi H, Yoshida T. Cloning and expression of cDNA for rat heme oxygenase. Proc Natl Acad Sci U S A.. 1985;82:7865-7869.[Abstract/Free Full Text]
176. Maines MD, Trakshel GM, Kutty RK. Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J Biol Chem.. 1986;261:411-419.[Abstract/Free Full Text]
177. McCoubrey WK, Jr, Huang TJ, Maines MD. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem.. 1997;247:725-732.[Medline]
178. Maines MD. The heme oxygenase system: a regulator of second messenger gases. Ann Rev Pharmacol Toxicol.. 1997;37:517-554.[Medline]
179. Abraham NG, Drummond GS, Lutton JD, Kappas A. The biological significance and physiological role of heme oxygenase. Cell Physiol Biochem.. 1996;6:129-168.
180. Calabrese V, Scapagnini G, Ravagna A, Fariello RG, Giuffrida Stella AM, Abraham NG. Regional distribution of heme oxygenase, HSP70, and glutathione in brain: relevance for endogenous oxidant/antioxidant balance and stress tolerance. J Neurosci Res.. 2002;68:65-75.[Medline]
181. Ewing JF, Maines MD. In Situ Hybridization and immunohistochemical localization of heme oxygenase-2 mRNA and protein in normal rat brain: differential distribution of isozyme 1 and 2. Mol Cell Neurosci.. 1992;3:559-570.
182. Scapagnini G, D'Agata V, Calabrese V, et al. Gene expression profiles of heme oxygenase isoforms in the rat brain. Brain Res.. 2002;954:51-59.[Medline]
183. Colombrita C, Calabrese V, Stella AM, Mattei F, Alkon DL, Scapagnini G. Regional rat brain distribution of heme oxygenase-1 and manganese superoxide dismutase mRNA: relevance of redox homeostasis in the aging processes. Exp Biol Med (Maywood).. 2003;228:517-524.[Abstract/Free Full Text]
184. Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snyder SH. Carbon monoxide: a putative neural messenger. Science.. 1993;259:381-384.[Abstract/Free Full Text]
185. Hon T, Hach A, Lee HC, Cheng T, Zhang L. Functional analysis of heme regulatory elements of the transcriptional activator Hap1. Biochem Biophys Res Commun.. 2000;273:584-591.[Medline]
186. McCoubrey WK, Jr, Huang TJ, Maines MD. Heme oxygenase-2 is a hemoprotein and binds heme through heme regulatory motifs that are not involved in heme catalysis. J Biol Chem.. 1997;272:12568-12574.[Abstract/Free Full Text]
187. Maines MD. The heme oxygenase system and its functions in the brain. Cell Mol Biol (Noisy-le-grand).. 2000;46:573-585.
188. Kazazian HH, Jr. Genetics. L1 retrotransposons shape the mammalian genome. Science.. 2000;289:1152-1153.[Free Full Text]
189. Mighell AJ, Smith NR, Robinson PA, Markham AF. Vertebrate pseudogenes. FEBS Lett.. 2000;468:109-114.[Medline]
190. Raju VS, McCoubrey WK, Jr, Maines MD. Regulation of heme oxygenase-2 by glucocorticoids in neonatal rat brain: characterization of a functional glucocorticoid response element. Biochim Biophys Acta.. 1997;1351:89-104.[Medline]
191. Liu N, Wang X, McCoubrey WK, Maines MD. Developmentally regulated expression of two transcripts for heme oxygenase-2 with a first exon unique to rat testis: control by corticosterone of the oxygenase protein expression. Gene.. 2000;241:175-183.[Medline]
192. Tyrrell R. Redox regulation and oxidant activation of heme oxygenase-1. Free Radic Res.. 1999;31:335-340.[Medline]
193. Motterlini R, Foresti R, Bassi R, Calabrese V, Clark JE, Green CJ. Endothelial heme oxygenase-1 induction by hypoxia. Modulation by inducible nitric-oxide synthase and S-nitrosothiols. J Biol Chem.. 2000;275:13613-13620.[Abstract/Free Full Text]
194. Hill-Kapturczak N, Sikorski E, Voakes C, Garcia J, Nick HS, Agarwal A. An internal enhancer regulates heme- and cadmium-mediated induction of human heme oxygenase-1. Am J Physiol Renal Physiol.. 2003;285:F515-F523.[Abstract/Free Full Text]
195. Fogg S, Agarwal A, Nick HS, Visner GA. Iron regulates hyperoxia-dependent human heme oxygenase 1 gene expression in pulmonary endothelial cells. Am J Respir Cell Mol Biol.. 1999;20:797-804.[Abstract/Free Full Text]
196. Scapagnini G, Foresti R, Calabrese V, Giuffrida Stella AM, Green CJ, Motterlini R. Caffeic acid phenethyl ester and curcumin: a novel class of heme oxygenase-1 inducers. Mol Pharmacol.. 2002;61:554-561.[Abstract/Free Full Text]
197. Balogun E, Hoque M, Gong P, et al. Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem J.. 2003;371:887-895.[Medline]
198. Chen K, Gunter K, Maines MD. Neurons overexpressing heme oxygenase-1 resist oxidative stress-mediated cell death. J Neurochem.. 2000;75:304-313.[Medline]
199. Dore S, Takahashi M, Ferris CD, Zakhary R, Hester LD, Guastella D, Snyder SH. Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury. Proc Natl Acad Sci U S A.. 1999;96:2445-2450.[Abstract/Free Full Text]
200. Le WD, Xie WJ, Appel SH. Protective role of heme oxygenase-1 in oxidative stress-induced neuronal injury. J Neurosci Res.. 1999;56:652-658.[Medline]
201. Dore S. Decreased activity of the antioxidant heme oxygenase enzyme: implications in ischemia and in Alzheimer's disease. Free Radic Biol Med.. 2002;32:1276-1282.[Medline]
202. Beschorner R, Adjodah D, Schwab JM, et al. Long-term expression of heme oxygenase-1 (HO-1, HSP-32) following focal cerebral infarctions and traumatic brain injury in humans. Acta Neuropathol (Berl).. 2000;100:377-384.[Medline]
203. Takeda A, Perry G, Abraham NG, et al. Overexpression of heme oxygenase in neuronal cells, the possible interaction with tau. J Biol Chem.. 2000;275:5395-5399.[Abstract/Free Full Text]
204. Takahashi M, Dore S, Ferris CD, et al. Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer's disease. Neuron.. 2000;28:461-473.[Medline]
205. Premkumar DR, Smith MA, Richey PL, et al. Induction of heme oxygenase-1 mRNA and protein in neocortex and cerebral vessels in Alzheimer's disease. J Neurochem.. 1995;65:1399-1402.[Medline]
206. Schipper HM, Chertkow H, Mehindate K, Frankel D, Melmed C, Bergman H. Evaluation of heme oxygenase-1 as a systemic biological marker of sporadic AD. Neurology.. 2000;54:1297-1304.[Abstract/Free Full Text]
207. Schipper HM, Liberman A, Stopa EG. Neural heme oxygenase-1 expression in idiopathic Parkinson's disease. Exp Neurol.. 1998;150:60-68.[Medline]
208. Yoo MS, Chun HS, Son JJ, et al. Oxidative stress regulated genes in nigral dopaminergic neuronal cells: correlation with the known pathology in Parkinson's disease. Brain Res Mol Brain Res.. 2003;110:76-84.[Medline]
209. Liu Y, Zhu B, Luo L, Li P, Paty DW, Cynader MS. Heme oxygenase-1 plays an important protective role in experimental autoimmune encephalomyelitis. Neuroreport.. 2001;12:1841-1845.[Medline]
210. Butterfield D, Castegna A, Pocernich C, Drake J, Scapagnini G, Calabrese V. Nutritional approaches to combat oxidative stress in Alzheimer's disease. J Nutr Biochem.. 2002;13:444-461.[Medline]
211. Piantadosi CA, Zhang J, Levin ED, Folz RJ, Schmechel DE. Apoptosis and delayed neuronal damage after carbon monoxide poisoning in the rat. Exp Neurol.. 1997;147:103-114.[Medline]
212. Graser T, Vedernikov YP, Li DS. Study on the mechanism of carbon monoxide induced endothelium-independent relaxation in porcine coronary artery and vein. Biomed Biochim Acta.. 1990;49:293-296.[Medline]
213. Glaum SR, Miller RJ. Zinc protoporphyrin-IX blocks the effects of metabotropic glutamate receptor activation in the rat nucleus tractus solitarii. Mol Pharmacol.. 1993;43:965-969.[Abstract]
214. White KA, Marletta MA. Nitric oxide synthase is a cytochrome P-450 type hemoprotein. Biochemistry.. 1992;31:6627-6631.[Medline]
215. Foresti R, Motterlini R. The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis. Free Radic Res.. 1999;31:459-475.[Medline]
216. Turcanu V, Dhouib M, Poindron P. Nitric oxide synthase inhibition by haem oxygenase decreases macrophage nitric-oxide-dependent cytotoxicity: a negative feedback mechanism for the regulation of nitric oxide production. Res Immunol.. 1998;149:741-744.[Medline]
217. Wang R, Wu L. Interaction of selective amino acid residues of K(ca) channels with carbon monoxide. Exp Biol Med (Maywood).. 2003;228:474-480.[Abstract/Free Full Text]
218. Coceani F, Kelsey L, Seidlitz E, et al. Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone. Br J Pharmacol.. 1997;120:599-608.[Medline]
219. Vijayasarathy C, Damle S, Lenka N, Avadhani NG. Tissue variant effects of heme inhibitors on the mouse cytochrome c oxidase gene expression and catalytic activity of the enzyme complex. Eur J Biochem.. 1999;266:191-200.[Medline]
220. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science.. 1987;235:1043-1046.[Abstract/Free Full Text]
221. Clark JE, Foresti R, Green CJ, Motterlini R. Dynamics of haem oxygenase-1 expression and bilirubin production in cellular protection against oxidative stress. Biochem J.. 2000;348 Pt 3:615-619.[Medline]
222. Dore S, Goto S, Sampei K, et al. Heme oxygenase-2 acts to prevent neuronal death in brain cultures and following transient cerebral ischemia. Neuroscience.. 2000;99:587-592.[Medline]
223. Kimpara T, Takeda A, Yamaguchi T, et al. Increased bilirubins and their derivatives in cerebrospinal fluid in Alzheimer's disease. Neurobiol Aging.. 2000;21:551-554.[Medline]
224. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A.. 1993;90:7915-7922.[Abstract/Free Full Text]
225. Rustin P, von Kleist-Retzow JC, Vajo Z, Rotig A, Munnich A. For debate: defective mitochondria, free radicals, cell death, aging-reality or myth-ochondria? Mech Ageing Dev.. 2000;114:201-206.[Medline]
226. Koubova J, Guarente L. How does calorie restriction work? Genes Dev.. 2003;17:313-321.[Free Full Text]
227. Kaeberlein M, McVey M, Guarente L. The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev.. 1999;13:2570-2580.[Abstract/Free Full Text]
228. Dilman VM. Growth hormone, age and the endometrium. N Engl J Med.. 1970;283:375.
229. Herrling T, Fuchs J, Rehberg J, Groth N. UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides. Free Radic Biol Med.. 2003;35:59-67.[Medline]
230. Weismann A. Ueber die Dauer des Lebens. Jena, Germany: Verlag von Gustav Fisher; 1982.
231. Olovnikov AM. Telomeres, telomerase, and aging: origin of the theory. Exp Gerontol.. 1996;31:443-448.[Medline]
232. Johnson S. The possible crucial role of iron accumulation combined with low tryptophan, zinc and manganese in carcinogenesis. Med Hypotheses.. 2001;57:539-543.[Medline]
233. Butterfield DA, Castegna A, Lauderback CM, Drake J. Evidence that amyloid beta-peptide-induced lipid peroxidation and its sequelae in Alzheimer's disease brain contribute to neuronal death. Neurobiol Aging.. 2002;23:655-664.[Medline]

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