Neurofilaments are the major neuronal target of hydroxynonenal-mediated protein cross-links.

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently binds amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated to be intimately associated with pathological lesions of Alzheimer's disease (AD), suggesting that oxidative stress is a major component in the disease. Here, we examined the HNE-cross-linking modifications by using an antibody specific for a lysine-lysine cross-link. Since in a prior study we noted no immunolabeling of neuritic plaques or neurofibrillary tangles but instead found strong labeling of axons, we focused this study on axons. Axonal labeling was examined in mouse sciatic nerve, and immunoblotting showed the cross-link was restricted to neurofilament heavy and medium subunits, which while altering migration, did not indicate larger NF aggregates, indicative of intermolecular cross-links. Examination of mice at various ages showed the extent of modification remaining relatively constant through the life span. These findings demonstrate lipid-cross-linking peroxidation primarily involves lysine-rich neurofilaments and is restricted to intramolecular cross-links.

Acrolein generation stimulates hypercontraction in isolated human blood vessels.

Increased risk of vasospasm, a spontaneous hyperconstriction, is associated with atherosclerosis, cigarette smoking, and hypertension-all conditions involving oxidative stress, lipid peroxidation, and inflammation. To test the role of the lipid peroxidation- and inflammation-derived aldehyde, acrolein, in human vasospasm, we developed an ex vivo model using human coronary artery bypass graft (CABG) blood vessels and a demonstrated acrolein precursor, allylamine. Allylamine induces hypercontraction in isolated rat coronary artery in a semicarbazide-sensitive amine oxidase activity (SSAO) dependent manner. Isolated human CABG blood vessels (internal mammary artery, radial artery, saphenous vein) were used to determine: (1) vessel responses and sensitivity to acrolein, allylamine, and H(2)O(2) exposure (1 microM-1 mM), (2) SSAO dependence of allylamine-induced effects using SSAO inhibitors (semicarbazide, 1 mM; MDL 72274-E, active isomer; MDL 72274-Z, inactive isomer; 100 microM), (3) the vasoactive effects of two other SSAO amine substrates, benzylamine and methylamine, and (4) the contribution of extracellular Ca(2+) to hypercontraction. Acrolein or allylamine but not H(2)O(2), benzylamine, or methylamine stimulated spontaneous and pharmacologically intractable hypercontraction in CABG blood vessels that was similar to clinical vasospasm. Allylamine-induced hypercontraction and blood vessel SSAO activity were abolished by pretreatment with semicarbazide or MDL 72274-E but not by MDL 72274-Z. Allylamine-induced hypercontraction also was significantly attenuated in Ca(2+)-free buffer. In isolated aorta of spontaneously hypertensive rat, allylamine-induced an SSAO-dependent contraction and enhanced norepinephrine sensitivity but not in Sprague-Dawley rat aorta. We conclude that acrolein generation in the blood vessel wall increases human susceptibility to vasospasm, an event that is enhanced in hypertension.

Catalytic turnover of benzylamine by a model for the lysine tyrosylquinone (LTQ) cofactor of lysyl oxidase.

Lysyl oxidase differs from other copper amine oxidases in that its active quinone cofactor reflects cross-linking of a lysyl residue into the tyrosine-derived quinone nucleus found in the plasma and other copper amine oxidases. A model for the lysyl oxidase cofactor (LTQ), 3,3-dimethyl-2,3-dihydroindole-5,6-quinone (4), was synthesized and found to be stable to both hydrolysis and oxidation events that prevent simpler models from functioning as turnover catalysts. We show that 4 catalyzes the aerobic oxidative deamination of benzylamine, though turnover eventually ceases on account of oxidation of the dihydrobenzoxazole tautomer of the "product Schiff base" to form a benzoxazole, a reaction that may be physiologically relevant. The mechanism of the overall reaction profile was elucidated by a combination of optical and NMR spectroscopy and O(2) uptake studies.

Active glycation in neurofibrillary pathology of Alzheimer disease: N(epsilon)-(carboxymethyl) lysine and hexitol-lysine.

Advanced glycation end products are a diverse class of posttranslational modifications, stemming from reactive aldehyde reactions, that have been implicated in the pathogenesis of a number of degenerative diseases. Because advanced glycation end products are accelerated by, and result in formation of, oxygen-derived free radicals, they represent an important component of the oxidative stress hypothesis of Alzheimer disease (AD). In this study, we used in situ techniques to assess N(epsilon)-(Carboxymethyl)lysine (CML), the predominant advanced glycation end product that accumulates in vivo, along with its glycation-specific precursor hexitol-lysine, in patients with AD as well as in young and aged-matched control cases. Both CML and hexitol-lysine were increased in neurons, especially those containing intracellular neurofibrillary pathology in cases of AD. The increase in hexitol-lysine and CML in AD suggests that glycation is an early event in disease pathogenesis. In addition, because CML can result from either lipid peroxidation or advanced glycation, while hexitol-lysine is solely a product of glycation, this study, together with studies demonstrating the presence of 4-hydroxy-2-nonenal adducts and pentosidine, provides evidence of two distinct oxidative processes acting in concert in AD neuropathology. Our findings support the notion that aldehyde-mediated modifications, together with oxyradical-mediated modifications, are critical pathogenic factors in AD.

Mechanisms by which metals promote events connected to neurodegenerative diseases.

Although the exact causative phenomenon responsible for the onset and progression of neurodegenerative disorders is at present unresolved, there are some clues as to the mechanisms underlying these chronic diseases. This review addresses mechanisms by which endogenous or environmental factors, through interaction with redox active metals, may initiate a common cascade of events terminating in neurodegeneration.

Chemistry and biochemistry of oxidative stress in neurodegenerative disease.

The age-related neurodegenerative diseases exemplified by Alzheimer&hyp;s disease (AD), Lewy body diseases such as Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington&hyp;s disease are characterized by the deposition of abnormal forms of specific proteins in the brain. Although several factors appear to underlie the pathological depositions, the cause of neuronal death in each disease appears to be multifactorial. In this regard, evidence in each case for a role of oxidative stress is provided by the finding that the pathological deposits are immunoreactive to antibodies recognizing protein side-chains modified either directly by reactive oxygen or nitrogen species, or by products of lipid peroxidation or glycoxidation. Although the source(s) of increased oxidative damage are not entirely clear, the findings of increased localization of redox-active transition metals in the brain regions most affected is consistent with their contribution to oxidative stress. It is tempting to speculate that free radical oxygen chemistry plays a pathogenetic role in all these neurodegenerative conditions, though it is as yet undetermined what types of oxidative damage occur early in pathogenesis, and what types are secondary manifestations of dying neurons. Delineation of the profile of oxidative damage in each disease will provide clues to how the specific neuronal populations are differentially affected by the individual disease conditions.

Temporary inactivation of plasma amine oxidase by alkylhydrazines. A combined enzyme/model study implicates cofactor reduction/reoxidation but cofactor deoxygenation and subsequent reoxygenation in the case of hydrazine itself.

It has been known for some time that hydrazine and its methyl and 1,1-dimethyl analogues induce inactivation of the copper-containing quinone-dependent plasma amine oxidase but that the activity recovers over time, suggesting metabolism of all three inhibitors. However, the mechanism responsible for loss and regain of activity has not been investigated. In this study a combination of enzyme studies under a controlled atmosphere along with model studies using 5-tert-butyl-2-hydroxy-1,4-benzoquinone to mimic the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor of the enzyme suggest that regain of enzyme activity represents two different O(2)-dependent processes. In the case of methylhydrazine and 1,1-dimethylhydrazine, we propose that the inactive methylhydrazone/azo form of the enzyme slowly rehydrates and eliminates MeN=NH to give the triol cofactor form, which instantly reoxidizes to the catalytically active quinone form in the presence of O(2). Metabolism of methylhydrazine represents its conversion to CH(4) and N(2), and of 1,1-dimethylhydrazine to CH(2)=O, CH(4), and N(2). In the case of hydrazine itself, however, we propose that the inactive hydrazone/azo form of the enzyme instead undergoes a slow decomposition, probably facilitated by the active-site copper, to give N(2) and a novel 5-desoxy resorcinol form of the cofactor. The latter undergoes a rapid, but noninstantaneous reoxygenation at C5 to restore the active cofactor form, also probably mediated by the active-site copper.

Novel 2,5-hexanedione analogues. Substituent-induced control of the protein cross-linking potential and oxidation susceptibility of the resulting primary amine-derived pyrroles.

The neurotoxic gamma-diketone, 2,5-hexanedione (2,5-HD), induces neurofilamentous swellings at prenodal sites in proximal axons as a consequence of pyrrolation of lysine epsilon-amino groups on neurofilament proteins. However, there is disagreement as to whether pyrrole formation and the associated alteration of noncovalent interactions is sufficient to cause neurofilament accumulation, or whether pyrrole autoxidation and subsequent protein-protein cross-linking is an obligatory event. To investigate gamma-diketones that might form pyrroles inert to autoxidative-induced cross-linking, we synthesized 1,1,1-trifluoro-2,5-hexanedione, 3-(trifluoromethyl)-2,5-hexanedione (3-TFMHD), and two 3-(dialkylaminocarbonyl)-2,5-diketones and assessed their rates of pyrrole formation with amines, the oxidation susceptibility of the resulting pyrroles, and the protein cross-linking potential in vitro, relative to those of 3-methyl-2,5-hexanedione. 1,1,1-Trifluoro-2,5-hexanedione does not form pyrroles, but the three 2,5-HD analogues with an electron-withdrawing 3-substituent all rapidly formed pyrroles that were inert to autoxidation. Although 3-TMFHD nonetheless still induced cross-linking of ribonuclease A, by a nonoxidative mechanism independent of the pyrrole, the two 3-(dialkylaminocarbonyl)-2,5-diketones did not exhibit any protein cross-linking. As these two gamma-diketones possess aqueous-organic partitioning properties similar to those of 2,5-HD, they should serve as useful mechanistic probes in further studies.

Catalytic turnover of substrate benzylamines by the quinone-dependent plasma amine oxidase leads to H2O2-dependent inactivation: evidence for generation of a cofactor-derived benzoxazole.

Incubation of bovine plasma amine oxidase (BPAO) with benzylamine and various p-substituted analogues results in a time-dependent inactivation that is attributable to buildup of the H(2)O(2)-turnover product on the basis of protection afforded by coincubation with catalase. The mechanism of inactivation is distinct from that effected by H(2)O(2) itself, which requires higher concentrations. Solution studies using models for the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor reveal a loss of catalytic activity arising from oxidation of the dihydrobenzoxazole tautomer of the product Schiff base, that competes with hydrolytic release of benzaldehyde product. The resulting stable benzoxazole exhibits a characteristic absorption depending on the nature of the benzylamine p-substituent. For benzylamine itself, the model benzoxazole absorbs at 313 nm, in an area of strong absorption by the enzyme, whereas for 4-nitrobenzylamine, the absorption of the model benzoxazole is sufficiently red-shifted (at 365 nm) to be discerned above the background enzyme absorption. Inactivation of BPAO by 4-nitrobenzylamine is accompanied by loss of the resting TPQ anion absorption at 480 nm concomitant with generation of a new absorption near 360 nm. Resonance Raman spectra of the inactivated enzyme show a close correspondence with those for the model 4-nitrobenzylamine-derived benzoxazole. Substrate-dependent inactivation is also observed for the other two mammalian enzymes examined, equine plasma amine oxidase and human kidney amine oxidase. Catalase provides complete protection in these instances as well. Benzoxazole formation may constitute a common mechanism of inactivation of quinone-dependent amine oxidases by normal substrates in vitro if the product H(2)O(2) is permitted to accumulate. More importantly, the results suggest that the benzoxazole inactivation pathway may be important physiologically and may have influenced the distribution of amine oxidases and catalase in cells.

Macrophage receptors responsible for distinct recognition of low density lipoprotein containing pyrrole or pyridinium adducts: models of oxidized low density lipoprotein.

Oxidation of low density lipoproteins (LDL) induced by incubation with Cu(2+) ions results in the formation of a heterogeneous group of aldehydic adducts on lysyl residues (Lys) of apolipoprotein B (apoB) that are thought to be responsible for the uptake of oxidized LDL (oxLDL) by macrophages. To define the structural and chemical criteria governing such cell recognition, we induced two modifications of lysines in LDL that mimic prototypic adducts present in oxLDL; namely, epsilon-amino charge-neutralizing pyrrolation by treatment with 2,5-hexanedione (hdLDL), and epsilon-amino charge-retaining pyridinium formation via treatment with 2,4,6-trimethylpyrylium (tmpLDL). Both modifications led to recognition by receptors on mouse peritoneal macrophages (MPM). To assess whether the murine scavenger receptor class A-I (mSR-A) was responsible for recognition of hdLDL or tmpLDL in MPM, we measured binding at 4 degrees C and degradation at 37 degrees C of these modified forms of (125)I-labeled LDL by mSR-A-transfected CHO cells. Although uptake and degradation of hdLDL by mSR-A-transfected CHO cells was quantitatively similar to that of the positive control, acLDL, tmpLDL was not recognized by these cells. However, both tmpLDL and hdLDL were recognized by 293 cells that had been transfected with CD36. In the human monocytic cell line THP-1 that had been activated with PMA, uptake of tmpLDL was significantly inhibited by blocking monoclonal antibodies to CD36, further suggesting recognition of tmpLDL by this receptor. Macrophage uptake and degradation of LDL oxidized by brief exposure to Cu(2+) was inhibited more effectively by excess tmpLDL and hdLDL than was more extensively oxidized LDL, consistent with the recognition of the former by CD36 and the latter primarily by SR-A.Collectively, these studies suggest that formation of specific pyrrole adducts on LDL leads to recognition by both the mSR-A and mouse homolog of CD36 expressed on MPM, while formation of specific pyridinium adducts on LDL leads to recognition by the mouse homolog of CD 36 but not by mSR-A. As such, these two modifications of LDL may represent useful models for dissecting the relative contributions of specific modifications on LDL produced during oxidation, to the cellular uptake of this heterogeneous ligand.

In Alzheimer's disease, heme oxygenase is coincident with Alz50, an epitope of tau induced by 4-hydroxy-2-nonenal modification.

In this study, we compared the neuronal induction of the antioxidant heme oxygenase-1 (HO-1) in Alzheimer's disease with abnormalities in tau marked by antibodies recognizing either phosphorylation (AT8) or conformational change (Alz50). The epitope recognized by Alz50 shows a complete overlap with HO-1-containing neurons, but AT8 recognized these neurons as well as neurons not displaying HO-1. These findings suggest that tau phosphorylation precedes the HO-1 response and that HO-1 is coincident with the Alz50 epitope. This led us to consider whether oxidative damage plays a role in forming the Alz50 epitope. We found that 4-hydroxy-2-nonenal (HNE), a highly reactive product of lipid peroxidation, reacts with normal tau and induces the Alz50 epitope in tau. It is important that the ability of HNE to create the Alz50 epitope not only is dependent on lysine residues of tau but also requires tau phosphorylation because neither methylated, recombinant, nor dephosphorylated tau reacts with HNE to create the Alz50 epitope. Supporting the in vivo relevance of this observation, endogenous paired helical filament-tau isolated from subjects with Alzheimer's disease was immunoreactive with an antibody to a stable HNE-lysine adduct, as were all vulnerable neurons in subjects with Alzheimer's disease but not in control individuals. Together, these findings support the involvement of oxidative damage early in neurofibrillary tangle formation in Alzheimer's disease and also suggest that HNE modification contributes to the generation of the tau conformation defining the Alz50 epitope. These findings provide evidence that an interplay between phosphorylation of tau and neuronal oxidative stress-induced pathology is important in the formation of neurofibrillary tangles.

Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by 2-phenyl-2-(1-piperidinyl)propane.

2-Phenyl-2-(1-piperidinyl)propane (PPP), an analog of phencyclidine, was tested for its ability to inactivate cytochrome P450s (P450s) 2B1 and 2B6. PPP inactivated the 7-(benzyloxy)resorufin O-dealkylation activity of liver microsomes obtained from phenobarbital-induced rats with a K(I) of 11 microM. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified rat liver P450 2B1 and expressed human P450 2B6 was inactivated by PPP in a reconstituted system containing NADPH-cytochrome P450 reductase and lipid. In the presence of NADPH, the loss of activity was time- and concentration-dependent, and followed pseudo first order kinetics. The rate of inactivation for P450 2B1 was 0.3 min(-1), and the concentration of PPP required to achieve half-maximal inactivation was 12 microM. The time for 50% of the P450 2B1 to become inactivated at saturating concentrations of PPP was 2.5 min. P450 2B6 was inactivated with a k(inact) of 0.07 min(-1), a K(I) of 1.2 microM, and a t(1/2) of 9.5 min. The inactivated P450s 2B1 and 2B6 lost about 25 and 15%, respectively, of their ability to form a CO-reduced complex, suggesting that the loss of activity was caused by a PPP modification of the apoprotein rather than the heme. The estimated partition ratio for P450s 2B1 and 2B6 with PPP was 31 and 15, respectively. The inactivation was not reversible and reductase activity was not affected. Coincubation of P450 2B1 and 2B6 with PPP and NADPH in the presence of an alternate substrate protected both enzymes from inactivation. The exogenous nucleophile GSH did not affect the rate of inactivation. PPP-inactivated P450s 2B1 and 2B6 were recognized on Western blots by an antibody generated to phencyclidine that had been conjugated to BSA. Stoichiometries of 1.4:1 and 0.7:1 were determined for the binding of a [3H]PPP metabolite to P450 2B1 and 2B6, respectively.

HNE-derived 2-pentylpyrroles are generated during oxidation of LDL, are more prevalent in blood plasma from patients with renal disease or atherosclerosis, and are present in atherosclerotic plaques.

Free radical oxidation of human plasma low-density lipoprotein (LDL) produces 2-pentylpyrrole epitopes that are generated by reaction of 4-hydroxy-2-nonenal (HNE), a product of lipid oxidation, with protein lysyl residues. The HNE-derived 2-pentylpyrrole ("HNE-pyrrole") epitopes were detected with an enzyme-linked immunosorbent assay (ELISA) using antibodies (ON-KLH) raised against protein-bound 2-pentylpyrrole obtained by the reaction of 2-oxononanal (ON) with keyhole limpet hemocyanin (KLH). HNE-pyrrole epitopes in human plasma are not associated primarily with LDL protein, apolipoprotein (apo) B, since only 15% of the total HNE-pyrrole immunoreactivity is removed by immunoprecipitation of apo B. The levels of ON-KLH immunoreactivity detected in human plasma were found to be significantly elevated in renal failure and atherosclerosis patients when compared to those in healthy volunteers. HNE-pyrrole immunoreactivity was also detected in atherosclerotic plaques. The highest levels were associated with extracellular connective tissue. Levels of ON-KLH immunoreactivity in human plasma far exceed levels of free HNE, presumably because of the rapid clearance of free relative to protein-bound HNE. Therefore, HNE-pyrrole epitopes provide a more indelible marker of oxidative injury than levels of free HNE.

Histochemical and immunocytochemical approaches to the study of oxidative stress.

We review an array of newly developed in situ detection methods that can be used for the qualitative and semi-quantitative measurement of various indices related to oxidative stress. The importance of in situ methods over bulk analysis cannot be overstated when considering the structural and cellular complexity of tissue and the effects of diseases thereof. Indeed, in situ detection allows detection of specific cell types affected or specific localization such that a process affecting only a small fraction of the tissue or cells can be readily visualized. Consequently, a positive signal in situ indicates real levels that cannot be masked by unrelated or compensatory responses in adjacent cells, and corrections can be easily made for the modifications to long-lived proteins during physiological aging. In fact, the damage to extracellular matrix proteins of major vessels, provides a cumulative record of long-term oxidative insult. Yet the same properties that make vessels ideal markers for aging limits their sensitivity to detect disease-specific changes unless in situ techniques are used.

Oxidative stress, antioxidants, and Alzheimer disease.

Recent evidence in the field of Alzheimer disease research has highlighted the importance of oxidative processes in its pathogenesis. Examination of cellular changes shows that oxidative stress is an event that precedes the appearance of neurofibrillary tangles, one of the hallmark pathologies of the disease. Although it is still unclear what the initial source of the oxidative stress is in Alzheimer disease, it is likely that the process is highly dependent on the presence of redox-active transition metals, such as iron and copper. Because of the proximal role that oxidative stress mechanisms seem to play in the pathogenesis of Alzheimer disease, further investigation in this realm may lead to novel therapeutic strategies.

The role of metals in neurodegenerative diseases.

There is increasing evidence in a number of neurodegenerative diseases that transition metal-mediated abnormalities play a crucial role in disease pathogenesis. In this treatise, we review the role of metal homeostasis as it pertains to alterations in brain function in neurodegenerative diseases. In fact, while there is documented evidence for alterations in transition metal homeostasis, redox-activity and localization, it is also important to realize that alterations in specific copper- and iron-containing metalloenzymes also appear to play a crucial role in the neurodegenerative process.

Polyclonal antibodies to a fluorescent 4-hydroxy-2-nonenal (HNE)-derived lysine-lysine cross-link: characterization and application to HNE-treated protein and in vitro oxidized low-density lipoprotein.

Oxidative modification of low-density lipoprotein (LDL) is thought to play a key role in the etiology of atherosclerosis. Oxidized LDL that accumulates in atherosclerotic plaques is known to exhibit a characteristic fluorescence with excitation and emission near 360 and 430 nm, respectively. (E)-4-Hydroxy-2-nonenal (HNE), formed during LDL oxidation, is capable of modifying LDL to generate the same fluorescent signature. The HNE-derived fluorophore was shown by us to possess a 2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one iminium (HPDPI) structure. We herein report the synthesis of the HPDPI-derived lysine-lysine cross-link needed as a standard reference for HPLC quantitation of the cross-link in protein hydrolysates. The main focus of the current work, however, is the design and development of two polyclonal antibodies against the HPDPI epitope. Utilizing these antibodies, levels of the HPDPI epitope were estimated in HNE-treated bovine serum albumin and in copper-oxidized LDL by an enzyme-linked immunosorbent assay. Our results are consistent with the premise that the fluorescent HPDPI cross-link is a key contributor to the fluorescence exhibited by atherosclerotic lesions.

In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer's disease: a central role for bound transition metals.

There is a great deal of evidence to support a pathogenic role of oxidative stress in Alzheimer's disease (AD), but the sources of reactive oxygen species have not been directly demonstrated. In this study, using a novel in situ detection system, we show that neurofibrillary tangles and senile plaques are major sites for catalytic redox reactivity. Pretreatment with deferoxamine or diethylenetriaminepentaacetic acid abolishes the ability of the lesions to catalyze the H2O2-dependent oxidation of 3,3'-diaminobenzidine (DAB), strongly suggesting the involvement of associated transition metal ions. Indeed, following chelated removal of metals, incubation with iron or copper salts reestablished lesion-dependent catalytic redox reactivity. Although DAB oxidation can also detect peroxidase activity, this was inactivated by H2O2 pretreatment before use of DAB, as shown by a specific peroxidase detection method. Model studies confirmed the ability of certain copper and iron coordination complexes to catalyze the H2O2-dependent oxidation of DAB. Also, the microtubule-associated protein tau, as an in vitro model for proteins relevant to AD pathology, was found capable of adventitious binding of copper and iron in a redox-competent manner. Our findings suggest that neurofibrillary tangles and senile plaques contain redox-active transition metals and may thereby exert prooxidant or possibly antioxidant activities, depending on the balance among cellular reductants and oxidants in the local microenvironment.