Lawrence M. Sayre

4‐Hydroxynonenal‐Derived Advanced Lipid Peroxidation End Products Are Increased in Alzheimer's Disease

Abstract: Recent studies have demonstrated oxidative damage is one of the salient features of Alzheimers disease (AD). In these studies, glycoxidation adduction to and direct oxidation of amino acid side chains have been demonstrated in the lesions and neurons of AD. To address whether lipid damage may also play an important pathogenic role, we raised rabbit antisera specific for the lysine‐derived pyrrole adducts formed by lipid peroxidation‐derived 4‐hydroxynonenal (HNE). These antibodies were used in immunocytochemical evaluation of brain tissue from AD and age‐matched control patients. HNE‐pyrrole immunoreactivity not only was identified in about half of all neurofibrillary tangles, but was also evident in neurons lacking neurofibrillary tangles in the AD cases. In contrast, few senile plaques were labeled, and then only the dystrophic neurites were weakly stained, whereas the amyloid‐β deposits were unlabeled. Age‐matched controls showed only background HNE‐pyrrole immunoreactivity in hippocampal or cortical neurons. In addition to providing further evidence that oxidative stress‐related protein modification is a pervasive factor in AD, the known neurotoxicity of HNE suggests that lipid peroxidation may also play a role in the neuronal death in AD that underlies cognitive deficits.

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 Parkinsons 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.

Radical AGEing in Alzheimer's disease

The pathological presentation of Alzheimers disease, the leading cause of senile dementia, involves regionalized neuronal death and an accumulation of intracellular and extracellular filamentous protein aggregates that form lesions termed neurofibrillary tangles and senile plaques, respectively. Several independent parameters have been suggested as the primary factor that is responsible for this pathogenesis, including apolipoprotein epsilon genotype, hyperphosphorylation of cytoskeletal proteins, or metabolism of amyloid beta. However, at present, no one theory explains adequately the host of complex biochemical and pathological facets of the disease. Recent findings suggest that age-related increases in oxidative stress and protein glycation either individually, or more probably in a synergistic manner, could, exclusive of the other theories or in concert with them, account for all aspects of Alzheimers disease.

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

Abstract: 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 τ, 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.

Protein Adducts Generated from Products of Lipid Oxidation: Focus on HNE and ONE

Modification of proteins in conditions of oxidative stress can contribute to protein dysfunction or tissue damage and disease progression. Bifunctional, most often α,β-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and acrolein, generated from oxidation of polyunsaturated fatty acids (PUFAs), readily bind to protein nucleophiles. Modification by bifunctional aldehydes can also lead to intramolecular or intermolecular protein crosslinking. Model studies are revealing the structure of adducts that can then be more readily identified in mass spectrometric studies on proteins exposed to the various pure aldehydes or to peroxidized PUFAs. Although simple Michael and Schiff base adducts are often formed initially, only some of these adducts, such as the HNE- and ONE-derived Michael adducts on Cys and His residues, are found to survive the conditions of proteolysis and HPLC-MS analysis. Reversibly formed adducts, such as the HNE-Lys Michael adduct, can be found on proteolytic peptides only if a NaBH4-reduction step is used prior to proteolysis. Initial adducts can evolve by tautomerization, oxidation, cyclization, dehydration, and sometimes condensation with a second aldehyde molecule (the same or different), to give stable advanced lipoxidation end products (ALEs) that can be found by mass spectrometry. These include the HNE-Lys-derived 2-pentylpyrrole, the ONE-Lys-derived 4-ketoamide, the ONE-derived His-Lys pyrrole crosslink, and a Lys-derived 3-formyl-4-pentylpyrrole that results from combined action of ONE and acrolein. Michael adducts of α,β-unsaturated aldehydes such as HNE and ONE can be derivatized by 2,4-dinitrophenylhydrazine (DNPH) and can thus constitute significant DNPH-detectable protein-bound carbonyl activity that serves as a key indicator of oxidative stress in tissues. It appears that lipid oxidation is a more important contributor to such activity than metal-catalyzed oxidation of protein side-chains.

Is oxidative damage the fundamental pathogenic mechanism of Alzheimer's and other neurodegenerative diseases?

In less than a decade, beginning with the demonstration by Floyd, Stadtman, Markesbery et al. of increased reactive carbonyls in the brains of patients with Alzheimers disease (AD), oxidative damage has been established as a feature of the disease. Here, we review the types of oxidative damage seen in AD, sites involved, possible origin, relationship to lesions, and compensatory changes, and we also consider other neurodegenerative diseases where oxidative stress has been implicated. Although much data remain to be collected, the broad spectrum of changes found in AD are only seen, albeit to a lesser extent, in normal aging with other neurodegenerative diseases showing distinct spectrums of change.

Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer's disease

Abstract: We present the first evidence for carbonyl‐related posttranslational modifications of neurofilaments in the neurofibrillary pathology of Alzheimers disease (AD). Two distinct monoclonal antibodies that consistently labeled neurofibrillary tangles (NFTs), neuropil threads, and granulovacuolar degeneration in sections of AD tissue also labeled the neurofilaments within axons of the white matter following modification by reducing sugars, glutaraldehyde, formaldehyde, or malondialdehyde. The epitope recognized by these two antibodies shows a strict dependency for carbonyl modification of the neurofilament heavy subunit. The in vivo occurrence of this neurofilament modification in the neurofibrillary pathology of AD suggests that carbonyl modification is associated with a generalized cytoskeletal abnormality that may be critical in the pathogenesis of neurofibrillary pathology. Furthermore, the data presented here support the idea that extensive posttranslational modifications, including oxidative stress‐type mechanisms, through the formation of cross‐links, might account for the biochemical properties of NFTs and their resistance to degradation in vivo.

Cytochemical demonstration of oxidative damage in Alzheimer disease by immunochemical enhancement of the carbonyl reaction with 2,4- dinitrophenylhydrazine

Formation of carbonyls derived from lipids, proteins, carbohydrates, and nucleic acids is common during oxidative stress. For example, metal-catalyzed, “site-specific” oxidation of several amino acid side-chains produces aldehydes or ketones, and peroxidation of lipids generates reactive aldehydes such as malondialdehyde and hydroxynonenal. Here, using in situ 2,4-dinitrophenylhydrazine labeling linked to an antibody system, we describe a highly sensitive and specific cytochemical technique to specifically localize biomacromolecule-bound carbonyl reactivity. When this technique was applied to tissues from cases of Alzheimer disease, in which oxidative events including lipoperoxidative, glycoxidative, and other oxidative protein modifications have been reported, we detected free carbonyls not only in the disease-related intraneuronal lesions but also in other neurons. In marked contrast, free carbonyls were not found in neurons or glia in age-matched control cases. Importantly, this assay was highly specific for detecting disease-related oxidative damage because the site of oxidative damage can be assessed in the midst of concurrent age-related increases in free carbonyls in vascular basement membrane that would contaminate biochemical samples subjected to bulk analysis. These findings demonstrate that oxidative imbalance and stress are key elements in the pathogenesis of Alzheimer disease.

Redox metals and neu rodegenerative disease

Multiple lines of evidence implicate redox-active transition metals as mediators of oxidative stress in neurodegenerative diseases. Among the recent research discoveries is the finding that transition metals bind to proteins associated with neurodegeneration, including the prion protein. Whereas binding in the latter case may serve an antioxidant function, adventitious binding of metals to other proteins appears to preserve their catalytic redox activity in a manner that disturbs free radical homeostasis. Alterations in the levels of copper- and iron-containing metalloenzymes, involved in processing partially reduced oxygen species, are also likely to contribute to altered redox balance in neurodegenerative diseases. Nonetheless, even in familial forms of amyotrophic lateral sclerosis linked to mutations in superoxide dismutase, it is unclear whether an altered enzyme activity or, indirectly, a disturbance in transition-metal homeostasis is involved in the disease pathogenesis.

In Alzheimer's Disease, Heme Oxygenase Is Coincident with Alz50, an Epitope of τ Induced by 4-Hydroxy-2-Nonenal Modification

Abstract: In this study, we compared the neuronal induction of the antioxidant heme oxygenase‐1 (HO‐1) in Alzheimers disease with abnormalities in τ 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 τ 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 τ and induces the Alz50 epitope in τ. It is important that the ability of HNE to create the Alz50 epitope not only is dependent on lysine residues of τ but also requires τ phosphorylation because neither methylated, recombinant, nor dephosphorylated τ reacts with HNE to create the Alz50 epitope. Supporting the in vivo relevance of this observation, endogenous paired helical filament‐τ isolated from subjects with Alzheimers disease was immunoreactive with an antibody to a stable HNE‐lysine adduct, as were all vulnerable neurons in subjects with Alzheimers disease but not in control individuals. Together, these findings support the involvement of oxidative damage early in neurofibrillary tangle formation in Alzheimers disease and also suggest that HNE modification contributes to the generation of the τ conformation defining the Alz50 epitope. These findings provide evidence that an interplay between phosphorylation of τ and neuronal oxidative stress‐induced pathology is important in the formation of neurofibrillary tangles.