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Detection and Localization of Markers of Oxidative Stress by In Situ Methods: Application in the Study of Alzheimer Disease

  • Paula I. Moreira
  • Lawrence M. Sayre
  • Xiongwei Zhu
  • Akihiko Nunomura
  • Mark A. Smith
  • George Perry
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 610)

Abstract

Oxidative stress is a key factor involved in the development and progression of Alzheimer disease (AD), and it is well documented that free radical oxidative damage, particularly of neuronal lipids, proteins, nucleic acids, and sugars, is extensive in brains of AD patients. The complex chemistry of peroxynitrite has been the subject of intense study and is now evident that there are two principal pathways for protein modification: the first one involves homolytic hydroxyl radical-like chemistry that results in protein-based carbonyls and the second involves electrophilic nitration of vulnerable side chains, in particular the electron-rich aromatic rings of Tyr and Trp. In the presence of buffering bicarbonate, peroxynitrite forms a CO2 adduct, which augments its reactivity. Formation of 3-nitrotyrosine by this route has become the classical protein marker specifically for the presence of peroxynitrite. Protein-based carbonyls can be detected by two methods: (i) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and detection of the protein-bound hydrazones using an enzyme-linked anti-2,4-dinitrophenyl antibody and (ii) derivatization with biotin-hydrazide and detection of the protein-bound acyl hydrazone with enzyme-linked avidin or streptavidin. Glycation of proteins by reducing sugars (Maillard reaction) results in a profile of time-dependent adduct evolution rendering susceptibility to oxidative elaboration. In addition, oxidative stress can result in oxidized sugar derivatives which can subsequently modify protein through a process known as glycoxidation. Of more general importance, oxidative stress results in lipid peroxidation and the production of a range of electrophilic and mostly bifunctional aldehydes that modify numerous proteins. The more important protein modifications are referred to as advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). Protein modification can result in both non-cross-link and cross-link AGEs and ALEs, the latter arising from the potential bifunctional reactivity, such as that of the lipid-derived modifiers 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA). Oxidative damage to nucleic acids results in base modification, substitutions, and deletions. Among the most common modifications, 8-hydroxyguanosine (8OHG) is considered a signature of oxidative damage to nucleic acid.

Cells are not passive to increased oxygen radical production but rather upregulate protective responses. In neurodegenerative diseases, heme oxygenase-1 (HO-1) induction is coincident with the formation of neurofibrillary tangles. This enzyme that converts heme, a prooxidant, to biliverdin/bilirubin (antioxidants) and free iron has been considered an antioxidant enzyme. But seen in the context of arresting apoptosis, HO-1 and tau may play a role in maintaining the neurons free from the apoptotic signal (cytochrome c), since tau has strong iron-binding sites. Given the importance of iron as a catalyst for the generation of reactive oxygen species, changes in proteins associated with iron homeostasis can be used as an index of cellular responses. One such class of proteins is the iron regulatory proteins (IRPs) that respond to cellular iron concentrations by regulating the translation of proteins involved in iron uptake, storage, and utilization. Therefore, IRPs are considered to be the central control components of cellular iron concentration.

Key words

Advanced glycation end products advanced lipoxidation end products glycation glycoxidation heme oxygenase-1 8-hydroxyguanosine 4-hydroxy-2-nonenal iron regulatory proteins malondialdehyde 3-nitrotyrosine protein carbonyls 

Notes

Acknowledgments

Work in the authors’ laboratories is supported by the National Institutes of Health, the Alzheimer’s Association, and Philip Morris USA and Philip Morris International.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Paula I. Moreira
    • 1
  • Lawrence M. Sayre
    • 2
  • Xiongwei Zhu
    • 3
  • Akihiko Nunomura
    • 4
  • Mark A. Smith
    • 3
  • George Perry
    • 3
    • 5
  1. 1.Center for Neuroscience and Cell Biology of Coimbra, University of CoimbraCoimbraPortugal
  2. 2.Department of ChemistryCase Western Reserve UniversityClevelandUSA
  3. 3.Department of PathologyCase Western Reserve UniversityClevelandUSA
  4. 4.Department of Psychiatry and NeurologyAsahikawa Medical CollegeAsahikawaJapan
  5. 5.College of SciencesUniversity of Texas at San AntonioSan AntonioUSA

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