Protein oxidation in the brain in Alzheimer's disease

doi:10.1016/S0306-4522(00)00580-7

Neuroscience

Volume 103, Issue 2, 14 March 2001, Pages 373-383

https://doi.org/10.1016/S0306-4522(00)00580-7 Get rights and content

Abstract

In this study we used immunohistochemistry and two-dimensional fingerprinting of oxidatively modified proteins (two-dimensional Oxyblot) together to investigate protein carbonyl formation in the Alzheimer's disease brain. Increased protein oxidation was detected in sections from the hippocampus and parahippocampal gyrus, superior and middle temporal gyri of six Alzheimer's disease and six age-matched control human subjects, but not in the cerebellum. In two brain regions severely affected by Alzheimer's disease pathology, prominent protein carbonyl immunoreactivity was localized in the cytoplasm of neurons without visual pathomorphological changes and degenerating neurons, suggesting that intracellular proteins might be significantly affected by oxidative modifications. Following two-dimensional electrophoresis the positions of some individual proteins were identified using specific antibodies, and immunoblot analysis for protein carbonyls was performed. These studies demonstrated the presence of protein carbonyl immunoreactivity in β-tubulin, β-actin and creatine kinase BB in Alzheimer's disease and control brain extracts. Protein carbonyls were undetectable in spots matching glial fibrillary acidic protein and tau isoforms. Specific protein carbonyl levels in β-actin and creatine kinase BB were significantly higher in Alzheimer's disease than in control brain extract. β-Tubulin did not demonstrate a significant increase in specific protein carbonyl content in Alzheimer's disease brains.

We suggest that oxidative stress-induced injury may involve the selective modification of different intracellular proteins, including key enzymes and structural proteins, which precedes and may lead to the neurofibrillary degeneration of neurons in the Alzheimer's disease brain.

Section snippets

Tissue samples

For protein carbonyl immunohistochemical analysis, specimens of the hippocampus and parahippocampal gyrus (HPG), superior and middle temporal gyri (SMT), and cerebellum from six AD patients (mean age 80.8±5.0 years, mean post mortem interval 3.0±0.3 h) and six age-matched control (mean age 78.8±4.8 years, mean post mortem interval 2.8±0.2 h) subjects were fixed in Methacarn (methanol:chloroform:acetic acid, 60:30:10) at 4°C for 24 h. Tissue was dehydrated through ascending ethanol solutions and

Results

Anti-DNP immunostaining of the AD brain sections showed increased protein carbonyl immunoreactivity in the HPG and SMT compared with the cerebellum. Severely affected brain regions in AD exhibited increased staining intensity compared with controls (Fig. 1).

Intense anti-DNP immunoreactivity localized in neuronal cell bodies was consistently found in HPG and SMT sections from AD patients. In control sections from the same brain regions, neurons with excessively oxidized proteins in the cytoplasm

Discussion

This study used immunohistochemical and 2D immunoblotting analyses together for the first time to study protein carbonyl formation in the brain of AD patients. We observed that in the severely affected regions of the AD brain, increased protein carbonyl immunoreactivity occurred in the cell bodies of neurons without visual pathomorphological changes and neurons with neurofibrillary abnormalities. Our results of the immunohistochemical analysis of protein carbonyls in brain regions severely

Conclusions

Immunohistochemical detection of oxidative damage markers demonstrates their preferential localization in neurons versus glia39., 43. in AD. In this study, we did not identify glial-specific GFAP as carbonyl-containing protein in AD or control brain extracts, which is consistent with the suggestion that glia are less sensitive to oxidative stress than neurons. Nevertheless, our results cannot imply that glial cells in the AD brain completely escape protein oxidation. Individual brain proteins,

Acknowledgements

This work was supported by NIH grants 5P01 AG05119, 5P50 AG05144, and grants from the Abercrombie Foundation and the Kleberg Foundation. The authors thank Dianna Tudor and Ela Patel for technical assistance, Jane Meara and Paula Thomason for assistance in manuscript preparation, and Cecil Runyons for subject demographic data. The authors also thank Elan Pharmaceutical for the gift of antibodies.

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Protein oxidation in the brain in Alzheimer's disease

Abstract

Section snippets

Tissue samples

Results

Discussion

Conclusions

Acknowledgements

Oxidation of cytosolic proteins and expression of creatine kinase BB in frontal lobe in different neurodegenerative disorders

Dement. Geriatr. Cogn. Disord.

Aging in a dish: age-dependent changes of neuronal survival, protein oxidation, and creatine kinase BB expression in long-term hippocampal cell culture

J. Neurosci. Res.

Glutamine synthetase-induced enhancement of β-amyloid peptide Aβ(1-40) accompanied by abrogation of fibril formation and Aβ fragmentation

J. Neurochem.

The expression of creatine kinase isoenzymes in neocortex of patients with neurodegenerative disorders: Alzheimer's and Pick's disease

Expl Neurol.

Oxidative modification of creatine kinase BB in Alzheimer's disease brain

J. Neurochem.

Histochemical detection of 4-hydroxynonenal protein in Alzheimer amyloid

J. neurol. Sci.

Alzheimer's disease and oxidative stress: implications for novel therapeutic approaches

Prog. Neurobiol.

Regions with abundant neurofibrillary pathology in human brain exhibit a selective reduction in levels of binding-competent tau and accumulation of abnormal tau-isoforms (A68 proteins)

Lab. Invest.

Protein oxidation processes in aging brain

Adv. Cell Aging Gerontol.

Biochemistry and pathology of radical-mediated protein oxidation

Biochem. J.

Age-related losses of cognitive function and motor skills in mice associated with oxidative protein damage in the brain

Proc. natn. Acad. Sci. USA

Immunocytochemical studies of actin localization in the central nervous system

J. Neurosci.

Carbonylated proteins in aging and exercise: immunoblot approaches

Mech. Agng Devl

Brain regional correspondence between Alzheimer's disease histopathology and biomarkers of protein oxidation

J. Neurochem.

Reactive oxygen species as causal agents in the neurotoxicity of the Alzheimer's disease-associated amyloid beta peptide

Ann. N.Y. Acad. Sci.

Immunohistochemical localization of advanced glycation end products, pentosidine, and carboxymethyllysine in lipofuscin pigments of Alzheimer's disease and aged neurons

Biochem. biophys. Res. Commun.

Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models

Proc. natn. Acad. Sci. USA

Redox regulation of cellular signalling

Cell. Signal.

An immunochemical study on tau glycation in paired helical filaments

Brain Res.

Epitopes that span the tau molecule are shared with paired helical filaments

Neuron

Cleavage of structural proteins during the assembly of the head of bacteriophage T4

Nature

3-Nitropropionic acid induced in vivo protein oxidation in striatal and cortical synaptosomes: insights into Huntington's disease

Brain Res.

Diffusible, nonfibrillar ligands derived from Aβ 1-42 are potent central nervous system neurotoxins

Proc. natn. Acad. Sci. USA

Analysis of microtubule-associated protein tau glycation in paired helical filaments

J. biol. Chem.

Carbonyl assays for determination of oxidatively modified proteins

Meth. Enzym.

Amyloid beta-peptide and oxidative injury in Alzheimer's disease

Molec. Neurobiol.