Protein oxidation in the brain in Alzheimer's disease

• Published in: Neuroscience Vol. 103; no. 2; pp. 373 - 383
• Main Authors: Aksenov, M.Y., Aksenova, M.V., Butterfield, D.A., Geddes, J.W., Markesbery, W.R.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.01.2001; Elsevier
• Subjects: 2D electrophoresis; 2D western blotting; Actins - analysis; Actins - metabolism; Aged; Aged, 80 and over; Alzheimer Disease - metabolism; Alzheimer's disease; Biological and medical sciences; Blotting, Western; Brain - metabolism; Cerebellum - chemistry; Cerebellum - metabolism; Creatine Kinase - analysis; Creatine Kinase - metabolism; Creatine Kinase, BB Form; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Electrophoresis, Gel, Two-Dimensional; Glial Fibrillary Acidic Protein - analysis; Glial Fibrillary Acidic Protein - metabolism; Hippocampus - chemistry; Hippocampus - metabolism; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Isoenzymes - analysis; Isoenzymes - metabolism; Medical sciences; Nerve Tissue Proteins - analysis; Nerve Tissue Proteins - metabolism; Neurology; Oxidation-Reduction; Parahippocampal Gyrus - chemistry; Parahippocampal Gyrus - metabolism; protein carbonyls; protein oxidation; tau Proteins - analysis; tau Proteins - metabolism; Tubulin - analysis; Tubulin - metabolism; GFAP, glial fibrillary acidic protein; HEPES, N-2-hydroxyethylpiperazine- N′-2-ethanesulphoric acid; HNE, 4-hydroxynonenal; TBS, Tris-buffered saline; CKBB, creatine kinase BB; protein carbonyls; HPG, hippocampus and parahippocampus gyrus; protein oxidation; ANOVA, analysis of variance; immunohistochemistry; Aβ, amyloid β-peptide; Alzheimer's disease; NFT, neurofibrillary tangles; BSA, bovine serum albumin; SDS–PAGE, sodium dodecylsulphate–polyacrylamide gel electrophoresis; SMT, superior and middle temporal gyri; NGS, normal goat serum; DNP, dinitrophenyl; DNPH, 2,4-dinitrophenylhydrazine; PMSF, phenylmethylsulfonyl fluoride; AD, Alzheimer's disease; AGE, advanced glycation endproducts; 2D western blotting; PHF, paired helical filaments; 2D electrophoresis; Human; Oxidative stress; Nervous system diseases; Alzheimer disease; Central nervous system disease; Central nervous system; Degenerative disease; Oxidation; Protein; Cerebral disorder; Brain (vertebrata)
• DOI: 10.1016/S0306-4522(00)00580-7
• ISSN: 0306-4522

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.