Quantitative alpha-ketoglutarate dehydrogenase activity staining in brain sections and in cultured cells.
The activity of a key mitochondrial enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in the brains of patients with neurodegenerative diseases such as Alzheimer's disease, as well as in thiamine-deficient (TD) animals. The decreased activity often occurs without a reduction in enzyme protein, which negates the use of immunocytochemistry to study cellular or regional changes in enzyme activity within the brain. To overcome this limitation, an activity staining method using nitroblue tetrazolium was developed. The histochemical activity staining was standardized in cultured cells. The assay was linear with time and was highly specific for KGDHC. The dark-blue reaction product (formazan) formed a pattern that was consistent with mitochondrial localization. Treatment of the cultured cells with both reversible and irreversible inhibitors decreased formazan production, whereas conventional enzyme assays on cell lysates only revealed loss of KGDHC activity with irreversible inhibitors. The activity staining was also linear with time and highly specific for KGDHC activity in mouse brain sections. Staining occurred throughout the brain, and discrete neuronal populations exhibited particularly intense staining. The pattern of staining differed markedly from the distribution of KGDHC protein by immunocytochemistry. Generalized decreases in the intensity of activity staining that occurred in the TD brains compared to controls were comparable with the loss of KGDHC activity by conventional enzyme assay. Thus, the present study introduces a new histochemical method to measure KGDHC activity at the cellular and regional level, which will be useful to determine changes of in situ enzyme activity.
Park
L C
LC
Department of Neurology, Weill Medical College of Cornell University at Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, New York, 10605, USA.
Calingasan
N Y
NY
Sheu
K F
KF
Gibson
G E
GE
D000818:Animals; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D004396:Coloring Agents; D006651:Histocytochemistry; D006801:Humans; D007150:Immunohistochemistry; D007655:Ketoglutarate Dehydrogenase Complex / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D009928:Organ Specificity; D014407:Tumor Cells, Cultured
D004396:Coloring Agents; D007655:Ketoglutarate Dehydrogenase Complex