Quantitative alpha-ketoglutarate dehydrogenase activity staining in brain sections and in cultured cells

The activity of a key mitochondrial enzyme, the alpha-ketoglutarate dehydro genase complex (KGDHC), declines in the brains of patients with neurodegene rative diseases such as Alzheimer's disease, as well as in thiamine-deficie nt (TD) animals. The decreased activity often occurs without a reduction in enzyme protein, which negates the use of immunocytochemistry to study cell ular or regional changes in enzyme activity within the brain, To overcome t his limitation, an activity staining method using nitroblue tetrazolium was developed. The histochemical activity staining was standardized in culture d cells. The assay was linear with time and was highly specific for KGDHC, The dark-blue reaction product (formazan) formed a pattern that was consist ent with mitochondrial localization. Treatment of the cultured cells with b oth reversible and irreversible inhibitors decreased formazan production, w hereas conventional enzyme assays on cell lysates only revealed loss of KGD HC activity with irreversible inhibitors. The activity staining was also li near with time and highly specific for KGDHC activity in mouse brain sectio ns. Staining occurred throughout the brain, and discrete neuronal populatio ns exhibited particularly intense staining, The pattern of staining differe d markedly from the distribution of KGDHC protein by immunocytochemistry. G eneralized 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 reg ional level, which will be useful to determine changes of in situ enzyme ac tivity. (C) 2000 Academic Press.