Subcellular localization of a glutathione-dependent dehydroascorbate reductase within specific rat brain regions

Recently, we described the occurrence of a dehydroascorbate reductase withi n the rat CNS. This enzyme regenerates ascorbate after it is; oxidized duri ng normal aerobic metabolism. In this work. we describe the neuronal compar tmentalization of the enzyme, using transmission electron microscopy of tho se brain areas in which the enzyme was most densely present when observed u nder light microscopy. In parallel biochemical studies. we performed immuno blotting and measured the enzyme activity of the cytoplasm and different nu clear fractions. Given the abundance of ascorbate in the caudate-putamen, w e focused mostly on the occurrence of dehydroascorbate reductase at the str iatal subcellular level, We also studied cerebellar Purkinje cells, hippoca mpal CA3 pyramidal cells and giant neurons in the magnocellular parr of the red nucleus. In addition to neurons. immunolabeling was found in striatal endothelial cells, in the basal membrane of blood vessels and in perivascul ar astrocytes. In neuronal cytosol, the enzyme was observed in a peri nucle ar position and on the nuclear membrane. In addition, in both the striatum and the cerebellum we found the enzyme within myelin sheets. Dehydroascorba te reductase was also present in the nucleus of neurons, as further indicat ed by measuring enzyme activity and by immunoblotting selected nuclear frac tions. Immunocytochemical labeling confirmed that the protein was present i n isolated pure nuclear fractions. Given the great amount of free radicals which are constantly generated in t he CNS, the discovery of a new enzyme with antioxidant properties which tra nslocates into neuronal nuclei appears to be a potential starting point to develop alternative strategies in neuroprotection. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.