Immunocytochemical localization of seleno-glutathione peroxidase in the adult mouse brain.

Cytoplasmic seleno-glutathione peroxidase, by reducing hydrogen peroxide and fatty acid hydroperoxides, may be a major protective enzyme against oxidative damage in the brain. Oxidative damage is strongly suspected to contribute to normal aging and neurodegenerative process of Alzheimer's and Parkinson's diseases. We report here an immunocytochemical analysis of the localization of glutathione peroxidase in the adult mouse brain, carried out with an affinity-purified polyclonal antibody. Most of the brain areas analysed showed weak to strong glutathione peroxidase immunoreactivity, expressed in both neurons and glial cells. The strongest immunoreactivity was found in the reticular thalamic and red nuclei. Highly immunoreactive neurons were observed in the cerebral cortex (layer II), the CA1, dentate gyrus and pontine nucleus. Other regions, such as the caudate-putamen, septum nuclei, diagonal band of Broca, hippocampus, thalamus and hypothalamus, showed moderate staining. This study provides original information about the wide distribution of glutathione peroxidase in the mouse brain. Double-staining experiments indicated that specific subsets of cholinergic neurons in septal and diagonal band nuclei were negative for this antigen. Similarly, many dopaminergic neurons of the substantia nigra pars compacta expressed low levels of glutathione peroxidase antigen, in contrast to the ventral tegmental area, wherein most catecholaminergic cells were strongly positive. A lack of glutathione peroxidase in subsets of dopaminergic or cholinergic neurons may thus confer a relative sensitivity of these cells to oxidative injury of various origins, including catecholamine oxidation, neurotoxins and excitotoxicity.

Transgenic glutathione peroxidase mouse models for neuroprotection studies.

Seleno-glutathione peroxidase (GSHPx) is considered to be the major enzymatic activity in charge of removing excess cytosolic and mitochondrial H2O2 in most tissues including brain. Intracellular GSHPx activity is therefore hypothesized to be one important factor that contributes to minimize hydroxyl radical formation via Fenton-type reactions. An animal model was developed to challenge this hypothesis in vivo and evaluate the role of GSHPx in hydroperoxide metabolism and oxidative stress homeostasis. Three lines of transgenic mice, homozygous for the integration of 1 to 3 GSHPx transgene copies, have been generated. The transgene was placed under transcriptional control of a metallothionein promoter (hMT-IIA). This promoter was chosen because metallothionein expression, normally low in most tissues, can be induced by several inflammatory cytokines, protein kinase C activators, and stress agents including heavy metals. The data reported here provide information on the constitutive expression of GSHPx mRNA and enzyme in various brain regions of healthy untreated adult tg-MT-GPx mice. Northern and/or Western analysis indicated that transgenic GSHPx was expressed constitutively in all brain regions investigated in tg-MT-GPx-6 mice, including the cerebral cortex, brainstem, hippothalamus, cerebellum, substantia nigra, and striatum. Similar results were obtained with the two other transgenic lines, tg-MT-GPx-11 and -13. Depending on the brain region, the GSHPx immunoreactivity detected in tissue extracts with an immunoaffinity-purified polyclonal antibody was about 2- to 5-fold stronger in transgenic extracts than in their non-tg counterparts (western blots). In contrast, the corresponding increase in GSHPx activity measured in these extracts was smaller, for example, about 1.5-fold in transgenic mesencephalon. Immunocytochemical data indicated that GSHPx-like staining was distinctly more intense in transgenic midbrain brain sections than in corresponding non-tg sections. Interestingly, only a subset of the cells displayed higher density staining that most likely reflects increased amounts of GSHPx protein. This observation suggests that the stained cells, not yet identified, may have larger GSHPx activity increments than the cell-average increments measured in tissue extracts. Current work is in progress to determine whether transgenic GSHPx expression may be induced by inflammatory processes or perturbations of heavy metal metabolism.