Jan Mirko Gutterer

The Glutathione System of Peroxide Detoxification Is Less Efficient in Neurons than in Astroglial Cells

Abstract: The ability of neurons to detoxify exogenously applied peroxides was analyzed using neuron‐rich primary cultures derived from embryonic rat brain. Incubation of neurons with H2O2 at an initial concentration of 100 μM (300 nmol/3 ml) led to a decrease in the concentration of the peroxide, which depended strongly on the seeding density of the neurons. When 3 × 106 viable cells were seeded per dish, the half‐time for the clearance by neurons of H2O2 from the incubation buffer was 15.1 min. Immediately after application of 100 μM H2O2 to neurons, glutathione was quickly oxidized. After incubation for 2.5 min, GSSG accounted for 48% of the total glutathione. Subsequent removal of H2O2 caused an almost complete regeneration of the original ratio of GSH to GSSG within 2.5 min. Compared with confluent astroglial cultures, neuron‐rich cultures cleared H2O2 more slowly from the incubation buffer. However, if the differences in protein content were taken into consideration, the ability of the cells to dispose of H2O2 was identical in the two culture types. The clearance rate by neurons for H2O2 was strongly reduced in the presence of the catalase inhibitor 3‐aminotriazol, a situation contrasting with that in astroglial cultures. This indicates that for the rapid clearance of H2O2 by neurons, both glutathione peroxidase and catalase are essential and that the glutathione system cannot functionally compensate for the loss of the catalase reaction. In addition, the protein‐normalized ability of neuronal cultures to detoxify exogenous cumene hydroperoxide, an alkyl hydroperoxide that is reduced exclusively via the glutathione system, was lower than that of astroglial cells by a factor of 3. These results demonstrate that the glutathione system of peroxide detoxification in neurons is less efficient than that of astroglial cells.

Aminopeptidase N mediates the utilization of the GSH precursor CysGly by cultured neurons.

Neurons in culture rely on the supply of exogenous cysteine for their glutathione synthesis. After application of cysteine to neuron‐rich primary cultures, the glutathione content was doubled after a 4‐hr incubation. The dipeptide cysteinylglycine (CysGly) was able to substitute for cysteine as exogenous glutathione precursor. In kidneys, the ectopeptidase aminopeptidase N (ApN) has been reported to hydrolyze CysGly. Expression of mRNA of ApN in rat brain and cultured rat neurons was demonstrated by reverse transcriptase polymerase chain reaction and sequencing of the cDNA fragment obtained. In addition, the presence of ApN protein in cultured neurons was demonstrated by its immunocytochemical localization. In the presence of an activity‐inhibiting antiserum against ApN the utilization of CysGly as neuronal glutathione precursor was completely prevented, whereas that of cysteine plus glycine was not affected. The data presented demonstrates that cultured rat neurons express ApN and that this ectopeptidase participates in the utilization of CysGly as precursor for neuronal glutathione.

Microglial cells in culture express a prominent glutathione system for the defense against reactive oxygen species.

To obtain information on the glutathione metabolism of microglial cells, the content of glutathione and activities of enzymes involved in the defense against peroxides were determined for microglia-rich cultures from rat brain. These cultures contain approximately 90% microglia cells as determined by immunocytochemical staining for glial markers, by the phagocytosis activity of the cells and by the production of superoxide after stimulation of the cells with phorbolester. For these cultures, a glutathione content of 41.2 ± 11.2 nmol/mg protein and a specific activity of glutathione reductase of 15.2 ± 3.2 nmol/(min × mg protein) were determined. These values are significantly higher than those found for astroglial or neuronal cultures. In addition, with 68.7 ± 23.5 nmol/(min × mg protein), the specific activity of glutathione peroxidase in microglial cultures was 78% higher than in cultured neurons. The specific catalase activity of microglial cultures was less than 40% that of astroglial or neuronal cultures. Microglial cultures contain only marginal amounts of oxidized glutathione. However, on application of oxidative stress by incubation of microglial cultures with hydrogen peroxide or with the superoxide-producing hypoxanthine/xanthine oxidase system, cellular glutathione was rapidly oxidized. These results demonstrate that microglial cells have a prominent glutathione system, which is likely to reflect the necessity for self-protection against reactive oxygen species when produced by these or surrounding brain cells.

Oligodendroglial cells in culture effectively dispose of exogenous hydrogen peroxide: comparison with cultured neurones, astroglial and microglial cells

To investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100u2003µm), the peroxide disappeared from the incubation medium following first order kinetics with a half‐time of approximately 18u2003min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5u2003±u200330.4 nmolu2003×u2003min−1u2003×u2003mg protein−1 and 27.5u2003±u20035.4u2003nmolu2003×u2003min−1u2003×u2003 mg protein−1 of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61u2003±u20030.54u2003min−1u2003×u2003mg protein−1. Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells.

Purification of Glutathione reductase from bovine brain, generation of an antiserum, and immunocytochemical localization of the enzyme in neural cells

Abstract : Glutathione reductase (GR) is an essential enzyme for the glutathione‐mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000‐fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate‐polycrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double‐labeling immunocytochemical staining in astroglia‐rich and neuron‐rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astrogliarich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron‐rich primary cultures, GAP43‐positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.

Glutathione reductase from bovine brain.

Publisher Summary Glutathione reductase (GR, NADPH:oxidized-glutathione oxidoreductase, EC1.6.4.2) catalyzes the reduction of glutathione disulfide (GSSG) during the glutathione redox cycling. GSSG is generated by the nonenzymatic reaction of radicals with reduced glutathione (GSH) and is product of the reactions catalyzed by glutathione peroxidases, which use GSH as an electron donor for the reduction of peroxides. Glutathione-dependent disposal of peroxides and radicals appears to be especially important for the brain, as this organ has an intense oxidative metabolism but is disadvantaged regarding the detoxification of reactive oxygen species. Enzymes such as glutathione peroxidases, superoxide dismutases, and catalase are present in brain in lower activities than in other tissues. This is also the case for GR.

Catalase in astroglia-rich primary cultures from rat brain: immunocytochemical localization and inactivation during the disposal of hydrogen peroxide

The expression of catalase in cells of astroglia-rich primary cultures derived from the brains of newborn rats was investigated by double-labelling immunocytochemical staining. Strong catalase immunoreactivity was found in cells positive for glial fibrillary acidic protein and galactocerebroside, cellular markers for astroglial and oligodendroglial cells, respectively. The cells of these cultures dispose of exogenously applied hydrogen peroxide (initial concentration 200 microM) quickly with first order kinetics. In contrast, after inhibition of glutathione peroxidases by mercaptosuccinate the rate of the catalase-dependent disposal of H(2)O(2) declined with time and after about 10 min the extracellular concentration of H(2)O(2) remained almost constant at a concentration of about 100 microM. Catalase activity after 10 min of incubation under these conditions was no longer detectable. In contrast, in the absence of mercaptosuccinate catalase activity was maintained during H(2)O(2) disposal. These results demonstrate that in astroglia-rich cultures catalase is strongly expressed in the predominant astroglial cells and in the minor population of oligodendroglial cells and that the enzyme is rapidly inactivated during the disposal of H(2)O(2), if the glutathione system of the cells is compromised.