Ralf Dringen

Metabolism and functions of glutathione in brain

The tripeptide glutathione is the thiol compound present in the highest concentration in cells of all organs. Glutathione has many physiological functions including its involvement in the defense against reactive oxygen species. The cells of the human brain consume about 20% of the oxygen utilized by the body but constitute only 2% of the body weight. Consequently, reactive oxygen species which are continuously generated during oxidative metabolism will be generated in high rates within the brain. Therefore, the detoxification of reactive oxygen species is an essential task within the brain and the involvement of the antioxidant glutathione in such processes is very important. The main focus of this review article will be recent results on glutathione metabolism of different brain cell types in culture. The glutathione content of brain cells depends strongly on the availability of precursors for glutathione. Different types of brain cells prefer different extracellular glutathione precursors. Glutathione is involved in the disposal of peroxides by brain cells and in the protection against reactive oxygen species. In coculture astroglial cells protect other neural cell types against the toxicity of various compounds. One mechanism for this interaction is the supply by astroglial cells of glutathione precursors to neighboring cells. Recent results confirm the prominent role of astrocytes in glutathione metabolism and the defense against reactive oxygen species in brain. These results also suggest an involvement of a compromised astroglial glutathione system in the oxidative stress reported for neurological disorders.

Synthesis of the Antioxidant Glutathione in Neurons: Supply by Astrocytes of CysGly as Precursor for Neuronal Glutathione

Deficiency of the antioxidant glutathione in brain appears to be connected with several diseases characterized by neuronal loss. To study neuronal glutathione metabolism and metabolic interactions between neurons and astrocytes in this respect, neuron-rich primary cultures and transient cocultures of neurons and astroglial cells were used. Coincubation of neurons with astroglial cells resulted within 24 hr of incubation in a neuronal glutathione content twice that of neurons incubated in the absence of astroglial cells. In cultured neurons, the availability of cysteine limited the cellular level of glutathione. During a 4 hr incubation in a minimal medium lacking all amino acids except cysteine, the amount of neuronal glutathione was doubled. Besides cysteine, also the dipeptides CysGly and γGluCys were able to serve as glutathione precursors and caused a concentration-dependent increase in glutathione content. Concentrations giving half-maximal effects were 5, 5, and 200 μm for cysteine, CysGly, and γGluCys, respectively. In the transient cocultures, the astroglia-mediated increase in neuronal glutathione was suppressed by acivicin, an inhibitor of the astroglial ectoenzyme γ-glutamyl transpeptidase, which generates CysGly from glutathione. These data suggest the following metabolic interaction in glutathione metabolism of brain cells: the ectoenzyme γ-glutamyl transpeptidase uses as substrate the glutathione released by astrocytes to generate the dipeptide CysGly that is subsequently used by neurons as precursor for glutathione synthesis.

Glutathione pathways in the brain.

Abstract The antioxidant glutathione (GSH) is essential for the cellular detoxification of reactive oxygen species in brain cells. A compromised GSH system in the brain has been connected with the oxidative stress occuring in neurological diseases. Recent data demonstrate that besides intracellular functions GSH has also important extracellular functions in brain. In this respect astrocytes appear to play a key role in the GSH metabolism of the brain, since astroglial GSH export is essential for providing GSH precursors to neurons. Of the different brain cell types studied in vitro only astrocytes release substantial amounts of GSH. In addition, during oxidative stress astrocytes efficiently export glutathione disulfide (GSSG). The multidrug resistance protein 1 participates in both the export of GSH and GSSG from astrocytes. This review focuses on recent results on the export of GSH and GSSG from brain cells as well as on the functions of extracellular GSH in the brain. In addition, implications of disturbed GSH pathways in brain for neurodegenerative diseases will be discussed.

Glycogen in astrocytes : possible function as lactate supply for neighboring cells

In order to contribute to the elucidation of the function of astrocyte glycogen in brain, studies on the fate of the glucosyl residues of glycogen were carried out on astroglia-rich primary cultures derived from the brains of newborn rats. On glucose deprivation astroglial cells rapidly deplete their glycogen. In contrast to the situation with hepatocytes, only lactate, but not glucose, is detectable in the medium surrounding the astroglial cells. Besides glucose, astroglial cultures can also use mannose as a substrate for the synthesis of glycogen and the generation of lactate. Although mannose-fed astroglial cells contain glucose-6-phosphate, they do not release a measurable amount of glucose into the culture medium. Instead of glucose the astroglial cells release high amounts of lactate into the culture medium. Gluconolactone or 2-deoxyglucose which prevent glycogen breakdown in astroglial cells after glucose deprivation, allow to discriminate between lactate generated from glycogen and lactate from other sources. The amount of lactate found in the medium in the absence of gluconolactone (or 2-deoxyglucose) exceeds the amount found in the presence of either compound by the lactate equivalents calculated to be contained in the cellular glycogen. In conclusion, glycogen in astrocytes can be considered as a store for lactate rather than for glucose.

Astrocytes: Glutamate producers for neurons

In order for the brain to use the common amino acid glutamate as a neurotransmitter, it has been necessary to introduce a series of innovations that greatly restrict the availability of glutamate, so that it cannot degrade the signal‐to‐noise ratio of glutamatergic neurons. The most far‐reaching innovations have been: i) to exclude the brain from access to glutamate in the systemic circulation by the blood‐brain barrier, thereby making the brain autonomous in the production and disposal of glutamate; ii) to surround glutamatergic synapses with glial cells and endow these cells with much more powerful glutamate uptake carriers than the neurons themselves, so that most released transmitter glutamate is rapidly inactivated by uptake in glial cells; iii) to restrict to glial cells a key enzyme (glutamine synthetase) that is involved in the return of accumulated glutamate to neurons by amidation to glutamine, which has no transmitter activity, and can be safely released to the extracellular space, returned to neurons and deaminated to glutamate; iv) to restrict to glial cells two key enzymes (pyruvate carboxylase and cytosolic malic enzyme) that are involved in, respectively, de novo synthesis (from glucose) of the carbon skeleton of glutamate, and the return of the carbon skeleton of excess glutamate to the metabolic pathway for glucose oxidation. As a consequence of these innovations, neurons constantly require new carbon skeletons from glial to sustain their TCA cycle. When these supplies are withdrawn, neurons are unable to generate amino acid transmitters and their rate of oxidative metabolism is impaired. Given the commensalism that exists between neurons and glia, it may be fruitful to view brain function not just as a series of interactions between neurons, but also as a series of interactions between neurons and their collaborating glial cells. J. Neurosci. Res. 57:417–428, 1999.

Peroxide detoxification by brain cells

Peroxides are generated continuously in cells that consume oxygen. Among the different peroxides, hydrogen peroxide is the molecule that is formed in highest quantities. In addition, organic hydroperoxides are synthesized as products of cellular metabolism. Generation and disposal of peroxides is a very important process in the human brain, because cells of this organ consume 20% of the oxygen used by the body. To prevent cellular accumulation of peroxides and damage generated by peroxide‐derived radicals, brain cells contain efficient antioxidative defense mechanisms that dispose of peroxides and protect against oxidative damage. Cultured brain cells have been used frequently to investigate peroxide metabolism of neural cells. Efficient disposal of exogenous hydrogen peroxide was found for cultured astrocytes, oligodendrocytes, microglial cells, and neurons. Comparison of specific peroxide clearance rates revealed that cultured oligodendrocytes dispose of the peroxide quicker than the other neural cell cultures. Both catalase and the glutathione system contribute to the clearance of hydrogen peroxide by brain cells. For efficient glutathione‐dependent reduction of peroxides, neural cells contain glutathione in high concentration and have substantial activity of glutathione peroxidase, glutathione reductase, and enzymes that supply the NADPH required for the glutathione reductase reaction. This article gives an overview on the mechanisms involved in peroxide detoxification in brain cells and on the capacity of the different types of neural cells to dispose of peroxides.

Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells

Fumarates suppress Th1 responses by blocking IL-12 and IL-23 production by dendritic cells via distinct pathways.

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.

Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay

Peroxides are often applied to cultured brain cells to investigate functions of these cells under oxidative stress. However, little is known about the ability of brain cells to detoxify peroxides. In order to investigate peroxide clearance of adherent cultured cells, the peroxide assay originally described for the determination of hydrogen peroxide production during experimental protein glycation by Jiang et al. [Z.-Y. Jiang, A.C.S. Woollard, S.P Wolff, Hydrogen peroxide production during experimental protein glycation, FEBS Lett. , 268 (1990) 69-71.] was adapted to microtiter plates. Besides hydrogen peroxide, with this assay organic hydroperoxides such as tertiary butylhydroperoxide (tBHP), and cumene hydroperoxide (CHP) can also be quantified. Up to an amount of 2.5 nmol of each peroxide per well of a plate the absorption measured was proportional to the concentration of the peroxide. Using the assay described the ability of astroglia-rich primary cultures to detoxify peroxides was monitored by measuring the peroxide content in 10 microliter samples collected at several time points from the peroxide-containing incubation buffer of one dish. If peroxides were applied at a concentration of 100 muM, hydrogen peroxide, tBHP, and CHP disappeared from the incubation buffer in reactions following first order kinetics with apparent half-times of 3.1 min, 2.9 min, and 4.2 min, respectively. In the absence of cells H2O2 and CHP were stable in the incubation buffer for at least 30 min, whereas tBHP decayed slowly in a spontaneous reaction. In conclusion, the method presented allows the determination of the rapid detoxification of various peroxides by cultured cells.

Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells

The ability of astroglial cells to detoxify exogenously applied hydrogen peroxide (H2O2) was tested using astroglia-rich primary cultures derived from the brains of newborn rats. Incubation of astroglial cells with 100 microM H2O2 in the absence of glucose led to a 66% oxidation of the cellular glutathione within 30 s. Under these conditions, the cells were unable to re-establish the original high ratio of GSH/GSSG within 30 min of incubation. In contrast, if glucose was present the amount of GSSG produced on incubation with H2O2 was smaller (45% of total glutathione after 30 s) and the original ratio of GSH/GSSG was almost completely re-established within 10 min. If 100 microM H2O2 was applied, H2O2 disappeared from the incubation buffer with an apparent half-life of approximately 4 min. After 15 min of incubation, no H2O2 was detectable any more. The apparent half-life of H2O2 in the incubation buffer increased slightly but significantly with increasing concentration of H2O2 or when the cells were starved of glucose. A small reduction in the capacity of the cells to detoxify H2O2 was also observed after depletion of the glutathione content to 14% of control level by a 24 h pre-incubation of the cells in culture medium containing buthionine sulfoximine, an inhibitor of glutathione synthesis. Incubation of astroglial cells with mercaptosuccinate or 3-aminotriazole, inhibitors of glutathione peroxidase and catalase, respectively, only marginally reduced the rate of disappearance of H2O2 from the incubation buffer. In contrast, the rate of H2O2 clearance was strongly reduced in the presence of both inhibitors. These results demonstrate that glutathione peroxidase and catalase are involved in the detoxification of H2O2 by astroglial cells and that both enzymes are able to substitute for each other in the detoxification of H2O2.