Hyperammonemia causes protein oxidation and enhanced proteasomal activity in response to mitochondria-mediated oxidative stress in rat primary astrocytes.

Hyperammonemia, as a consequence of severe liver failure, is strongly associated with the neurological syndrome hepatic encephalopathy (HE) whereby excessive ammonia is metabolized by astrocytes, followed by cell and brain swelling in vivo. In the present study we were able to show that ammonia treatment of primary astrocytes in vitro is followed by cell swelling and a loss of cell viability at higher ammonia concentrations. Lower ammonia concentrations are accompanied by mitochondria-derived oxidative stress, as demonstrated by using inhibitors of mitochondrial glutaminase I, 143B-rho (0) cells and isolated mitochondria. The oxidative stress generated by mitochondria is accompanied by protein oxidation. In further studies we could show, that an activation of the proteasomal system takes place during ammonia exposure and protects cells. The proteasome acitvation can be blocked by antioxidants or by inhibitors of enzymes of glutamine metabolism. We conclude that oxidative stress-mediated proteasomal activation is important for survival of astroglial cells.

Postanoxic damage of microglial cells is mediated by xanthine oxidase and cyclooxygenase.

Brain ischemia and the following reperfusion are important causes for brain damage and leading causes of brain morbidity and human mortality. Numerous observations exist describing the neuronal damage during ischemia/reperfusion, but the outcome of such conditions towards glial cells still remains to be elucidated. Microglia are resident macrophages in the brain. In this study, we investigated the anoxia/reoxygenation caused damage to a microglial cell line via determination of energy metabolism, free radical production by dichlorofluorescein fluorescence and nitric oxide production by Griess reagent. Consequences of oxidant production were determined by measurements of protein oxidation and lipid peroxidation, as well. By using site-specific antioxidants and inhibitors of various oxidant-producing pathways, we identified major sources of free radical production in the postanoxic microglial cells. The protective influences of these compounds were tested by measurements of cell viability and apoptosis. Although, numerous free radical generating systems may contribute to the postanoxic microglial cell damage, the xanthine oxidase- and the cyclooxygenase-mediated oxidant production seems to be of major importance.

Protein oxidation and degradation during aging: role in skin aging and neurodegeneration.

During aging, the products of oxidative processes accumulate and might disturb cellular metabolism. Among them are oxidized proteins and protein aggregates. On the other hand, in a functioning metabolic system oxidized proteins are degraded, mainly by the proteasome. During aging, however, proteasome activity declines. Therefore, the ability to degrade oxidized proteins is attenuated. The following review summarises the accumulation of oxidized proteins and the decline of the proteasomal system during skin and brain aging including some age-related neurodegenerative processes. The role of protein aggregates will be discussed as a potential reason for the accelerated dysfunction of tissue during aging.

Ferritin levels in microglia depend upon activation: modulation by reactive oxygen species.

Iron is one of the trace elements playing a key role in the normal cellular metabolism. Since an excess of free iron is catalyzing the Fenton reaction, most of the intracellular iron is sequestered in the iron storage protein ferritin. The binding of iron into ferritin is well described for physiological conditions, however, under certain pathophysiological situations, the efficiency of this process is unknown. In the brain, microglial cells are among others the cell population most importantly responsible for the maintenance of the extracellular environment. These cells might undergo activation, and little is known about the expression of ferritin during activation of microglial cells. Therefore, we tested the microglial model cell line RAW264.7 for the expression of ferritin after LPS activation. A significant decrease in the levels of the ferritin H-chain during activation and a significant increase in the early recovery phase were found. We were able to demonstrate that reactive oxygen species are responsible for a suppression of the H-chain of ferritin, whereas iNOS expression and NO synthesis are counteracting the reactive oxygen species effect. The balance of reactive oxygen species and NO production are, therefore, determining expression levels of the ferritin H-chain during activation of microglial cells.

Tocopherol-mediated modulation of age-related changes in microglial cells: turnover of extracellular oxidized protein material.

Proteins accumulate during aging and form insoluble protein aggregates. Microglia are responsible for their removal from the brain. During aging, changes within the microglia might play a crucial role in the malfunctioning of these cells. Therefore, we isolated primary microglial cells from adult rats and compared their activation status and their ability to degrade proteins to that of microglial cells isolated from newborn animals. The ability of adult microglial cells to degrade proteins is substantially decreased. However, the preincubation of microglial cells with vitamin E improves significantly the degradation of such modified proteins. The degradation of proteins from apoptotic vesicles is decreased in microglia isolated from adult rats. This might be the result of a suppression of the CD36 receptor due to vitamin E treatment. We concluded that microglial cells isolated from adult organisms have different metabolic properties and seem to be a more valuable model to study age-related diseases.

Degradation of glycated bovine serum albumin in microglial cells.

Glycated protein products are formed upon binding of sugars to lysine and arginine residues and have been shown to accumulate during aging and in pathologies such as Alzheimer disease and diabetes. Often these glycated proteins are transformed into advanced glycation end products (AGEs) by a series of intramolecular rearrangements. In the study presented here we tested the ability of microglial cells to degrade BSA-AGE formed by glycation reactions of bovine serum albumin (BSA) with glucose and fructose. Microglial cells are able to degrade BSA-AGEs to a certain degree by proteasomal and lysosomal pathways. However, the proteasome and lysosomal proteases are severely inhibited by cross-linked BSA-AGEs. BSA-AGEs are furthermore able to activate microglial cells. This activation is accompanied by an enhanced degradation of BSA-AGE. Therefore, we conclude that microglial cells are able to degrade glycated proteins, although cross-linked protein-AGEs have an inhibitory effect on proteolytic systems in microglial cells.

Iron uptake of the normoxic, anoxic and postanoxic microglial cell line RAW 264.7.

Iron is one of the trace elements playing a key role in the normal brain metabolism. An excess of free iron on the other hand is catalyzing the iron-mediated oxygen radical production. Such a condition might be a harmful event leading perhaps to serious tissue damage and degeneration. Therefore, during evolution a complex iron sequestering apparatus developed, minimizing the amount of redox-reactive free iron. However, this system might be severely disturbed under pathophysiological conditions including hypoxia or anoxia. Since little is known about the non-transferrin-mediated iron metabolism of the brain during anoxia/reoxygenation, we tested the ability of the microglial cell line RAW 264.7 to take up iron independently of transferrin under various oxygen concentrations. Microglial cells are thought to be the major player in the maintenance of the extracellular homeostasis in the brain. Therefore, we investigated the iron metabolism of microglial cells employing radiolabeled ferric chloride. We tested the uptake of iron under normoxic, anoxic and postanoxic conditions. Furthermore, the amount of ferritin was measured by immunoblotting. We were able to show that iron enters the microglial cell line in the absence of extracellular transferrin under normoxic, anoxic and postanoxic conditions. Interestingly, the amount of ferritin is decreasing in the early reoxygenation phase. Therefore, we concluded that microglia is able to contribute to the brain iron homeostasis under anoxic and postanoxic conditions.