Dringen, Ralf
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Dringen, Ralf
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Dringen, Ralf
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Item-typ:Veröffentlichung, Uptake and Metabolism of Iron Oxide Nanoparticles in Cultured Brain Cells(2015-02-20); ; ; Iron oxide nanoparticles (IONPs) are used in various biomedical applications and are already applied in human therapy. Since IONPs can reach the brain, detailed knowledge on uptake and effects of IONPs on neural cells is required. In the present thesis, IONPs were synthesized and fluorescently labeled by attaching the green fluorescent dye BODIPY to the coating material dimercaptosuccinate. When 1.5% of the thiol groups of dimercaptosuccinate were functionalized, IONPs had identical physicochemical properties to the non-fluorescent version of the IONPs and were therefore considered as suitable fluorescent tool to study uptake and intracellular localization of dimercaptosuccinate-coated IONPs. Cellular uptake, localization and potential toxic effects of IONPs were investigated in cell culture models of the four major neural cell types (neurons, astrocytes, microglia, oligodendrocytes). In general, neural cell cultures efficiently accumulated IONPs which led to an increase of the specific cellular iron contents to maximal levels of up to 3000 nmol iron per mg protein. The uptake of IONPs in cultured brain cells strongly depended on experimental conditions such as time of incubation, IONP concentration, temperature and on the absence or presence of serum. In the presence of serum, the accumulation of IONPs was decreased by 80-90% in all cell types investigated compared to serum-free conditions. Dependent on the cell type investigated, IONP uptake in presence of serum was strongly lowered by known inhibitors of endocytotic processes suggesting involvement of clathrin-mediated endocytosis and/or macropinocytosis. In contrast, for IONP uptake in absence of serum the pathways involved remain to be elucidated. A direct comparison of cultured astrocytes, neurons and microglia revealed that microglia were most efficient in IONP accumulation but also highly vulnerable to IONP exposure. Microglial cell death was prevented by neutralizing lysosomes or by chelating iron ions, suggesting that toxicity is mediated by rapid transfer of IONPs to lysosomes and fast IONP degradation in the acidic environment which resulted in microglial death by iron-mediated oxidative stress. In contrast to microglia, primary astrocytes, neurons and oligodendroglial OLN-93 cells were not acutely damaged within hours upon IONP exposure. However, at least neurons which had accumulated substantial amounts of IONPs during a short time exposure suffered from delayed toxicity after removal of exogenous IONPs. The data presented in this thesis reveal that brain cells deal well with low amounts of IONPs. However, higher iron contents after IONP exposure cause acute or delayed toxicity in some neural cell types. Among the different cell types, especially microglia were vulnerable to IONPs. Hence, concerning biomedical application of IONPs to the brain one should consider protecting microglia from IONP-derived stress to reduce or prevent potential adverse effects to the brain.Dissertation459 194 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Formaldehyde Metabolism and Formaldehyde-induced Alterations in Glucose and Glutathione Metabolism of Cultured Brain Cells(2013-01-16); ; ; Formaldehyde is an environmental pollutant that is also generated in the body during normal metabolic processes. Interestingly, several pathological conditions are associated with an increase in formaldehyde-generating enzymes in the body. The level of formaldehyde in the brain is elevated with increasing age and in neurodegenerative conditions which may contribute to lowered cognitive functions. Although the neurotoxic potential of formaldehyde is well established, the molecular mechanisms involved remain, to a great extent, obscure. Also, the ability of the different types of brain cells to metabolize formaldehyde has not been reported so far. This thesis investigated the capacity of cultured brain cells to metabolize formaldehyde and studied the effects of a formaldehyde exposure on the glucose and the glutathione metabolism by using primary cultures of cerebellar granule neurons or astrocytes as well as the oligodendroglial cell-line OLN-93 as model systems. These cultured cells were remarkably resistant towards acute toxicity of formaldehyde and expressed the mRNAs for enzymes that are known to be involved in formaldehyde generation and disposal, suggesting that brain cells are able to metabolize this aldehyde. Furthermore, all three types of cultures cleared exogenously applied formaldehyde with almost identical rates, but differed in the extent of the formation of the formaldehyde oxidation product, formate. Since formate is a known inhibitor of the cytochrome c oxidase of the mitochondrial respiratory chain and since the metabolism of formaldehyde involves the important antioxidant glutathione, the effect of an exposure of cultured brain cells to formaldehyde on their glucose and glutathione metabolism was also investigated. Formaldehyde application accelerated the export of glycolysis-derived lactate and induced a rapid multidrug-resistance protein 1-mediated export of glutathione from cultured brain cells. These formaldehyde-induced alterations in metabolic pathways of brain cells may contribute to the known impairments in memory and learning that have been reported for neurodegenerative conditions and for formaldehyde-exposed animals.Dissertation273 168 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Metabolism of iron and iron oxide nanoparticles in glial cells(2011-08-16); ; ; Iron is an essential metal for mammalian cells catalyzing redox reactions in various metabolic pathways. However, iron can also induce cellular damage due to increased formation of reactive oxygen species (ROS). Among the different brain cell types, oligodendrocytes produce and maintain the myelin sheaths around neuronal axons whereas brain astrocytes participate in a variety of different brain functions such as synaptic signal transduction, regulation of metal homeostasis and detoxification of xenobiotics. In brain, these cells may encounter iron oxide nanoparticles (Fe-NP), since Fe-NP are extensively investigated for biomedical applications. This thesis investigated the metabolism of iron and Fe-NP in glial cells. The oligodendroglial OLN-93 cells express the mRNAs of the protein transferrin, transferrin-receptor and divalent metal transporter 1 for iron uptake as well as the iron storage protein ferritin. The proliferation of these cells depended on the availability of extracellular iron and can be inhibited by iron chelators. Furthermore, OLN-93 cells accumulated substantial amounts of iron from low molecular weight iron salts and Fe-NP. The cell viability was not compromised despite of high intracellular iron concentrations. Moreover, exposure to Fe-NP hardly affected the metabolism of OLN-93 cells. Intracellularly, iron was mobilized from Fe-NP by OLN-93 cells as demonstrated by the increase in proliferation following iron restriction, by the upregulation of ferritin and by the inhibition of Fe-NP-dependent ROS formation by a cell-membrane-permeable iron chelator. Also primary astrocytes took up Fe-NP as shown by increased cellular iron contents and electron microscopy. Both OLN-93 cells and astrocytes accumulated iron from Fe-NP in comparable amounts, showed similar time- and concentration-dependencies of iron accumulation and stored iron in ferritin. These observations suggest that the uptake and the cellular fate of Fe-NP are similar in OLN-93 cells and astrocytes.Dissertation296 149 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Eisenaufnahme in Astroglia-reichen Primärkulturen(2012-05-04); ; ; Fe is essential for the normal functioning of cells but can also generate toxic reactive oxygen species. The metabolism of Fe has to be tightly regulated. Astrocytes are considered to play an important role in Fe-homeostasis of the brain, yet the mechanisms involved in the uptake of Fe into astrocytes remain elusive. To investigate the uptake of Fe into astrocytes, I had applied FAC to rat astrocyte-rich primary cultures (APCs). The results of this thesis show that Fe-accumulationstudies in present of phosphat can lead to artefacts, because of the formation of Fe-precipitates. In serum and phosphat-free media APCs do accumulate Fe from FAC in a Tf-independent pathway, which follows a Michaelis-Menten-Kinetic that was found to be temperature-dependent and sensitive to the extracell. concentration of divalent cations. Further experimental results suggest that the here descript Fe-accumulation pathway is most likely independent form any known Fe-transporter reported in literature.Dissertation264 153 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Copper metabolism and copper-mediated alterations in the metabolism of cultured astrocytes(2012-06-22); ; ; Copper is an essential element that is required for a variety of important cellular functions. Since not only copper deficiency, but also excess of copper can seriously affect cellular functions, cellular copper metabolism is tightly regulated. Disturbances of copper homeostasis are the underlying defect of the inherited diseases Menkes and Wilson s disease and have also been linked to several neurodegenerative diseases including Alzheimer s disease and Parkinson s disease. Known astrocytes features strongly suggest a pivotal role of theses cells in the metal metabolism of the brain. Using astrocyte-rich primary cultures as model system, this thesis investigated the copper metabolism as well as copper-mediated alterations in the metabolism of astrocytes. Cultured astrocytes efficiently accumulated copper with saturable kinetics. The characteristics of the observed copper accumulation suggest that both copper transporter receptor 1 (Ctr1) and a Ctr1-independent mechanism are involved in astrocytic copper accumulation. Cultured astrocytes were also found to release copper in a time-, concentration- and temperature-dependent manner. Copper export from these cells most likely involves the copper-ATPase ATP7A. Thus, with being capable of both taking up and exporting copper, astrocytes possess the cellular machinery required to transport copper from the blood-brain barrier to the brain parenchyma. Cultured astrocytes were remarkable resistance against copper-induced toxicity. Nevertheless, prolonged copper treatment led to profound alterations in their metabolism. For example, copper accumulation by cultured astrocytes was accompanied by a stimulation of glycolytic flux, an increase in the cellular glutathione content and an acceleration of glutathione export. Such copper-mediated alterations in the metabolism of astrocytes may also occur in vivo, for example in copper overload conditions such in Wilson s disease and could either contribute to disease progression or serve as compensatory response to protect the brain against the toxic effects of an excess of copper.Dissertation408 320 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Effects of Antiretroviral Drugs on the Glutathione and Glucose Metabolism of Cultured Brain Cells(2014-06-19); ; ; The highly active antiretroviral therapy (HAART) has been successfully used for 30 years to treat the aquired immuno deficiency syndrome (AIDS) and the human immuno deficiency virus (HIV)-associated dementia. However, as minor neurocognitive deficits persist in treated HIV patients potential adverse consequences of antiretroviral drugs on brain cells are currently intensively discussed, but lack sufficient experimental proof. To address such questions, this thesis investigated the acute effects of various antiretroviral drugs from the classes of reverse transcriptase (RT) inhibitors and protease inhibitors on viability, glutathione (GSH) metabolism and glycolytic flux of brain cells, using primary cultures of astrocytes and neurons as model systems. A treatment with protease and RT inhibitors did not acutely damage cultured brain cells. However, the incubation of viable cultured astrocytes or neurons with protease inhibitors, but not with RT inhibitors, strongly stimulated cellular GSH release. The protease inhibitor-induced acceleration of GSH export was completely blocked by an inhibitor of the multidrug resistance protein 1 (Mrp1), suggesting that this exporter mediates the protease inhibitor-induced GSH depletion of brain cells. Protease inhibitors or RT inhibitors did not modulate the glycolytic flux of cultured astrocytes. In contrast, 8-hydroxy efavirenz (8-OH-efv), the primary metabolite of the frequently used RT inhibitor efavirenz, accelerated the glycolysis-derived lactate release from viable cultured astrocytes. However, in contrast to respiratory chain inhibitors, a direct inhibition of mitochondrial respiration by 8-OH-efv appears not to be the mechanism underlying the 8-OH-efv-mediated stimulation of glycolytic flux in astrocytes. As the lifelong treatment of HIV patients with antiretroviral drugs establishes a chronic exposure of brain cells to such compounds or their metabolites, alterations in basic metabolism of brain cells, such as those reported in this thesis, should be considered to contribute to the reported mild neurocognitive impairments of treated HIV patients.Dissertation256 155 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Accumulation of Iron Oxide and Silver Nanoparticles in Cultured Glial Cells(2013-04-05); ; ; Iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs) are frequently used in everyday products as well as for biomedical applications. As nanoparticles (NPs) are known to cross the intact or damaged blood brain barrier, brain cells have to deal with NPs and NP-derived metal ions. Astrocytes and microglia are the first brain cells that are discussed to encounter NPs which reach the brain, but at the start of this thesis only little was known about the effects of AgNPs or IONPs on those cell types. The consequences of an exposure of astrocytes to AgNPs were studied on astrocyte-rich primary cultures as model systems. These cells efficiently took up AgNPs by endocytotic mechanisms and were neither acutely impaired in their viability nor during prolonged presence of accumulated AgNPs. Neither silver nor AgNPs was exported from the cells but presence of AgNPs in the cells was accompanied by an upregulation of metallothioneins that may safely store AgNP-derived silver ions, thereby protecting astrocytes against the potential toxicity of silver ions. These results are in line with the view that in brain astrocytes efficiently accumulate potentially toxic metals and metal-containing NPs and thereby provide protection for other brain cells. Primary microglial cultures were established and characterized as cell culture model of microglial cells and used to study the effects of an exposure of microglia to IONPs. Fluorescently labelled IONPs were applied to visualize the uptake and intracellular localization of IONPs. These NPs were rapidly taken up by microglia into lysosomal compartments via endocytotic mechanisms. Viable microglia appeared not to suffer from oxidative stress as the cellular glutathione levels remained stable, however, in contrast to astrocytes that had been treated with comparable IONPs, microglia only tolerated moderate concentrations of accumulated IONPs for a few hours before their viability was impaired which may be a consequence of a liberation of iron ions from the accumulated IONPs. The data presented in this thesis support the described differences regarding toxicity and uptake of NPs in astrocytes and microglia. This allows the assumption that astrocytes, due to their high capacity to take up NPs without impairment in their viability, may provide protection for microglial cells which efficiently accumulate NPs but are already damaged by the accumulation of relatively low concentrations.Dissertation340 120 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Konsequenzen von oxidativem Stress auf oligodendrogliale Zellen(2011-07-13); ; ; Oligodendrocytes are the myelinating cells of the central nervous system. Due to their high metabolic activity and lipid content antioxidative defence mechanisms are essential, as oxidative stress is a hallmark of demyelinating diseases. The present work focussed on the investigation of protective mechanisms of oligodendroglial cells against oxidative stress and its impact on cellular metabolism using the oligodendroglial cell line OLN-93 and secondary oligodendrocytes. Peroxide detoxification capacities as well as cellular glutathione and iron contents of OLN-93 cells were determined and potential heat shock protein 27 (HSP27) mediated modulations of these were studied. The obtained data show that an over expression of HSP27 did not affect those components and might therefore not be associated with protection against oxidative stress in oligodendroglial cells. Fumaric acid esters are currently being investigated as therapeutic agents for multiple sclerosis in clinical studies. Fumaric acid dialkyl esters were shown to be non-toxic, but depleted the cellular glutathione content of both OLN-93 cells and secondary oligodendrocytes, probably by conjugate formation of glutathione with these substances or their metabolites. Cellular glutathione content was restored via new synthesis subsequent to treatment with fumaric acid dialkyl esters and was accompanied by induction of heme oxygenase 1. Studies on the long term effects of non-lethal peroxide treatment showed consequences in cellular glucose metabolism and resulted in a cell type specific increased lactate release by OLN-93 cells. As lactate is essential for neurons, an increased lactate release of oligodendroglial cells might hint at a protective mechanism for neurons under oxidative stress.Dissertation282 517