Michaela C. Hohnholt

Uptake of dimercaptosuccinate-coated magnetic iron oxide nanoparticles by cultured brain astrocytes

Magnetic iron oxide nanoparticles (Fe-NP) are currently considered for various diagnostic and therapeutic applications in the brain. However, little is known on the accumulation and biocompatibility of such particles in brain cells. We have synthesized and characterized dimercaptosuccinic acid (DMSA) coated Fe-NP and have investigated their uptake by cultured brain astrocytes. DMSA-coated Fe-NP that were dispersed in physiological medium had an average hydrodynamic diameter of about 60 nm. Incubation of cultured astrocytes with these Fe-NP caused a time- and concentration-dependent accumulation of cellular iron, but did not lead within 6 h to any cell toxicity. After 4 h of incubation with 100-4000 µM iron supplied as Fe-NP, the cellular iron content reached levels between 200 and 2000 nmol mg⁻¹ protein. The cellular iron content after exposure of astrocytes to Fe-NP at 4 °C was drastically lowered compared to cells that had been incubated at 37 °C. Electron microscopy revealed the presence of Fe-NP-containing vesicles in cells that were incubated with Fe-NP at 37 °C, but not in cells exposed to the nanoparticles at 4 °C. These data demonstrate that cultured astrocytes efficiently take up DMSA-coated Fe-NP in a process that appears to be saturable and strongly depends on the incubation temperature.

Glutathione-Dependent Detoxification Processes in Astrocytes

Astrocytes have a pivotal role in brain as partners of neurons in homeostatic and metabolic processes. Astrocytes also protect other types of brain cells against the toxicity of reactive oxygen species and are considered as first line of defence against the toxic potential of xenobiotics. A key component in many of the astrocytic detoxification processes is the tripeptide glutathione (GSH) which serves as electron donor in the GSH peroxidase-catalyzed reduction of peroxides. In addition, GSH is substrate in the detoxification of xenobiotics and endogenous compounds by GSH-S-transferases which generate GSH conjugates that are efficiently exported from the cells by multidrug resistance proteins. Moreover, GSH reacts with the reactive endogenous carbonyls methylglyoxal and formaldehyde to intermediates which are substrates of detoxifying enzymes. In this article we will review the current knowledge on the GSH metabolism of astrocytes with a special emphasis on GSH-dependent detoxification processes.

Treatment with iron oxide nanoparticles induces ferritin synthesis but not oxidative stress in oligodendroglial cells.

Magnetic iron oxide nanoparticles (IONPs) have been used for a variety of neurobiological applications, although little is yet known as to the fate of such particles in brain cells. To address these questions, we have exposed oligodendroglial OLN-93 cells to dimercaptosuccinate-coated IONPs. Treatment of the cells strongly increased the specific cellular iron content proportional to the IONP concentrations applied (0-1000 μM total iron as IONPs) up to 300-fold, but did not cause any acute cytotoxicity or induce oxidative stress. To investigate the potential of OLN-93 cells to liberate iron from the accumulated IONPs, we have studied the upregulation of the iron storage protein ferritin and the cell proliferation as cellular processes that depend on the availability of low-molecular-weight iron. The presence of IONPs caused a concentration-dependent increase in the amount of cellular ferritin and partially bypassed the inhibition of cell proliferation by the iron chelator deferoxamine. These data demonstrate that viable OLN-93 cells efficiently take up IONPs and suggest that these cells are able to use iron liberated from accumulated IONPs for their metabolism.

Ferritin up-regulation and transient ROS production in cultured brain astrocytes after loading with iron oxide nanoparticles

To investigate the cellular consequences of a prolonged cellular presence of large amounts of iron oxide nanoparticles (IONPs) as well as the fate of such particles in brain cells, cultured primary astrocytes were loaded for 4h with dimercaptosuccinate-coated IONPs. Subsequently, the IONP-treated cells were incubated for up to 7 days in IONP-free medium and the cell viability, metabolic parameters and iron metabolism of the cells were investigated. Despite an up to 100-fold elevated specific cellular iron content, IONP-loaded cells remained viable throughout the 7 day main incubation and did not show any substantial alteration in glucose and glutathione metabolism. During the incubation, the high cellular iron content of IONP-loaded astrocytes remained almost constant. Electron microscopy revealed that after 7 days of incubation most of the cellular iron was still present in IONP-filled vesicles. However, the transient appearance of reactive oxygen species (ROS) as well as a strong increase in cellular levels of the iron storage protein ferritin suggest that at least some low-molecular-weight iron was liberated from the accumulated IONPs. These results demonstrate that even the prolonged presence of large amounts of accumulated IONPs does not harm astrocytes and that these cells store IONP-derived iron in ferritin.

Handling of Iron Oxide and Silver Nanoparticles by Astrocytes

Metal-containing nanoparticles (NPs) are currently used for various biomedical applications. Since such NPs are able to enter the brain, the cells of this organ have to deal with NPs and with NP-derived metal ions. In brain, astrocytes are considered to play a key function in regulating metal homeostasis and in protecting other brain cells against metal toxicity. Thus, among the different types of brain cells, especially astrocytes are of interest regarding the uptake and the handling of metal-containing NPs. This article summarizes the current knowledge on the consequences of an exposure of astrocytes to NPs. Special focus will be given to magnetic iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs), since the biocompatibility of these NPs has been studied for astrocytes in detail. Cultured astrocytes efficiently accumulate IONPs and AgNPs in a time-, concentration- and temperature-dependent manner by endocytotic processes. Astrocytes are neither acutely damaged by the exposure to high concentrations of NPs nor by the prolonged intracellular presence of large amounts of accumulated NPs. Although metal ions are liberated from accumulated NPs, NP-derived iron and silver ions are not exported from astrocytes but are rather stored in proteins such as ferritin and metallothioneins which are synthesized in NP-treated astrocytes. The efficient accumulation of large amounts of metal-containing NPs and the upregulation of proteins that safely store NP-derived metal ions suggest that astrocytes protect the brain against the potential toxicity of metal-containing NPs.

Uptake and metabolism of iron and iron oxide nanoparticles in brain astrocytes

Astrocytes are considered key regulators of the iron metabolism of the brain. These cells are able to rapidly accumulate iron ions and various iron-containing compounds, store iron efficiently in ferritin and also export iron. The present short review summarizes our current knowledge of the molecular mechanisms involved in the handling of iron by astrocytes. Cultured astrocytes efficiently take up iron as ferrous or ferric iron ions or as haem by specific iron transport proteins in their cell membrane. In addition, astrocytes accumulate large amounts of iron oxide nanoparticles by endocytotic mechanisms. Despite the rapid accumulation of high amounts of iron from various iron-containing sources, the viability of astrocytes is hardly affected. A rather slow liberation of iron from accumulated haem or iron oxide nanoparticles as well as the strong up-regulation of the synthesis of the iron storage protein ferritin are likely to contribute to the high resistance of astrocytes to iron toxicity. The efficient uptake of extracellular iron by cultured astrocytes as well as their strong up-regulation of ferritin after iron exposure also suggests that brain astrocytes deal well with an excess of iron and protect the brain against iron-mediated toxicity.

Formaldehyde metabolism and formaldehyde‐induced stimulation of lactate production and glutathione export in cultured neurons

Formaldehyde is endogenously produced in the human body and brain levels of this compound are elevated in neurodegenerative conditions. Although the toxic potential of an excess of formaldehyde has been studied, little is known on the molecular mechanisms underlying its neurotoxicity as well as on the ability of neurons to metabolize formaldehyde. To address these topics, we have used cerebellar granule neuron cultures as model system. These cultures express mRNAs of various enzymes that are involved in formaldehyde metabolism and were remarkably resistant toward acute formaldehyde toxicity. Cerebellar granule neurons metabolized formaldehyde with a rate of around 200 nmol/(h × mg) which was accompanied by significant increases in the cellular and extracellular concentrations of formate. In addition, formaldehyde application significantly increased glucose consumption, almost doubled the rate of lactate release from viable neurons and strongly accelerated the export of the antioxidant glutathione. The latter process was completely prevented by inhibition of the known glutathione exporter multidrug resistance protein 1. These data indicate that cerebellar granule neurons are capable of metabolizing formaldehyde and that the neuronal glycolysis and glutathione export are severely affected by the presence of formaldehyde.

Multiassay analysis of the toxic potential of hydrogen peroxide on cultured neurons

To clarify discrepancies in the literature on the adverse effects of hydrogen peroxide on neurons, this study investigated the application of this peroxide to cultured cerebellar granule neurons with six assays frequently used to test for viability. Cultured neurons efficiently cleared exogenous H2O2. Although viability was not affected by exposure to 10 µM hydrogen peroxide, an exposure to the peroxide in higher concentrations rapidly lowered, within 15 min, the cellular 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltertrazolium bromide (MTT) reduction capacity to 53% ± 1% (100 µM) and 31% ± 1% (1,000 µM) and the 3‐amino‐7‐dimethylamino‐2‐methyl‐phenazine hydrochloride (neutral red; NR) uptake to 84% ± 6% (100 µM) and 33% ± 1% (1,000 µM) of control cells. The release of glycolytically generated lactate was stopped within 30 min in neurons treated with 1,000 µM peroxide. In contrast, even hours after peroxide application, the cell morphology, the number of propidium iodide‐positive cells, and the extracellular activity of the cytosolic enzyme lactate dehydrogenase (LDH) were not significantly altered. The rapid loss in MTT reduction and NR uptake after exposure of neurons to H2O2 for 5 or 15 min correlated well with a strongly compromised MTT reduction and a very high extracellular LDH activity observed after further incubation in peroxide‐free medium for a total incubation period of 24 hr. These data demonstrate that cultured neurons do not recover from damage that is inflicted by a short exposure to H2O2 and that the rapid losses in the capacities of neurons for MTT reduction and NR uptake are good predictors of delayed cell damage.

Short time exposure to hydrogen peroxide induces sustained glutathione export from cultured neurons.

Hydrogen peroxide is a normal by-product of cellular metabolism that in higher concentrations can cause oxidative stress. Cultured cerebellar granule neurons efficiently disposed of micromolar concentrations of hydrogen peroxide with half-times in the minute range in a process that predominately involved catalase. Application of up to 100 µM hydrogen peroxide did not affect the cell viability for up to 4h, but caused a time- and concentration-dependent increase in the extracellular glutathione (GSH) content that was accompanied by a matching decrease in the cellular GSH content. Hydrogen peroxide at 100 µM stimulated maximally the GSH export from viable neurons, but did not affect GSH export from cultured astrocytes. The peroxide-induced extracellular GSH accumulation from neurons was lowered by 70% in the presence of MK571, an inhibitor of multidrug resistance protein (Mrp) 1. The extracellular GSH content determined after 4h of incubation was already significantly increased after a 5-min exposure of neurons to hydrogen peroxide and became maximal after 15 min of peroxide application. These data demonstrate that just a short exposure of viable cerebellar granule neurons to micromolar concentrations of hydrogen peroxide stimulates a prolonged Mrp1-mediated export of cellular GSH. This process may compromise the antioxidative potential of neurons and increase their sensitivity toward drugs and toxins.

Glutamate dehydrogenase is essential to sustain neuronal oxidative energy metabolism during stimulation

The enzyme glutamate dehydrogenase (GDH; Glud1) catalyzes the (reversible) oxidative deamination of glutamate to α-ketoglutarate accompanied by a reduction of NAD+ to NADH. GDH connects amino acid, carbohydrate, neurotransmitter and oxidative energy metabolism. Glutamine is a neurotransmitter precursor used by neurons to sustain the pool of glutamate, but glutamine is also vividly oxidized for support of energy metabolism. This study investigates the role of GDH in neuronal metabolism by employing the Cns-Glud1−/− mouse, lacking GDH in the brain (GDH KO) and metabolic mapping using 13C-labelled glutamine and glucose. We observed a severely reduced oxidative glutamine metabolism during glucose deprivation in synaptosomes and cultured neurons not expressing GDH. In contrast, in the presence of glucose, glutamine metabolism was not affected by the lack of GDH expression. Respiration fuelled by glutamate was significantly lower in brain mitochondria from GDH KO mice and synaptosomes were not able to increase their respiration upon an elevated energy demand. The role of GDH for metabolism of glutamine and the respiratory capacity underscore the importance of GDH for neurons particularly during an elevated energy demand, and it may reflect the large allosteric activation of GDH by ADP.