Elise Rundén-Pran

Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.

BACKGROUND High extracellular glutamate concentrations have been identified as a likely trigger of epileptic seizures in mesial temporal lobe epilepsy (MTLE), but the underlying mechanism remains unclear. We investigated whether a deficiency in glutamine synthetase, a key enzyme in catabolism of extracellular glutamate in the brain, could explain the perturbed glutamate homoeostasis in MTLE. METHODS The anteromedial temporal lobe is the focus of the seizures in MTLE, and surgical resection of this structure, including the hippocampus, leads to resolution of seizures in many cases. By means of immunohistochemistry, western blotting, and functional enzyme assays, we assessed the distribution, quantity, and activity of glutamine synthetase in the MTLE hippocampus. FINDINGS In western blots, the expression of glutamine synthetase in the hippocampus was 40% lower in MTLE than in non-MTLE samples (median 44 [IQR 30-58] vs 69 [56-87]% of maximum concentration in standard curve; p=0.043; n=8 and n=6, respectively). The enzyme activity was lower by 38% in MTLE vs non-MTLE (mean 0.0060 [SD 0.0031] vs 0.0097 [0.0042] U/mg protein; p=0.045; n=6 and n=9, respectively). Loss of glutamine synthetase was particularly pronounced in areas of the MTLE hippocampus with astroglial proliferation, even though astrocytes normally have high content of the enzyme. Quantitative immunoblotting showed no significant change in the amount of EAAT2, the predominant glial glutamate transporter in the hippocampus. INTERPRETATION A deficiency in glutamine synthetase in astrocytes is a possible molecular basis for extracellular glutamate accumulation and seizure generation in MTLE. Further studies are needed to define the cause, but the loss of glutamine synthetase may provide a new focus for therapeutic interventions in MTLE.

Widespread distribution of DNA glycosylases removing oxidative DNA lesions in human and rodent brains

High metabolic activity and low levels of antioxidant enzymes make neurons particularly prone to damage by reactive oxygen species. Thus, repair of oxidative DNA damage is essential for normal brain function. Base excision repair is the major pathway for repair of oxidative DNA damage, and is initiated by DNA glycosylases recognizing and removing the damaged base. In mammalian cells at least five different DNA glycosylases with overlapping substrate specificity, NEIL1, NEIL2, NEIL3, OGG1 and NTH1, remove oxidative DNA base lesions. Here we report mRNA expression and distribution of these five DNA glycosylases in human and rodent brains using in situ hybridization and Northern blotting supported by glycosylase activity assays. NEIL1, NEIL2, OGG1 and NTH1 showed widespread expression at all ages. In situ hybridization studies in mouse brain showed that expression of mNeil1 increased with age. In newborn mouse brain, mNeil3 revealed a discrete expression pattern in brain regions known to harbour stem cell populations, i.e., the subventricular zone, the rostral migratory stream, and the hilar region of the hippocampal formation. Expression of mNeil3 decreased with age, and in old mice brains could be detected only in layer V of neocortex. MNth1 was constitutively expressed during lifespan. In Northern blots, mOgg1 expression showed a transient decrease followed by an increase after 8 weeks of age. Assays for faPy DNA glycosylase activity revealed increased activity level with age in all brain regions analyzed. The widespread but differential expression of the DNA glycosylases recognizing oxidative base lesions suggests distinct and age dependent roles of these enzymes in genome maintenance in brain. The distribution of mNeil3 is particularly intriguing and points to a specific role of this enzyme in stem cell differentiation.

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles

Abstract Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, 3H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.

Neuronal Ca2+-activated K+ channels limit brain infarction and promote survival.

Neuronal calcium-activated potassium channels of the BK type are activated by membrane depolarization and intracellular Ca2+ ions. It has been suggested that these channels may play a key neuroprotective role during and after brain ischemia, but this hypothesis has so far not been tested by selective BK-channel manipulations in vivo. To elucidate the in vivo contribution of neuronal BK channels in acute focal cerebral ischemia, we performed middle cerebral artery occlusion (MCAO) in mice lacking BK channels (homozygous mice lacking the BK channel alpha subunit, BK−/−). MCAO was performed in BK−/− and WT mice for 90 minutes followed by a 7-hour-reperfusion period. Coronal 1 mm thick sections were stained with 2,3,5-triphenyltetrazolium chloride to reveal the infarction area. We found that transient focal cerebral ischemia by MCAO produced larger infarct volume, more severe neurological deficits, and higher post-ischemic mortality in BK−/− mice compared to WT littermates. However, the regional cerebral blood flow was not significantly different between genotypes as measured by Laser Doppler (LD) flowmetry pre-ischemically, intra-ischemically, and post-ischemically, suggesting that the different impact of MCAO in BK−/− vs. WT was not due to vascular BK channels. Furthermore, when NMDA was injected intracerebrally in non-ischemic mice, NMDA-induced neurotoxicity was found to be larger in BK−/− mice compared to WT. Whole-cell patch clamp recordings from CA1 pyramidal cells in organotypic hippocampal slice cultures revealed that BK channels contribute to rapid action potential repolarization, as previously found in acute slices. When these cultures were exposed to ischemia-like conditions this induced significantly more neuronal death in BK−/− than in WT cultures. These results indicate that neuronal BK channels are important for protection against ischemic brain damage.

Increased expression of phosphate-activated glutaminase in hippocampal neurons in human mesial temporal lobe epilepsy

Patients with mesial temporal lobe epilepsy (MTLE) have increased basal concentrations of extracellular glutamate in the epileptogenic versus the non-epileptogenic hippocampus. Such elevated glutamate levels have been proposed to underlie the initiation and maintenance of recurrent seizures, and a key question is what causes the elevation of glutamate in MTLE. Here, we explore the possibility that neurons in the hippocampal formation contain higher levels of the glutamate synthesizing enzyme phosphate-activated glutaminase (PAG) in patients with MTLE versus patients with other forms of temporal lobe epilepsy (non-MTLE). Increased PAG immunoreactivity was recorded in subpopulations of surviving neurons in the MTLE hippocampal formation, particularly in CA1 and CA3 and in the polymorphic layer of the dentate gyrus. Immunogold analysis revealed that PAG was concentrated in mitochondria. Double-labeling experiments indicated a positive correlation between the mitochondrial contents of PAG protein and glutamate, as well as between PAG enzyme activity and PAG protein as determined by Western blots. These data suggest that the antibodies recognize an enzymatically active pool of PAG. Western blots and enzyme activity assays of hippocampal homogenates revealed no change in PAG between MTLE and non-MTLE, despite a greatly (>50%) reduced number of neurons in the MTLE hippocampal formation compared to non-MTLE. Thus, the MTLE hippocampal formation contains an increased concentration and activity of PAG per neuron compared to non-MTLE. This increase suggests an enhanced capacity for glutamate synthesis—a finding that might contribute to the disrupted glutamate homeostasis in MTLE.

Towards an alternative testing strategy for nanomaterials used in nanomedicine: Lessons from NanoTEST

Abstract In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project (www.nanotest-fp7.eu) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

Silver nanoparticles induce premutagenic DNA oxidation that can be prevented by phytochemicals from Gentiana asclepiadea

Among nanomaterials, silver nanoparticles (AgNPs) have the broadest and most commercial applications due to their antibacterial properties, highlighting the need for exploring their potential toxicity and underlying mechanisms of action. Our main aim was to investigate whether AgNPs exert toxicity by inducing oxidative damage to DNA in human kidney HEK 293 cells. In addition, we tested whether this damage could be counteracted by plant extracts containing phytochemicals such as swertiamarin, mangiferin and homoorientin with high antioxidant abilities. We show that AgNPs (20 nm) are taken up by cells and localised in vacuoles and cytoplasm. Exposure to 1, 25 or 100 µg/ml AgNPs leads to a significant dose-dependent increase in oxidised DNA base lesions (8-oxo-7,8-dihydroguanine or 8-oxoG) detected by the comet assay after incubation of nucleoids with 8-oxoG DNA glycosylase. Oxidised DNA base lesions and strand breaks caused by AgNPs were diminished by aqueous and methanolic extracts from both haulm and flower of Gentiana asclepiadea.

Neuroprotective effects of inhibiting N-methyl-D-aspartate receptors, P2X receptors and the mitogen-activated protein kinase cascade: a quantitative analysis in organotypical hippocampal slice cultures subjected to oxygen and glucose deprivation.

Cell death was assessed by quantitative analysis of propidium iodide uptake in rat hippocampal slice cultures transiently exposed to oxygen and glucose deprivation, an in vitro model of brain ischemia. The hippocampal subfields CA1 and CA3, and fascia dentata were analyzed at different stages from 0 to 48 h after the insult. Cell death appeared at 3 h and increased steeply toward 12 h. Only a slight additional increase in propidium iodide uptake was seen at later intervals. The mitogen-activated protein kinases extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 were activated immediately after oxygen and glucose deprivation both in CA1 and in CA3/fascia dentata. Inhibition of the specific mitogen-activated protein kinase activator mitogen-activated protein kinase kinase by PD98059 or U0126 offered partial protection against oxygen and glucose deprivation-induced cell damage. The non-selective P2X receptor antagonist suramin gave neuroprotection of the same magnitude as the N-methyl-D-aspartate channel blocker MK-801 (approximately 70%). Neuroprotection was also observed with the P2 receptor blocker PPADS. Immunogold data indicated that hippocampal slice cultures (like intact hippocampi) express several isoforms of P2X receptors at the synaptic level, consistent with the idea that the effects of suramin and PPADS are mediated by P2X receptors. Virtually complete neuroprotection was obtained by combined blockade of N-methyl-D-aspartate receptors, P2X receptors, and mitogen-activated protein kinase kinase. Both P2X receptors and N-methyl-D-aspartate receptors mediate influx of calcium. Our results suggest that inhibition of P2X receptors has a neuroprotective potential similar to that of inhibition of N-methyl-D-aspartate receptors. In contrast, our comparative analysis shows that only partial protection can be achieved by inhibiting the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase cascade, one of the downstream pathways activated by intracellular calcium overload.

In vitro genotoxicity testing of four reference metal nanomaterials, titanium dioxide, zinc oxide, cerium oxide and silver: towards reliable hazard assessment

There is serious concern about the potential harmful effects of certain nanomaterials (NMs), on account of their ability to penetrate cell membranes and the increased reactivity that results from their increased surface area compared with bulk chemicals. To assess the safety of NMs, reliable tests are needed. We have investigated the possible genotoxicity of four representative NMs, derived from titanium dioxide, zinc oxide, cerium oxide and silver, in two human cell lines, A549 alveolar epithelial cells and lymphoblastoid TK6 cells. A high-throughput version of the comet assay was used to measure DNA strand beaks (SBs) as well as oxidised purines (converted to breaks with the enzyme formamidopyrimidine DNA glycosylase). In parallel, cytotoxicity was measured with the alamarBlue® assay, and the ability of NM-treated cells to survive was assessed by their colony-forming efficiency. TiO2 and CeO2 NMs were only slightly cytotoxic by the alamarBlue® test, and had no long-term effect on colony-forming efficiency. However, both induced DNA damage at non-cytotoxic concentrations; the damage decreased from 3 to 24-h exposure, except in the case of CeO2-treated A549 cells. ZnO and Ag NMs affected cell survival, and induced high levels of DNA damage at cytotoxic concentrations. At lower concentrations, there was significant damage, which tended to persist over 24 h. The implication is that all four reference metal NMs tested—whether cytotoxic or not—are genotoxic. A full assessment of NM toxicity should include tests on different cell types, different times of incubation and a wide range of (especially non-cytotoxic) concentrations; a test for cell viability should be performed in parallel. Inclusion of Fpg in the comet assay allows detection of indirect genotoxic effects via oxidative stress.

Base excision repair activities in organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation

The capacity for DNA repair is likely to be one of the factors that determine the vulnerability of neurons to ischemic stress and may influence the pathological outcome of stroke. In this report, initiation of base excision repair (BER) was assessed by analysis of enzyme activity and gene expression level of DNA glycosylases and AP-endonucleases in rat organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) - an in vitro model of stroke. Under basal conditions, AP-endonuclease activity and base removal of ethenoadenine and 8-oxoguanine (8-oxoG) were higher (by approximately 20-35 %) in CA3/fascia dentata (FD) than in CA1. Base removal of uracil did not differ between the two hippocampal regions, while removal of 5-hydroxyuracil (5-OHU) was slightly less efficient in CA3/FD than in CA1. Analyses performed immediately after 30 min of OGD revealed a decreased AP-endonuclease activity (by approximately 20%) in CA1 as well as CA3/FD, and an increased ethenoadenine activity (by approximately 25%) in CA1. Activities for 8-oxoG, 5-OHU and uracil showed no significant changes at this time point. At 8h after OGD, none of the enzyme activities differed from control values. Real-time RT-PCR showed that transcription of DNA glycosylases, including Ogg1, Nth1, Ung, Aag, Neil1 and Neil2 were not changed in response to OGD treatment (t=0 h). The hippocampal expression of Neil2 was low compared with the other DNA glycosylases. These data indicate that CA1 has a lower capacity than CA3/FD for removal of base lesions under basal conditions. The relatively low capacity for BER in basal conditions and the apparent failure to upregulate repair of oxidative damage after OGD might contribute to the high vulnerability of CA1 to ischemic injury.