Aleksandra Stafiej

Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30 min in the Mg(2+)-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24h later using propidium iodide staining. D,L-Homocysteine given alone appeared to be a weak neurotoxin, with EC(50) of 17.4mM, whereas EC(50) for L-glutamate was 0.17 mM. Addition of 50 microM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5 microM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45Ca uptake by granule cells incubated in the presence of 25 mM D,L-homocysteine with 50 microM glycine was only 3% of the net uptake evoked by 1mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250 microM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca(2+) to neurones.

Metabotropic glutamate receptors (mGluRs) are involved in early phase of memory formation: possible role of modulation of glutamate release.

Metabotropic glutamate receptors (mGluRs) groups I and II are involved in the cellular processes of long-term potentiation (LTP) and learning and memory formation. I.c.v. injection of the mGluRs agonist 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) can impair memory formation in some types of learning task. The role of mGluRs in neurotransmitters release and production of second messengers has been suggested. The aim of the present study was to determine the effect of i.c.v. administration of the new potent mGluRs agonist ABHxD-I and compare its effect with that of ACPD. We studied the effect of both agonists on acquisition and memory for a one-trial passive avoidance learning task in day-old chicks and on the training related glutamate (Glu) release. ACPD or ABHxD-I (50 nmole per chick, i.c.v. injection) were administered at different times before or after training and chicks were tested at various times after training. Chicks injected with ABHxD-I 30 min before training showed amnesia when tested 30 min or 3h after training. The amnestic effect of ACPD was significant only 30 min after training. Glu release evoked by 70 mM KCl was measured in slices prepared from the IMHV of chick brain isolated from animals injected with either ACPD or ABHxD-I 30 min before training and tested 30 min after training. Glu concentration was measured using HPLC. Both ACPD and ABHxD-I significantly increased Glu release in slices isolated from untrained chicks (30 and 48% compare to control, respectively, P<0.05). Training itself increased Glu release (41% compared to control, P<0.01) and no additional effect of either ACPD or ABHxD-I was observed. These results suggest that mGluRs groups I and II are involved in the early stages of memory formation and that application of either of the studied mGluRs agonists may interfere with that process. The amnestic effect of ABHxD-I seems to be stronger and longer lasting. Although the mechanism of this effect still remains unclear, our results suggest that disregulation of Glu release by mGluR agonists may participate in this process.

Excitotoxic neuronal injury in acute homocysteine neurotoxicity: Role of calcium and mitochondrial alterations

In this study we tested if calcium imbalance and mitochondrial dysfunction, which have been implicated in the conventional mechanisms of excitotoxicity induced by glutamate (Glu), are also involved in homocysteine (Hcy) neurotoxicity. Primary cultures of rat cerebellar granule cells were incubated for 30 min in the presence of 25 mM D,L-Hcy or 1mM Glu. At these concentrations both amino acids induced comparable neurodegeneration and chromatin condensation, evaluated after 24 h using the propidium iodide and Hoechst 33258 staining. These effects were partially prevented by cyclosporin A (CsA), but not FK506. Hcy-induced release of [(3)H]inositol phosphates and increase in intracellular calcium level (evaluated with fluo-3 fluorescent probe) were weakly expressed. Hcy- and Glu-induced mitochondrial swelling was visualized under electron microscope, and the release of Cytochrome c was evaluated using immunocytochemical method and confocal microscopy. Comparing to Glu, the effects of Hcy were slightly less expressed and less sensitive to CsA, while FK506 did not modify mitochondrial alterations. These data indicate that mitochondrial alterations play a similar role in acute Hcy and Glu neurotoxicity, although the mechanisms triggering Glu- and Hcy-evoked mitochondrial dysfunction seem to differ, Hcy toxicity being less dependent on calcium.

The role of the glutamatergic NMDA receptor in nanosilver-evoked neurotoxicity in primary cultures of cerebellar granule cells.

Nanoparticles are known to enter the vertebrate brain, but little is known about their neurotoxicity. The aim of this study is to investigate mechanisms of the contribution of AgNPs to neuronal cell death using primary cultures of rat cerebellar granule cells (CGCs). We tested the role of glutamatergic N-methyl-d-aspartate receptors (NMDA) in AgNP-evoked neurotoxicity using MK-801, a noncompetitive inhibitor of NMDAR. We used commercially available 0.2% PVP-coated AgNPs <100 nm in a concentration range of 2.5-75 μg/ml sonicated with fetal calf serum. After a 10 min incubation period, a dose-dependent increase in the uptake of (45)Ca(2+) into neurons was observed in the presence of 25-75 μg/mL AgNPs which was completely abolished by addition of MK-801. Using the fluorescent dye fluo3 AM we observed an increase in the intracellular calcium level by 87% compared to control. ROS production was found to increase by about 30% over control after a 30-min incubation with 75 μg/mL AgNPs. Further, we observed a significant decrease in the mitochondrial potential during a 30-min incubation with AgNPs. Administration of MK-801 was found to provide a protective effect. Our results show that excitotoxicity via activation of NMDA receptor, followed by calcium imbalance, destabilization of mitochondrial function and ROS production, indicate an important mechanism involved in neurotoxicity evoked by AgNPs in cultured neurons.

Homocysteine-Evoked 45Ca Release in the Rabbit Hippocampus Is Mediated by Both NMDA and Group I Metabotropic Glutamate Receptors: In Vivo Microdialysis Study

This in vivo microdialysis study compared the effects of NMDA and d,l-homocysteine (Hcy) administered via dialysis medium on 45Ca efflux from prelabeled rabbit hippocampus. Application of these agonists evoked dose-dependent, and sensitive to MK-801, opposite effects: NMDA decreased the 45Ca radioactivity in the dialysate, whereas Hcy induced the release of 45Ca. The latter effect was potentiated by glycine, inhibited by the antagonist of group I metabotropic glutamate receptors (mGluR) LY367385, and mimicked by t-ADA, an agonist of these receptors. Electron microscopic examination of pyramidal neurones in the CA1 sector of the hippocampus in the vicinity of the microdialysis probe after NMDA application demonstrated swelling of mitochondria, which was prevented by cyclosporin A. This study shows, for the first time, Hcy-induced activation of both group I mGluR and NMDA receptors, which may play a role in acute Hcy neurotoxicity. We present new applications of brain microdialysis in studies on excitotoxicity and neuroprotection.

Early changes in extracellular amino acids and calcium concentrations in rabbit hippocampus following complete 15-min cerebral ischemia

The effect of cerebral ischemia on extracellular amino acids and calcium content and on the permeability of the blood-brain barrier was studied by in vivo dialysis of rabbit hippocampus. This was combined with physiological and neurophysiological measurements. It was found that immediately after 15-min ischemia extracellular concentrations of glutamate, aspartate and taurine increased 3-, 2- and 6-fold, respectively, whereas a maximal, 7-fold increase of phosphoethanolamine and persistent elevation of glutamate were observed 45 min after ischemia. Extracellular calcium concentration, monitored with 45Ca2+, increased by 10% during the initial phase of ischemia, and decreased to approx. 74% of the basal level 10 min after ischemia. Recovery of extracellular calcium content was not attained until 45 min of recirculation, at which time the first signs of return of bioelectric activity were noted. Increased permeability of the blood-brain barrier to fluoresceine developed immediately after ischemia and persisted up to 2 h of recirculation. The obtained results are discussed in reference to the noted simultaneity of changes in extracellular excitatory amino acids and calcium concentrations and of brain bioelectric activity during and after ischemia. Causal relations between these effects are suggested.

Low molecular weight thiols reduce thimerosal neurotoxicity in vitro: Modulation by proteins

Thimerosal (TH), an ethylmercury complex of thiosalicylic acid has been used as preservative in vaccines. In vitro neurotoxicity of TH at high nM concentrations has been reported. Although a number of toxicological experiments demonstrated high affinity of mercury to thiol groups of the extracellular amino acids and proteins that may decrease concentration of free TH in the organism, less is known about the role of interactions between proteins and amino acids in protection against TH neurotoxicity. In the present study we examined whether the presence of serum proteins and of l-cysteine (Cys), d,l-homocysteine (Hcy), N-acetyl cysteine (NAC), l-methionine (Met) and glutathione (GSH) in the incubation medium affects the TH-induced changes in the viability, the intracellular levels of calcium and zinc and mitochondrial membrane potential in primary cultures of rat cerebellar granule cells. The cells were exposed to 500 nM TH for 48 h or to 15-25 μM TH for 10 min. Our results demonstrated a decrease in the cells viability evoked by TH, which could be prevented partially by serum proteins, albumin or in a dose-dependent manner by 60, 120 or 600 μM Cys, Hcy, NAC and GSH, but not by Met. This neuroprotection was less pronounced in the presence of proteins. Incubation of neurons with TH also induced the rise in the intracellular calcium and zinc concentration and decrease in mitochondrial membrane potential, and these effects were abolished by all the sulfur containing compounds studied and administered at 600 μM concentration, except Met. The loss of the ethylmercury moiety from TH as a result of interaction with thiols studied was monitored by (1)H NMR spectroscopy. This extracellular process may be responsible for the neuroprotection seen in the cerebellar cell cultures, but also provides a molecular pathway for redistribution of TH-derived toxic ethylmercury in the organism. In conclusion, these results confirmed that proteins and sulfur-containing amino acids applied separately reduce TH neurotoxicity, while their combination modulates in more complex way neuronal survival in the presence of TH.

Synergistic neurotoxicity of oxygen-glucose deprivation and tetrabromobisphenol A in vitro: role of oxidative stress

BACKGROUND Tetrabromobisphenol A (TBBPA) is a toxic brominated flame retardant. Previous studies have demonstrated that exposure of primary cultures of rat cerebellar granule cells (CGC) to ≥ 10 μM TBBPA induces toxicity and excitotoxicity, and the underlying mechanism may involve calcium imbalance and oxidative stress. Here we examined whether the application of TBBPA at subtoxic concentrations may exacerbate acute damage of CGC challenged with oxygen-glucose deprivation (OGD), and evaluated with fluorescent indicators the involvement of calcium imbalance, mitochondrial depolarization and oxidative stress. METHODS Survival of CGC was assessed 24 h after OGD/TBBPA using fluorescent dyes. An OGD challenge lasting for 45, 60 or 75 min induced a duration-dependent injury to the neurons. RESULTS Application of 2.5, 5 or 7.5 μM TBBPA for 45 min to normoxic and glucose-containing incubation medium did not reduce the viability of cultured CGC, but this compound exacerbated the toxic effects of OGD in a concentration-dependent way. Moreover, TBBPA had a slight effect on calcium homeostasis and mitochondrial membrane potential, but significantly activated the production of reactive oxygen species in CGC. The application of H(2)O(2) at 5, 10 and 25 μM mimicked the effects of TBBPA on OGD toxicity, while 0.1 mM ascorbic acid or 1 mM glutathione ameliorated this toxicity. CONCLUSION These results suggest the involvement of oxidative stress in the synergistic neurotoxic effects of TBBPA and OGD.

Synthetic Bastadins Modify the Activity of Ryanodine Receptors in Cultured Cerebellar Granule Cells

Although the interactions of several natural bastadins with the RyR1 isoform of the ryanodine receptor in sarcoplasmic reticulum has been described, their structure-dependent interference with the RyR2 isoform, mainly expressed in cardiac muscle and brain neurons, has not been studied. In this work, we examined calcium transients induced by natural bastadin 10 and several synthetic bastadins in cultured cerebellar granule cells known to contain RyR2. The fluorescent calcium indicator fluo-3 and confocal microscopy were used to evaluate changes in the intracellular Ca2+ concentration (Cai), and the involvement of ryanodine receptors was assessed using pharmacological tools. Our results demonstrate that apart from the inactive BAST218F6 (a bisdebromo analogue of bastadin 10), synthetic bastadin 5, and synthetic analogues BAST217B, BAST240 and BAST268 (at concentrations >20 µM) increased Cai in a concentration-dependent, ryanodine- and FK-506-sensitive way, with a potency significantly exceeding that of 20 mM caffeine. Moreover, the same active bastadins at a concentration of 5 µM in the presence of ryanodine prevented a thapsigargin-induced increase in Cai. These results indicate that bastadins, acting in a structure-dependent manner, modify the activity of RyR2 in primary neuronal culture and provide new information about structure-related pharmacological properties of bastadins.

Contents Vol. 15, 2006–2007

40 Croucher Advanced Study Institute (ASI): Signaling in Cell Growth and Differentiation. Organized by: Department of Biochemistry, The Hong Kong University of Science and Technology