E Zieminska

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.

The mechanism of 1,2,3,4‐tetrahydroisoquinolines neuroprotection: the importance of free radicals scavenging properties and inhibition of glutamate‐induced excitotoxicity

1‐Methyl‐1,2,3,4‐tetrahydroisoquinoline (1MeTIQ), unlike several other tetrahydroisoquinolines, displays neuroprotective properties. To elucidate this action we compared the effects of 1MeTIQ with 1,2,3,4‐tetrahydroisoquinoline (TIQ), a compound sharing many activities with 1MeTIQ (among them reducing free radicals formed during dopamine catabolism), but offering no clear neuroprotection. We found that the compounds similarly inhibit free‐radical generation in an abiotic system, as well as indices of neurotoxicity (caspase‐3 activity and lactate dehydrogenase release) induced by glutamate in mouse embryonic primary cell cultures (a preparation resistant to NMDA toxicity). However, in granular cell cultures obtained from 7‐day‐old rats, 1MeTIQ prevented the glutamate‐induced cell death and 45Ca2+ influx, whereas TIQ did not. This suggested a specific action of 1MeTIQ on NMDA receptors, which was confirmed by the inhibition of [3H]MK‐801 binding by 1MeTIQ. Finally, we demonstrated in an in vivo microdialysis experiment that 1MeTIQ prevents kainate‐induced release of excitatory amino acids from the rat frontal cortex. Our results indicate that 1MeTIQ, in contrast to TIQ, offers a unique and complex mechanism of neuroprotection in which antagonism to the glutamatergic system may play a very important role. The results suggest the potential of 1MeTIQ as a therapeutic agent in various neurodegenarative illnesses of the central nervous system.

Neuroprotection by co-treatment and post-treating with calcitriol following the ischemic and excitotoxic insult in vivo and in vitro.

Several in vivo and in vitro studies have demonstrated the neuroprotective potential of pretreatment with 1alpha,25-dihydroxyvitamin D3 (calcitriol). The aim of the present study was to determine the effectiveness of calcitriol administered in vivo after a brain ischemic episode in the rat model of perinatal asphyxia, or when co-applied with or without delay during 24-h exposure of mouse hippocampal, neocortical and cerebellar neuronal cultures to glutamate on their 7th and 12th day in vitro (7 DIV and 12 DIV, respectively). Calcitriol was also administered after acute exposure of rat cerebellar neurons to glutamate. In 7-day-old rat pups subjected to hypoxia-ischemia, acute application of calcitriol in a single dose of 2 microg/kg, 30 min after termination of the insult, or subchronic, 7-day post-treatment with calcitriol, effectively reduced brain damage. The level of neuroprotection exceeded that achieved by hypoxic preconditioning used as the reference neuroprotective method. The results of in vitro experiments revealed the ability of calcitriol to reduce excitotoxicity in a manner dependent on the origin of the neuronal cells, their stage of maturation in culture and the duration of exposure to the excitotoxic insult before calcitriol application. Calcitriol was neuroprotective when it was administered together with glutamate or even after a delay of up to 6h during 24-h excitotoxic challenge of hippocampal and neocortical, but not cerebellar neuronal cultures. Application of calcitriol to cultured cerebellar granule neurons after acute exposure to glutamate was ineffective. In 12 DIV hippocampal cell cultures, 50 nM calcitriol inhibited glutamate-induced caspase-3 activity, while only 100 nM concentrations were effective in 7 DIV cultures. We ascribe the protective effects of calcitriol to the rapid modulation of mechanisms that are instrumental in the direct anti-apoptotic, neuroprotective action of this compound.

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.

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.

Involvement of multiple protein kinases in cPLA2 phosphorylation, arachidonic acid release, and cell death in in vivo and in vitro models of 1‐methyl‐4‐phenylpyridinium‐induced parkinsonism – the possible key role of PKG

The study was aimed at investigating in vivo and in vitro the involvement of the cGMP/cGMP‐dependent protein kinase (PKG) signaling pathway in MPP+‐induced cytosolic phospholipase A2 (cPLA2) activation of dopaminergic neurons. MPP+ activated neuronal nitric oxide synthase (NOS)/soluble guanylyl cyclase/cGMP pathway in mouse midbrain and striatum, and in pheochromocytoma cell line 12 cells, and caused an upward shift in [Ca2+]i level in the latter. The activation was accompanied by increases in total and phosphorylated cPLA2, and increased arachidonic acid release. Effects of selective inhibitors [2‐oxo‐1,1,1‐trifluoro‐6,9‐12,15‐heneicosatetraene (AACOCF3), (E)‐6‐(bromomethylene)tetrahydro‐3‐(1‐naphthalenyl)2h‐pyran‐2‐one (BEL)] indicated the main impact of cPLA2 on arachidonic acid release in pheochromocytoma cell line 12 cells. Treatment of the cells with the protein kinase inhibitors GF102610x, UO126, and KT5823, and with the nitric oxide synthase (NOS) inhibitor NNLA revealed the involvement of protein kinase C (PKC) and extracellular signal‐regulated kinases 1 and 2 (ERK 1/2), with the possible key role of PKG, in cPLA2 phosphorylation at Ser505. Inhibitors of cPLA2 and PKG increased viability and reduced MPP+‐induced apoptosis of the cells. Our results indicate that the neuronal NOS/cGMP/PKG pathway stimulates cPLA2 phosphorylation at Ser505 by activating PKC and ERK1/2, and suggest that up‐regulation of this pathway in experimental models of Parkinson’s disease may mediate dopaminergic neuron degeneration and death through activation of cPLA2.

Analysis of glutamine accumulation in rat brain mitochondria in the presence of a glutamine uptake inhibitor, histidine, reveals glutamine pools with a distinct access to deamidation.

Rat cerebral nonsynaptic mitochondria were incubated in medium containing 2 mM glutamine (Gln) or 2 mM glutamate (Glu), in the presence of a Gln uptake inhibitor histidine (His) as well as other basic amino acids, lysine and arginine (Lys, Arg) not inhibiting Gln uptake. Subsequently, the mitochondrial contents of Glu and Gln were determined by HPLC. Incubation in the presence of Glu alone increased the Glu content from ∼3.5 to 15 nmol/mg protein, without affecting the Gln content. On the other hand, incubation with Gln increased the content of Gln from ∼1.5 to ∼12 nmol/mg, and that of Glu to 10 nmol/mg. As expected, addition of His did not alter the Glu and Gln content resulting from incubation with Glu. However, His significantly decreased to almost the preincubation level the content of Glu in mitochondria incubated with Gln, without affecting the content of Gln. No other amino acid had any effect on these parameters. The results point to the existence of distinct Gln pools, one of which is accessible to external Gln via a His-sensitive transporter and is accessible for deamidation in the mitochondria.

Dantrolene inhibits NMDA-induced 45Ca uptake in cultured cerebellar granule neurons.

Dantrolene is an inhibitor of a skeletal muscle subtype of ryanodine receptors that stabilizes intracellular calcium concentrations and exerts neuroprotective effects in neurons submitted to excitotoxic challenges. The mechanisms of dantrolene-induced neuroprotection are not clear. In this study, using a model of cultured rat cerebellar granule neurons, we demonstrated that dantrolene inhibits NMDA-evoked 45Ca uptake, indicating that this drug may inhibit the activity of NMDA receptor channels. Primary neuronal cultures were incubated for 10 min in Mg(2+)-free ionic medium with NMDA and 45Ca in the presence of different concentrations of dantrolene, then radioactivity in neurons was measured by liquid scintillation spectroscopy. The results demonstrated that dantrolene, applied at micromolar concentrations, inhibits NMDA-evoked 45Ca uptake in neurons in a dose-dependent manner. DMSO, a vehicle to dantrolene, in concentrations used in this study had no effect on NMDA-evoked 45Ca uptake. These results, indicating that dantrolene inhibits activation of the NMDA receptors, might at least partially explain the mechanisms of a dantrolene-evoked protection of neurons against excitotoxicity mediated by agonists of NMDA receptors.