Metabolomics and metabolites in ischemic stroke.

Stroke is a major reason for disability and the second highest cause of death in the world. When a patient is admitted to a hospital, it is necessary to identify the type of stroke, and the likelihood for development of a recurrent stroke, vascular dementia, and depression. These factors could be determined using different biomarkers. Metabolomics is a very promising strategy for identification of biomarkers. The advantage of metabolomics, in contrast to other analytical techniques, resides in providing low molecular weight metabolite profiles, rather than individual molecule profiles. Technically, this approach is based on mass spectrometry and nuclear magnetic resonance. Furthermore, variations in metabolite concentrations during brain ischemia could alter the principal neuronal functions. Different markers associated with ischemic stroke in the brain have been identified including those contributing to risk, acute onset, and severity of this pathology. In the brain, experimental studies using the ischemia/reperfusion model (IRI) have shown an impaired energy and amino acid metabolism and confirmed their principal roles. Literature data provide a good basis for identifying markers of ischemic stroke and hemorrhagic stroke and understanding metabolic mechanisms of these diseases. This opens an avenue for the successful use of identified markers along with metabolomics technologies to develop fast and reliable diagnostic tools for ischemic and hemorrhagic stroke.

Disabling phosphorylation at the homer ligand of the metabotropic glutamate receptor 5 alleviates complete Freund's adjuvant-induced inflammatory pain.

Metabotropic glutamate receptor 5 (mGluR5) has been reported to contribute to inflammatory pain. The intracellular C-terminal domain has a Homer-binding motif that can form an mGluR5/Homer complex. Phosphorylation of mGluR5 at the Homer binding domain enhances the mGluR5/Homer interaction and modulates intracellular signal transduction. However, the characteristics of this interaction have not been fully elucidated in inflammatory pain. We aimed to evaluate the effects of CFA-induced phosphorylation of mGluR5 at the Homer binding domain on the mGluR5/Homer interaction. Von-frey filaments and thermal latency were used to monitor the development of inflammatory pain. Spinal mGluR5 phosphorylation at Ser1126 and mGluR5/Homer crosslinking were detected. Mutant mGluR5 that could not be phosphorylated at Thr1123 or Ser1126 was evaluated in inflammatory pain. CFA-induced inflammatory pain resulted in obvious phosphorylation at Ser1126 of mGluR5. Moreover, increased phosphorylation at the Homer-binding motif enhanced crosslinking between mGluR5 and Homer. Mutations at Thr1123 and Ser1126 of mGluR5 blocked the development of CFA-induced inflammatory pain. Overall, our findings showed that disruption of the phosphorylation of mGluR5 Thr1123 and Ser1126 alleviated CFA-induced inflammatory pain.

High Concentration of Ketone Body ß-Hydroxybutyrate Modifies Synaptic Vesicle Cycle and Depolarizes Plasma Membrane of Rat Brain Synaptosomes.

Ketoacidosis is a dangerous complication of diabetes mellitus in which plasma levels of ketone bodies can reach 20-25 mM. This condition is life-threatening. In contrast, a ketogenic diet, achieving plasma levels of ketone bodies of about 4-5 mM, can be used for treating different brain diseases. However, the factors leading to the conversion of the neuroprotective ketone bodies' action to the neurotoxic action during ketoacidosis are still unknown. We investigated the influence of high concentration (25 mM) of the main ketone body, ß-hydroxybutyrate (BHB), on intrasynaptosomal pH (pHi), synaptic vesicle cycle, plasma membrane, and mitochondrial potentials. Using the fluorescent dye BCECF-AM, it was shown that BHB at concentrations of 8 and 25 mM did not influence pHi in synaptosomes. By means of the fluorescent dye acridine orange, it was demonstrated that 25 mM of BHB had no effect on exocytosis but inhibited compensatory endocytosis by 5-fold. Increasing buffer capacity with 25 mM HEPES did not affect endocytosis. Glucose abolished BHB-induced endocytosis inhibition. Using the fluorescent dye DiSC3(5), it was shown that 25 mM of BHB induced a significant plasma membrane depolarization. This effect was not impacted by glucose. Using the fluorescent dye rhodamine-123, it was shown that BHB alone (25 mМ) did not alter the potential of intrasynaptosomal mitochondria.Importantly, the high concentration of BHB (25 mМ) causes the depolarization of the plasma membrane and stronger inhibition of endocytosis compared with the intermediate concentration (8 mM).

Ketogenic diet versus ketoacidosis: what determines the influence of ketone bodies on neurons?

Glucose is the main energy substrate for neurons, however, at certain conditions, e.g. in starvation, these cells could also use ketone bodies. This approach is used in clinical conditions as the ketogenic diet. The ketogenic diet is actually a biochemical model of fasting. It includes replacing carbohydrates by fats in daily meal. Synthesis of ketone bodies ß-hydroxubutirate, acetoacetate and acetone begins once glycogen stores have depleted in the liver. The ketogenic diet can be used to treat clinical conditions, primarily epilepsy. The mechanism of neuroprotective action of ketogenic diet is not very clear. It is shown that ketone bodies influence neurons at three different levels, namely, metabolic, signaling and epigenetic levels. Ketone bodies are not always neuroprotective. Sometimes they can be toxic for the brain. Ketoacidosis which is a very dangerous complication of diabetes mellitus or alcoholism can be taken as an example. The exact mechanism of how neuroprotective properties of ketone bodies reverse to neurotoxic is yet to be established.

Metabolic regulation of synaptic activity.

Brain tissue is bioenergetically expensive. In humans, it composes approximately 2% of body weight and accounts for approximately 20% of calorie consumption. The brain consumes energy mostly for ion and neurotransmitter transport, a process that occurs primarily in synapses. Therefore, synapses are expensive for any living creature who has brain. In many brain diseases, synapses are damaged earlier than neurons start dying. Synapses may be considered as vulnerable sites on a neuron. Ischemic stroke, an acute disturbance of blood flow in the brain, is an example of a metabolic disease that affects synapses. The associated excessive glutamate release, called excitotoxicity, is involved in neuronal death in brain ischemia. Another example of a metabolic disease is hypoglycemia, a complication of diabetes mellitus, which leads to neuronal death and brain dysfunction. However, synapse function can be corrected with "bioenergetic medicine". In this review, a ketogenic diet is discussed as a curative option. In support of a ketogenic diet, whereby carbohydrates are replaced for fats in daily meals, epileptic seizures can be terminated. In this review, we discuss possible metabolic sensors in synapses. These may include molecules that perceive changes in composition of extracellular space, for instance, ketone body and lactate receptors, or molecules reacting to changes in cytosol, for instance, KATP channels or AMP kinase. Inhibition of endocytosis is believed to be a universal synaptic mechanism of adaptation to metabolic changes.

Calcium release from intracellular stores is involved in mitochondria depolarization after lowering extracellular pH in rat brain synaptosomes.

In the brain, pH can be lowered in both healthy and disease states. Previously, we showed that moderate extracellular acidification (down to pHo 7.0), but not intracellular acidification, leads to mitochondrial depolarization in synaptosomes. This indicates that the plasma membranes of neuronal presynaptic endings have proton receptors that can induce mitochondrial dysfunction when activated. In the present paper we attempt to identify this hypothetical receptor. First, we have demonstrated that lowering pHo to 7.0 does not induce sodium influx as monitored by the fluorescent dye Sodium Green. This fact, in conjunction with the absence of calcium influx in the same conditions - demonstrated previously, excludes ion channels as possible receptors. However, we showed that acidification-induced mitochondrial depolarization is sensitive to thapsigargin - an inhibitor of calcium release from intracellular stores, U73122 - an inhibitor of phospholipase C, as well as Cu2+ and Zn2+, which can block the metabotropic proton receptor ovarian cancer G protein-coupled receptor 1 (OGR1). Furthermore, using fluorescent dye Fluo-3 we have demonstrated that moderate extracellular acidification induces a cytosolic calcium increase. Excess calcium was scavenged by mitochondria (monitored by fluorescent dye Rhod-2). Our results suggest that the metabotropic OGR1 is a hypothetical presynaptic receptor for low pH. Its activation leads to phospholipase C activation and calcium release from the endoplasmic reticulum followed by accumulation in mitochondria, which likely causes a decrease in mitochondrial membrane potential.

Biogenetic and morphofunctional heterogeneity of mitochondria: the case of synaptic mitochondria.

The mitochondria of different cells are different in their morphological and biochemical properties. These organelles generate free radicals during activity, leading inevitably to mitochondrial DNA damage. It is not clear how this problem is addressed in long-lived cells, such as neurons. We propose the hypothesis that mitochondria within the same cell also differ in lifespan and ability to divide. According to our suggestion, cells have a pool of 'stem' mitochondria with low metabolic activity and a pool of 'differentiated' mitochondria with significantly shorter lifespans and high metabolic activity. We consider synaptic mitochondria as a possible example of 'differentiated' mitochondria. They are significantly smaller than mitochondria from the cell body, and they are different in key enzyme activity levels, proteome, and lipidome. Synaptic mitochondria are more sensitive to different damaging factors. It has been established that neurons have a sorting mechanism that sends mitochondria with high membrane potential to presynaptic endings. This review describes the properties of synaptic mitochondria and their role in the regulation of synaptic transmission.

ß-Hydroxybutyrate supports synaptic vesicle cycling but reduces endocytosis and exocytosis in rat brain synaptosomes.

The ketogenic diet is used as a prophylactic treatment for different types of brain diseases, such as epilepsy or Alzheimer's disease. In such a diet, carbohydrates are replaced by fats in everyday food, resulting in an elevation of blood-borne ketone bodies levels. Despite clinical applications of this treatment, the molecular mechanisms by which the ketogenic diet exerts its beneficial effects are still uncertain. In this study, we investigated the effect of replacing glucose by the ketone body ß-hydroxybutyrate as the main energy substrate on synaptic vesicle recycling in rat brain synaptosomes. First, we observed that exposing presynaptic terminals to nonglycolytic energy substrates instead of glucose did not alter the plasma membrane potential. Next, we found that synaptosomes were able to maintain the synaptic vesicle cycle monitored with the fluorescent dye acridine orange when glucose was replaced by ß-hydroxybutyrate. However, in presence of ß-hydroxybutyrate, synaptic vesicle recycling was modified with reduced endocytosis. Replacing glucose by pyruvate also led to a reduced endocytosis. Addition of ß-hydroxybutyrate to glucose-containing incubation medium was without effect. Reduced endocytosis in presence of ß-hydroxybutyrate as sole energy substrate was confirmed using the fluorescent dye FM2-10. Also we found that replacement of glucose by ketone bodies leads to inhibition of exocytosis, monitored by FM2-10. However this reduction was smaller than the effect on endocytosis under the same conditions. Using both acridine orange in synaptosomes and the genetically encoded sensor synaptopHluorin in cortical neurons, we observed that replacing glucose by ß-hydroxybutyrate did not modify the pH gradient of synaptic vesicles. In conclusion, the nonglycolytic energy substrates ß-hydroxybutyrate and pyruvate are able to support synaptic vesicle recycling. However, they both reduce endocytosis. Reduction of both endocytosis and exocytosis together with misbalance between endocytosis and exocytosis could be involved in the anticonvulsant activity of the ketogenic diet.

Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

Role of iron, zinc and reduced glutathione in oxidative stress induction by low pH in rat brain synaptosomes.

Brain ischemia leads to a decrease in pHo. We have shown previously in synaptosomes that the extracellular acidification induces depolarization of mitochondria followed by synthesis of superoxide anions and oxidative stress. Here, we investigated the effects of lowered pHo on oxidative stress and membrane potentials in synaptosomes treated by the iron chelator deferoxamine and zinc chelator TPEN. We demonstrated that chelating of metals has no impact on superoxide anion synthesis and intrasynaptosomal mitochondria depolarization. Meanwhile, deferoxamine was able to inhibit oxidative stress induced by low pHo and hydrogen peroxide application. Compared to deferoxamine, TPEN was less effective but it decreased the DCF fluorescence induced by pHo 6.0 which had no effects in other oxidative stress models. We found that the chelators were able to inhibit slightly plasma membrane depolarization. Synaptosomes preincubation at low pHo caused no effects on the reduced glutathione level. Depletion of glutathione by CDNB produced no additional increase in the DCF fluorescence induced by pHo 7.0. Our results suggest that free iron is crucial for the development of oxidative stress elicited by acidification in synaptosomes. Chelating of this metal seems to be a promising strategy for protecting the neuronal presynaptic terminals against oxidative stress developed at stroke.

Piracetam induces plasma membrane depolarization in rat brain synaptosomes.

Piracetam is a cyclic derivative of γ-aminobutyric acid (GABA). It was the first nootropic drug approved for clinical use. However, mechanism of its action is still not clear. In present paper, I investigated effects of piracetam on neurotransmitter release, plasma membrane potential monitored by fluorescent dye DiSC3(5) and chloride transport monitored by fluorescent dye SPQ in rat brain synaptosomes. It was shown that piracetam (1 mM) induces slow weak plasma membrane depolarization. This effect was decreased on 43% and 58% by both AMPA/kainate receptor blockers NBQX (10 µM) and CNQX (100 µM), respectively, on 84% by GABA ionotropic receptor blocker picrotoxin (50 µM) and on 91% upon withdrawal of HCO(3-) ions from incubation medium. GABA (1 mM) and kainate (100 µM) were found not to produce changes of plasma membrane potential. Also, it was found that piracetam induces chloride efflux which seems to be the reason of depolarization. Thereby, piracetam induces depolarization of plasma membrane of isolated neuronal presynaptic endings by picrotoxin-sensitive way.

Influence of intra- and extracellular acidification on free radical formation and mitochondria membrane potential in rat brain synaptosomes.

Brain ischemia is accompanied by lowering of intra- and extracellular pH. Stroke often leads to irreversible damage of synaptic transmission by unknown mechanism. We investigated an influence of lowering of pH(i) and pH(o) on free radical formation in synaptosomes. Three models of acidosis were used: (1) pH(o) 6.0 corresponding to pH(i) decrease down to 6.04; (2) pH(o) 7.0 corresponding to the lowering of pH(i) down to 6.92: (3) 1 mM amiloride corresponding to pH(i) decrease down to 6.65. We have shown that both types of extracellular acidification, but not intracellular acidification, increase 2',7'-dichlorodihydrofluorescein diacetate fluorescence that reflects free radical formation. These three treatments induce the rise of the dihydroethidium fluorescence that reports synthesis of superoxide anion. However, the impact of amiloride on superoxide anion synthesis was less than that induced by moderate extracellular acidification. Superoxide anion synthesis at pH(o) 7.0 was almost completely eliminated by mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. Furthermore, using fluorescent dyes JC-1 and rhodamine-123, we confirmed that pH(o) lowering, but not intracellular acidification, led to depolarization of intrasynaptosomal mitochondria. We have shown that pH(o) but not pH(i) lowering led to oxidative stress in neuronal presynaptic endings that might underlie the long-term irreversible changing in synaptic transmission.

Glutamate-induced free radical formation in rat brain synaptosomes is not dependent on intrasynaptosomal mitochondria membrane potential.

Glutamate induces reactive oxygen species formation (ROS) in neurons. Free radicals can potentially be synthesized by NADPH oxidase or mitochondria. The primary source of ROS origin has yet to be identified. In addition, pro-oxidant action of glutamate receptors on neuronal presynaptic terminals is still not characterized. We investigated the influence of glutamate and agonists of its ionotropic receptors on ROS formation detected by fluorescent dye DCFDA in rat brain synaptosomes. Glutamate in concentration 10 and 100µM led to an increase of probe fluorescence pointing to free radical accumulation. This effect was mimicked by 100µM of NMDA or 100µM of kainate. Glutamate-induced ROS formation was sensitive to NMDA inhibitors MK-801 (10µM), NO synthase (NOS) inhibitor l-NAME (100µM) and NADPH oxidase inhibitors DPI (30µM) and not affected by mitochondrial uncoupler CCCP (10µM) and mitochondrial toxins rotenone (10µM)+oligomycin (5µg/ml). We also showed that 100µM of glutamate leads to a decrease of intrasynaptosomal mitochondrial potential monitored by fluorescent dye Rhodamine-123. Hence, the depolarization of intrasynaptosomal mitochondria is not a primary cause of glutamate-induced ROS formation in neuronal presynaptic terminals. Activation of NMDA receptors might be responsible for a certain part of glutamate pro-oxidant action. Most likely, sources of glutamate-induced ROS formation in neuronal presynaptic terminals are NADPH oxidase and NOS activation.

Ca2+ nanodomain-mediated component of swelling-induced volume-sensitive outwardly rectifying anion current triggered by autocrine action of ATP in mouse astrocytes.

The volume-sensitive outwardly rectifying (VSOR) anion channel provides a major pathway for anion transport during cell volume regulation. It is typically activated in response to cell swelling, but how the channel senses the swelling remains unclear. Meanwhile, we recently found that in mouse astrocytes the channel is activated by an inflammatory chemical mediator, bradykinin, without cell swelling and that the activation is regulated via high concentration regions of intracellular Ca(2+) ([Ca(2+)](i)) in the immediate vicinity of open Ca(2+)-permeable channels, so-called Ca(2+) nanodomains. Here we investigated whether a similar mechanism is involved in the swelling-induced VSOR channel activation in the astrocytes. A hypotonic stimulus (25% reduction in osmolality) caused the [Ca(2+)](i) rises in the astrocytes, and the rises were abolished in the presence of an ATP-degrading enzyme, apyrase (10 U/ml). Application of ATP (100 µM) under isotonic conditions generated the current through VSOR channels via Ca(2+) nanodomains, as bradykinin does. The current induced by the hypotonic stimulus was suppressed by ~40% in the Ca(2+)-depleted condition where the ATP-induced VSOR current was totally prevented. Thus the swelling-induced VSOR channel activation in mouse astrocytes is partly regulated via Ca(2+) nanodomains, whose generation is triggered by an autocrine action of ATP.

Presynaptic glycine receptors influence plasma membrane potential and glutamate release.

Glycine is a classical inhibitory neurotransmitter however presynaptic glycine receptors have rather depolarizing action. Reasons for latter phenomenon are unknown. In the present paper we have investigated how glycine influences cytosolic chloride level monitored by fluorescent dye SPQ, membrane potential monitored by fluorescent dye DiSC3(5) and [(14)C]-glutamate release in synaptosomes. We estimated that cytosolic chloride concentration in synaptosomes was about 52 +/- 1 mM. Glycine (1 mM) induced chloride efflux and caused slow plasma membrane depolarization. Chloride efflux was almost completely blocked by 100 microM strychnine whilst glycine-induced depolarization was only partially. We also showed that 1 mM glycine induced [(14)C]-glutamate release via a strychnine-insensitive pathway. Hence we have concluded that glycine was able to induce two independent effects in synaptosomes: (1) Chloride efflux with following depolarization. This efflux was sensitive to strychnine and thereby is probably conducted through glycine-gated ion channels. (2) Glutamate release seems to be mediated by glycine transporters.

Are synapses targets of nanoparticles?

The last few years have been marked by real breakthroughs in the field of nanotechnology. Application of nanoparticles was proposed for diagnosis and treatment of different central nervous system diseases. Exposure to nanoparticles in vivo increases the risk of onset of neurodegenerative diseases and nanoparticles are apparently able to kill neurons in vitro. We suggested that presynaptic terminals of neurons are another target for nanoparticles, beyond the already established microglial cells. Ferritin was chosen as a prototypic nanoparticle model. We found that even a high concentration of ferritin, 800 microg/ml, was not able to induce spontaneous release of [(14)C]glutamate. In contrast, [(14)C]glutamate uptake was inhibited by ferritin in a dose-dependent fashion. As a next step, the influence of ferritin on the formation of reactive oxygen species was monitored using the fluorescent dye DCFH-DA (2',7'-dichlorofluorescein diacetate). It was shown that ferritin leads to a dose-dependent formation of free radicals. We found that the ferritin-mediated changes in glutamatergic neurotransmission at presynaptic endings can result in neuronal damage and finally neurodegeneration.

Ferritin, a protein containing iron nanoparticles, induces reactive oxygen species formation and inhibits glutamate uptake in rat brain synaptosomes.

Nanoparticles are currently used in medicine as agents for targeted drug delivery and imaging. However it has been demonstrated that nanoparticles induce neurodegeneration in vivo and kill neurons in vitro. The cellular and molecular bases of this phenomenon are still unclear. We have used the protein ferritin as a nanoparticle model. Ferritin contains iron particles (Fe(3+)) with size 7 nm and a protein shell. We investigated how ferritin influences uptake and release of [(14)C]glutamate and free radical formation as monitored by fluorescent dye DCFDA in rat brain synaptosomes. We found that even a high concentration of ferritin (800 microg/ml) did not induce spontaneous [(14)C]glutamate release. In contrast the same concentration of this protein inhibited [(14)C]glutamate uptake two fold. Furthermore ferritin induced intrasynaptosomal ROS (reactive oxygen species) formation in a dose-dependent manner. This process was insensitive to 30 microM DPI, an inhibitor of NADPH oxidase and to 10 microM CCCP, a mitochondrial uncoupler. These results indicate that iron-based nanoparticles can cause ROS and decreased glutamate uptake, potentially leading to neurodegeneration.

Influence of integrin-blocking peptide on gadolinium- and hypertonic shrinking-induced neurotransmitter release in rat brain synaptosomes.

Polyvalent cations and hypertonic shrinking of presynaptic endings lead to calcium-independent exocytosis in various synapses. In the present study we have investigated the contribution of integrins to this phenomenon. It was found that hypertonic shrinking, polyvalent cations ruthenium red and gadolinium results in dose-dependent calcium-independent neurotransmitter release in rat brain synaptosomes. The exocytotic mechanism of neurotransmitter release induced by 300 microM gadolinium was additionally verified by the fluorescent dye FM2-10. We found that 200 microM of RGDS peptide, an inhibitor of integrins, decreased polyvalent gadolinium-induced [3H]D: -aspartate release by 26%. This compound had no effect upon hypertonicity-induced release. The peptide RGES, a negative control for RGDS; genistein, an inhibitor of tyrosine kinases; and citrate, an inhibitor of lanthanides-induced aggregation were ineffective in both cases. Therefore, we have shown that integrins did not influence hypertonicity-evoked [3H]D: -aspartate release, but partially mediated that evoked by gadolinium ions.