Institute of Biophysics and Cell Engineering

Minsk, Belarus

Institute of Biophysics and Cell Engineering

Minsk, Belarus
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Ivanov A.I.,Aix - Marseille University | Malkov A.E.,Aix - Marseille University | Malkov A.E.,Institute Theoretical and Experimental Biophysics | Waseem T.,Aix - Marseille University | And 8 more authors.
Journal of Cerebral Blood Flow and Metabolism | Year: 2014

Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-Triggered energy metabolism. These effects are not induced by changes in ATP content, pH i, Ca 2+ i or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices - instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes. © 2014 ISCBFM All rights reserved.


Mukhtarov M.,Aix - Marseille University | Mukhtarov M.,Kazan Federal University | Liguori L.,National Research Council Italy | Waseem T.,Aix - Marseille University | And 6 more authors.
Frontiers in Molecular Neuroscience | Year: 2013

Monitoring of the intracellular concentrations of Cl- and H+ requires sensitive probes that allow reliable quantitative measurements without perturbation of cell functioning. For these purposes the most promising are genetically encoded fluorescent biosensors, which have become powerful tools for non-invasive intracellular monitoring of ions, molecules and enzymatic activity. A ratiometric CFP/YFP-based construct with a relatively good sensitivity to Cl- has been developed (Markova et al., 2008; Waseem et al., 2010). Recently, a combined Cl-/pH sensor (ClopHensor) opened the way for simultaneous ratiometric measurement of these two ions (Arosio et al., 2010). ClopHensor was obtained by fusion of a red-fluorescent protein (DsRed-monomer) to the E2GFP variant that contains a specific Cl--binding site. This construct possesses pKa = 6.8 for H+ and Kd in the 40-50 mM range for Cl- at physiological pH (~7.3) As in the majority of cell types the intracellular Cl- concentration ([Cl-]i) is about 10 mM, the development of sensors with higher sensitivity is highly desirable. Here we report the intracellular calibration and functional characterization of ClopHensor and its two derivatives: the membrane targeting PalmPalm-ClopHensor and the H148G/V224L mutant with improved Cl- affinity, reduced pH dependence and pKa shifted to more alkaline values. For functional analysis, constructs were expressed in CHO cells and [Cl-]i was changed by using pipettes with different Cl- concentrations during whole-cell recordings. Kd values for Cl- measured at 33°C and pH ~ 7.3 were, respectively, 39 mM, 47 mM and 21 mM for ClopHensor, PalmPalm-ClopHensor and the H148G/V224L mutant. PalmPalm-ClopHensor resolved responses to activation of Cl--selective glycine receptor channels better than did ClopHensor. Our observations indicate that these different ClopHensor constructs are promising tools for non- invasive measurement of [Cl-]i in various living cells. © 2013 Mukhtarov, Liguori, Waseem, Rocca, Buldakova, Arosio and Bregestovski.


Hrynevich S.V.,Institute of Biophysics and Cell Engineering | Pekun T.G.,Institute of Biophysics and Cell Engineering | Waseem T.V.,Institute of Biophysics and Cell Engineering | Waseem T.V.,Aix - Marseille University | Fedorovich S.V.,Institute of Biophysics and Cell Engineering
Neurochemical Research | Year: 2015

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 Ca2+-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. © 2015, Springer Science+Business Media New York.


PubMed | Aix - Marseille University and Institute of Biophysics and Cell Engineering
Type: | Journal: SpringerPlus | Year: 2014

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.


Malkov A.,French Institute of Health and Medical Research | Malkov A.,Russian Academy of Sciences | Ivanov A.I.,French Institute of Health and Medical Research | Popova I.,French Institute of Health and Medical Research | And 8 more authors.
Journal of Cerebral Blood Flow and Metabolism | Year: 2014

Excessive accumulation of reactive oxygen species (ROS) underlies oxidative damage. We find that in hippocampal slices, decreased activity of glucose-based antioxidant system induces a massive, abrupt, and detrimental change in cellular functions. We call this phenomenon metabolic collapse (MC). This collapse manifested in long-lasting silencing of synaptic transmission, abnormal oxidation of NAD(P)H and FADH2 associated with immense oxygen consumption, and massive neuronal depolarization. MC occurred without any preceding deficiency in neuronal energy supply or disturbances of ionic homeostasis and spread throughout the hippocampus. It was associated with a preceding accumulation of ROS and was largely prevented by application of an efficient antioxidant Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). The consequences of MC resemble cortical spreading depression (CSD), a wave of neuronal depolarization that occurs in migraine, brain trauma, and stroke, the cellular initiation mechanisms of which are poorly understood. We suggest that ROS accumulation might also be the primary trigger of CSD. Indeed, we found that Tempol strongly reduced occurrence of CSD in vivo, suggesting that ROS accumulation may be a key mechanism of CSD initiation. © 2014 ISCBFM All rights reserved.


Waseem T.V.,Institute of Biophysics and Cell Engineering | Fedorovich S.V.,Institute of Biophysics and Cell Engineering
Neurochemical Research | Year: 2010

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 [14C]-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 μM strychnine whilst glycine-induced depolarization was only partially. We also showed that 1 mM glycine induced [14C]-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. © 2010 Springer Science+Business Media, LLC.


Lysenko E.A.,RAS Timiryazev Institute of Plant Physiology | Klaus A.A.,RAS Timiryazev Institute of Plant Physiology | Pshybytko N.L.,Institute of Biophysics and Cell Engineering | Kusnetsov V.V.,RAS Timiryazev Institute of Plant Physiology
Photosynthesis Research | Year: 2015

Data on cadmium accumulation in chloroplasts of terrestrial plants are scarce and contradictory. We introduced CdSO4 in hydroponic media to the final concentrations 80 and 250 μM and studied the accumulation of Cd in chloroplasts of Hordeum vulgare and Zea mays. Barley accumulated more Cd in the chloroplasts as compared to maize, whereas in the leaves cadmium accumulation was higher in maize. The cadmium content in the chloroplasts of two species varied from 49 to 171 ng Cd/mg chlorophyll, which corresponds to one Cd atom per 728-2,540 chlorophyll molecules. Therefore, Mg2+ can be substituted by Cd2+ in a negligible amount of antenna chlorophylls only. The percentage of chloroplastic cadmium can be estimated as 0.21-1.32 % of all the Cd in a leaf. Photochemistry (Fv/Fm, ΦPSII, qP) was not influenced by Cd. Non-photochemical quenching of chlorophyll-excited state (NPQ) was greatly reduced in barley but not in maize. The decrease in NPQ was due to its fast relaxing component; the slow relaxing component rose slightly. In chloroplasts, Cd did not affect mRNA levels, but content of some photosynthetic proteins was reduced: slightly in the leaves of barley and heavily in the leaves of maize. In all analyzed C3-species, the effect of Cd on the content of photosynthetic proteins was mild or absent. This is most likely the first evidence of severe reduction of photosynthetic proteins in leaves of a Cd-treated C4-plant. © 2014 Springer Science+Business Media.


PubMed | Institute of Biophysics and Cell Engineering
Type: Journal Article | Journal: Neurochemical research | Year: 2015

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 compounds fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 M) and oligomycin (5 g/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.


PubMed | University of Lausanne and Institute of Biophysics and Cell Engineering
Type: | Journal: Neurochemistry international | Year: 2016

The ketogenic diet is used as a prophylactic treatment for different types of brain diseases, such as epilepsy or Alzheimers 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 -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 -hydroxybutyrate. However, in presence of -hydroxybutyrate, synaptic vesicle recycling was modified with reduced endocytosis. Replacing glucose by pyruvate also led to a reduced endocytosis. Addition of -hydroxybutyrate to glucose-containing incubation medium was without effect. Reduced endocytosis in presence of -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 -hydroxybutyrate did not modify the pH gradient of synaptic vesicles. In conclusion, the nonglycolytic energy substrates -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.


PubMed | Institute of Biophysics and Cell Engineering
Type: Journal Article | Journal: Neuroscience letters | Year: 2012

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 100M led to an increase of probe fluorescence pointing to free radical accumulation. This effect was mimicked by 100M of NMDA or 100M of kainate. Glutamate-induced ROS formation was sensitive to NMDA inhibitors MK-801 (10M), NO synthase (NOS) inhibitor l-NAME (100M) and NADPH oxidase inhibitors DPI (30M) and not affected by mitochondrial uncoupler CCCP (10M) and mitochondrial toxins rotenone (10M)+oligomycin (5g/ml). We also showed that 100M 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.

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