Roman Sivko

Cholesterol depletion attenuates tonic release but increases the ambient level of glutamate in rat brain synaptosomes.

The low level of ambient glutamate is important for the brains spontaneous activity and proper synaptic transmission. Cholesterol deficiency has been implicated in the pathogenesis of several neurodegenerative disorders. It was examined whether membrane cholesterol modulated the extracellular glutamate level in nerve terminals and the processes responsible for its maintenance. The ambient L-[(14)C]glutamate level, being an equilibrium between Na(+)-dependent uptake and tonic release, was increased from 0.193+/-0.013 nmol/mg protein to 0.282+/-0.013 (extracellular endogenous glutamate-from 6.9+/-2.0 to 16.6+/-2.0, respectively) in rat brain synaptosomes treated with a cholesterol acceptor methyl-beta-cyclodextrin (MbetaCD). This alteration was not due to the change in the activity of glutamine synthetase that was shown with the specific blocker L-methionine sulfoximine. In the presence of DL-threo-beta-benzyloxyaspartate, which significantly reduced the contribution of glutamate transporters, net tonic release of L-[(14)C]glutamate was decreased by 38% and release in low-Na(+) medium was attenuated by 41% after cholesterol extraction. Also, cholesterol-deficient synaptosomes showed a reduced content of cytosolic L-[(14)C]glutamate and a lower initial velocity of L-[(14)C]glutamate uptake. We suggested that cholesterol deficiency altered the intra-to-extracellular glutamate ratio by the reduction of the cytosolic level of the neurotransmitter and the augmentation of the ambient glutamate level, thereby favoring a decrease in tonic glutamate release. Thus, increased extracellular glutamate in cholesterol-deficient nerve terminals was not a result of the changes in tonic release and/or glutamine synthetase activity, but was set by lack function of glutamate transporters.

Presynaptic malfunction: the neurotoxic effects of cadmium and lead on the proton gradient of synaptic vesicles and glutamate transport.

Exposure to Cd(2+) and Pb(2+) has neurotoxic consequences for human health and may cause neurodegeneration. The study focused on the analysis of the presynaptic mechanisms underlying the neurotoxic effects of non-essential heavy metals Cd(2+) and Pb(2+). It was shown that the preincubation of rat brain nerve terminals with Cd(2+) (200 μM) or Pb(2+) (200 μM) resulted in the attenuation of synaptic vesicles acidification, which was assessed by the steady state level of the fluorescence of pH-sensitive dye acridine orange. A decrease in L-[(14)C]glutamate accumulation in digitonin-permeabilized synaptosomes after the addition of the metals, which reflected lowered L-[(14)C]glutamate accumulation by synaptic vesicles inside of synaptosomes, may be considered in the support of the above data. Using isolated rat brain synaptic vesicles, it was found that 50 μM Cd(2+) or Pb(2+) caused dissipation of their proton gradient, whereas the application of essential heavy metal Mn(2+) did not do it within the range of the concentration of 50-500 μM. Thus, synaptic malfunction associated with the influence of Cd(2+) and Pb(2+) may result from partial dissipation of the synaptic vesicle proton gradient that leads to: (1) a decrease in stimulated exocytosis, which is associated not only with the blockage of voltage-gated Ca(2+) channels, but also with incomplete filling of synaptic vesicles; (2) an attenuation of Na(+)-dependent glutamate uptake.

Neuromodulatory properties of fluorescent carbon dots: effect on exocytotic release, uptake and ambient level of glutamate and GABA in brain nerve terminals.

Carbon dots (C-dots), a recently discovered class of fluorescent nano-sized particles with pure carbon core, have great bioanalytical potential. Neuroactive properties of fluorescent C-dots obtained from β-alanine by microwave heating were assessed based on the analysis of their effects on the key characteristics of GABA- and glutamatergic neurotransmission in isolated rat brain nerve terminals. It was found that C-dots (40-800 μg/ml) in dose-dependent manner: (1) decreased exocytotic release of [(3)H]GABA and L-[(14)C]glutamate; (2) reduced acidification of synaptic vesicles; (3) attenuated the initial velocity of Na(+)-dependent transporter-mediated uptake of [(3)H]GABA and L-[(14)C]glutamate; (4) increased the ambient level of the neurotransmitters, nevertheless (5) did not change significantly the potential of the plasma membrane of nerve terminals. Almost complete suppression of exocytotic release of the neurotransmitters was caused by C-dots at a concentration of 800 μg/ml. Fluorescent and neuromodulatory features combined in C-dots create base for their potential usage for labeling and visualization of key processes in nerve terminals, and also in theranostics. In addition, natural presence of carbon-containing nanoparticles in the human food chain and in the air may provoke the development of neurologic consequences.

Cholesterol Depletion from the Plasma Membrane Impairs Proton and Glutamate Storage in Synaptic Vesicles of Nerve Terminals

We report that cholesterol depletion with methyl-β-cyclodextrin (MβCD) acutely applied to rat brain synaptosomes is accompanied by an immediate increase in transporter-mediated glutamate release and decrease in exocytotic release. To clarify the possible mechanisms underlying these phenomena, we investigated the influence of MβCD on synaptic vesicle acidification and exo/endocytotic process in nerve terminals. As shown by acridine orange fluorescence measurements, the application of MβCD to synaptosomes, as well as to isolated synaptic vesicles, led to the gradual leakage of the protons from the vesicles, whereas the application of MβCD complexed with cholesterol stimulated additional vesicle acidification and an increase in Ca2+-dependent exocytotic response. It was found that the treatment of nerve terminals with MβCD did not block Ca2+-triggered vesicle recycling. We suggest that cholesterol depletion of the plasma membrane with MβCD induces the removal of cholesterol from the membrane of synaptic vesicles resulting in immediate dissipation of synaptic vesicle proton gradient and redistribution of the neurotransmitter between the vesicular and cytosolic pools. The latter appears to be the main cause of a dramatic decrease in exocytotic and considerable increase in transporter-mediated release of l-[14C]glutamate.

Manipulation of isolated brain nerve terminals by an external magnetic field using D-mannose-coated γ-Fe2O3 nano-sized particles and assessment of their effects on glutamate transport

Summary The manipulation of brain nerve terminals by an external magnetic field promises breakthroughs in nano-neurotechnology. D-Mannose-coated superparamagnetic nanoparticles were synthesized by coprecipitation of Fe(II) and Fe(III) salts followed by oxidation with sodium hypochlorite and addition of D-mannose. Effects of D-mannose-coated superparamagnetic maghemite (γ-Fe2O3) nanoparticles on key characteristics of the glutamatergic neurotransmission were analysed. Using radiolabeled L-[14C]glutamate, it was shown that D-mannose-coated γ-Fe2O3 nanoparticles did not affect high-affinity Na+-dependent uptake, tonic release and the extracellular level of L-[14C]glutamate in isolated rat brain nerve terminals (synaptosomes). Also, the membrane potential of synaptosomes and acidification of synaptic vesicles was not changed as a result of the application of D-mannose-coated γ-Fe2O3 nanoparticles. This was demonstrated with the potential-sensitive fluorescent dye rhodamine 6G and the pH-sensitive dye acridine orange. The study also focused on the analysis of the potential use of these nanoparticles for manipulation of nerve terminals by an external magnetic field. It was shown that more than 84.3 ± 5.0% of L-[14C]glutamate-loaded synaptosomes (1 mg of protein/mL) incubated for 5 min with D-mannose-coated γ-Fe2O3 nanoparticles (250 µg/mL) moved to an area, in which the magnet (250 mT, gradient 5.5 Т/m) was applied compared to 33.5 ± 3.0% of the control and 48.6 ± 3.0% of samples that were treated with uncoated nanoparticles. Therefore, isolated brain nerve terminals can be easily manipulated by an external magnetic field using D-mannose-coated γ-Fe2O3 nanoparticles, while the key characteristics of glutamatergic neurotransmission are not affected. In other words, functionally active synaptosomes labeled with D-mannose-coated γ-Fe2O3 nanoparticles were obtained.

Neurotoxic Potential of Lunar and Martian Dust: Influence on Em, Proton Gradient, Active Transport, and Binding of Glutamate in Rat Brain Nerve Terminals

The harmful effects of lunar dust (LD) on directly exposed tissues are documented in the literature, whereas researchers are only recently beginning to consider its effects on indirectly exposed tissues. During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and transported to the central nervous system. The neurotoxic potential of LD and martian dust (MD) has not yet been assessed. Glutamate is the main excitatory neurotransmitter involved in most aspects of normal brain function, whereas disturbances in glutamate homeostasis contribute to the pathogenesis of major neurological disorders. The research was focused on the analysis of the effects of LD/MD simulants (JSC-1a/JSC, derived from volcanic ash) on the key characteristics of glutamatergic neurotransmission. The average size of LD and MD particles (even minor fractions) before and after sonication was determined by dynamic light scattering. With the use of radiolabeled l-[(14)C]glutamate, it was shown that there is an increase in l-[(14)C]glutamate binding to isolated rat brain nerve terminals (synaptosomes) in low [Na(+)] media and at low temperature in the presence of LD. MD caused significantly lesser changes under the same conditions, whereas nanoparticles of magnetite had no effect at all. Fluorimetric experiments with potential-sensitive dye rhodamine 6G and pH-sensitive dye acridine orange showed that the potential of the plasma membrane of the nerve terminals and acidification of synaptic vesicles were not altered by LD/MD (and nanoparticles of magnetite). Thus, the unique effect of LD to increase glutamate binding to the nerve terminals was shown. This can have deleterious effects on extracellular glutamate homeostasis in the central nervous system and cause alterations in the ambient level of glutamate, which is extremely important for proper synaptic transmission. During a long-term mission, a combination of constant irritation due to dust particles, inflammation, stress, low gravity and microgravity, radiation, UV, and so on may consequently change the effects of the dust and aggravate neurological consequences.

Vitamin D3 deficiency in puberty rats causes presynaptic malfunctioning through alterations in exocytotic release and uptake of glutamate/GABA and expression of EAAC-1/GAT-3 transporters

Recent experimental and epidemiologic investigations have revealed that the central nervous system is a target for vitamin D3 action and also linked vitamin D3 deficiency to Alzheimers and Parkinsons disease, autism and dementia. Abnormal homeostasis of glutamate and GABA and signaling disbalance are implicated in the pathogenesis of major neurological diseases. Here, key transport characteristics of glutamate and GABA were analysed in presynaptic nerve terminals (synaptosomes) isolated from the cortex of vitamin D3 deficient (VDD) rats. Puberty rats were kept at the VDD diet up to adulthood. VDD caused: (i) a decrease in the initial rates of L-[14C]glutamate and [3H]GABA uptake by plasma membrane transporters of nerve terminals; (ii) a decrease in exocytotic release of L-[14C]glutamate and [3H]GABA; (iii) changes in expression of glutamate (EAAC-1) and GABA (GAT-3) transporters. Whereas, the synaptosomal ambient levels and Ca2+-independent transporter-mediated release of L-[14C]glutamate and [3H]GABA were not significantly altered in VDD. Vitamin D3 is a potent neurosteroid and its nutritional deficiency can provoke development of neurological consequences changing glutamate/GABA transporter expressions and excitation/inhibition balance. Also, changes in glutamate transport can underlie lower resistance to hypoxia/ischemia, larger infarct volumes and worsened outcomes in ischemic stroke patients with VDD.