L-type Ca2+ Channels at Low External Calcium Differentially Regulate Neurotransmitter Release in Proximal-Distal Compartments of the Frog Neuromuscular Junction.

L-type Ca2+ channels (LTCCs) are key elements in electromechanical coupling in striated muscles and formation of neuromuscular junctions (NMJs). However, the significance of LTCCs in regulation of neurotransmitter release is still far from understanding. Here, we found that LTCCs can increase evoked neurotransmitter release (especially asynchronous component) and spontaneous exocytosis in two functionally different compartment of the frog NMJ, namely distal and proximal parts. The effects of LTCC blockage on evoked and spontaneous release as well as timing of exocytotic events were prevented by inhibition of either protein kinase C (PKC) or P2Y receptors (P2Y-Rs). Hence, endogenous signaling via P2Y-R/PKC axis can sustain LTCC activity. Application of ATP, a co-neurotransmitter able to activate P2Y-Rs, suppressed both evoked and spontaneous exocytosis in distal and proximal parts. Surprisingly, inhibition of LTCCs (but not PKC) decreased the negative action of exogenous ATP on evoked (only in distal part) and spontaneous exocytosis. Lipid raft disruption suppressed (1) action of LTCC antagonist on neurotransmitter release selectively in distal region and (2) contribution of LTCCs in depressant effect of ATP on evoked and spontaneous release. Thus, LTCCs can enhance and desynchronize neurotransmitter release at basal conditions (without ATP addition), but contribute to ATP-mediated decrease in the exocytosis. The former action of LTCCs relies on P2Y-R/PKC axis, whereas the latter is triggered by exogenous ATP and PKC-independent. Furthermore, relevance of lipid rafts for LTCC function as well as LTCCs for ATP effects is different in distal and proximal part of the NMJ.

[Efficacy and safety of Kinezia (fampridine) in the complex therapy of multiple sclerosis]. / Effektivnost' i bezopasnost' preparata Kinezia (fampridin) v kompleksnoi terapii rasseyannogo skleroza.

OBJECTIVE: To analyze the efficacy and safety of fampridine** (Valenta Pharm, Russia) in the complex therapy of multiple sclerosis (MS). MATERIAL AND METHODS: One hundred and twenty-six patients with MS were double blind randomized to receive fampridine (n=60) or placebo (n=66). Fampridine was administered in prolonged-release form (film-coated tablets, 10 mg) at a dose of 10 mg (1 tablet) 2 times a day, for 24 weeks. The placebo group was treated in the same way. From the 12th week, all patients in the placebo group were transferred to therapy with fampridine, 10 mg 2 times a day, for another 12 weeks. Concomitant standard therapy for MS was allowed in both groups (concomitant disease-modifying medications and other treatment). The primary outcome in the study was the proportion of patients with reduced t25fw test time (determining walking speed on a 25-foot path) on at least two out of three visits compared to baseline. The mean change in Multiple Sclerosis Functional Composite (MSFC) scores from baseline was assessed at visits 4-7 (8-24 weeks). RESULTS: The proportion of patients with reduced t25fw test time compared to the baseline level was 31.7% in the fampridine group, which is higher than in the placebo group - 3.0% (p<0.001). The overall result of the Multiple Sclerosis Functional Composite (MSFC) reflected a gradual improvement in the patient's condition during treatment period. The dynamics of MSFC result relative to the baseline level significantly differed (p<0.05) between the fampridine and placebo groups in favor of the fampridine group during all treatment periods. In the fampridine group, adverse events (AE) associated with disorders of the nervous system were more common: headache, dizziness, and coordination disorders. CONCLUSIONS: Fampridine improves walking performance in MS patients. The Russian product fampridine has demonstrated a favorable safety profile.

Cadmium desynchronizes neurotransmitter release in the neuromuscular junction: Key role of ROS.

Cd2+ is one of the most widespread environmental pollutants and its accumulation in central and peripheral nervous systems leads to neurotoxicity as well as aggravation of common neurodegenerative diseases. Mechanism of the Cd2+ toxicity is far from being resolved. Here, using microelectrode recordings of postsynaptic responses and fluorescent redox indicators we studied the effect of Cd2+ in the submicromolar range on timing of neurotransmitter release and oxidative status in two functionally different compartments of the same frog motor nerve terminal. Cd2+ (0.1-1 µM) acting as typical voltage-gated Ca2+channel (VGCC) antagonist decreased neurotransmitter release in both distal and proximal parts of the nerve terminal, but in contrast to the VGCC blockers Cd2+(0.1-0.5 µM) desynchronized the release selectively in the distal region. The latter action of Cd2+ was completely prevented by inhibitor of NADPH-oxidase and antioxidants, including mitochondrial specific, as well as redox-sensitive TRPV1 channel blocker. Cd2+ markedly increased levels of mitochondrial reactive oxygen species (ROS) in both the distal and proximal compartments of the nerve terminal, which was associated with lipid peroxidation mainly in the distal region. Zn2+, whose transport systems translocate Cd2+, markedly enhanced the effects of Cd2+ on both the mitochondrial ROS levels and timing of neurotransmitter release. Furthermore, in the presence of Zn2+ ions, Cd2+ also desynchronized the neurotransmitter release in the proximal region. Thus, in synapses Cd2+ at very low concentrations can increase mitochondrial ROS, lipid peroxidation and disturb the timing of neurotransmitter release via a ROS/TRPV-dependent mechanism. Desynchronization of neurotransmitter release and synaptic oxidative stress could be early events in Cd2+ neurotoxicity.

Intracellular Acidification Suppresses Synaptic Vesicle Mobilization in the Motor Nerve Terminals.

Intracellular protons play a special role in the regulation of presynaptic processes, since the functioning of synaptic vesicles and endosomes depends on their acidification by the H+-pump. Furthermore, transient acidification of the intraterminal space occurs during synaptic activity. Using microelectrode recording of postsynaptic responses (an indicator of neurotransmitter release) and exo-endocytic marker FM1-43, we studied the effects of intracellular acidification with propionate on the presynaptic events underlying neurotransmitter release. Cytoplasmic acidification led to a marked decrease in neurotransmitter release during the first minute of a 20-Hz stimulation in the neuromuscular junctions of mouse diaphragm and frog cutaneous pectoris muscle. This was accompanied by a reduction in the FM1-43 loss during synaptic vesicle exocytosis in response to the stimulation. Estimation of the endocytic uptake of FM1-43 showed no disruption in synaptic vesicle endocytosis. Acidification completely prevented the action of the cell-membrane permeable compound 24-hydroxycholesterol, which can enhance synaptic vesicle mobilization. Thus, the obtained results suggest that an increase in [H+]in negatively regulates neurotransmission due to the suppression of synaptic vesicle delivery to the sites of exocytosis at high activity. This mechanism can be a part of the negative feedback loop in regulating neurotransmitter release.

Cholesterol in the Pathogenesis of Alzheimer's, Parkinson's Diseases and Autism: Link to Synaptic Dysfunction.

In our previous review, we described brain cholesterol metabolism in control conditions and in the case of some rare neurological pathologies linked to defects in the genes which are directly involved in the synthesis and/or traffic of cholesterol. Here, we have analyzed disruptions in cholesterol homeostasis in widespread neurodegenerative diseases (Alzheimer's and Parkinson's diseases) and autism spectrum disorders. We particularly focused on the synaptic dysfunctions that could arise from changes in both membrane cholesterol availability and oxysterol production. Notably, alterations in the brain cholesterol metabolism and neurotransmission occur in the early stages of these pathologies and the polymorphism of the genes associated with cholesterol homeostasis and synaptic communication affects the risk of onset and severity of these diseases. In addition, pharmacological and genetic manipulations of brain cholesterol homeostasis in animal models frequently have marked effects on the progression of neurodegenerative diseases. Thus, the development of Alzheimer's, Parkinson's and autism spectrum disorders may be partially associated with an imbalance of cholesterol homeostasis that leads to changes in the membrane cholesterol and oxysterol levels that, in turn, modulates key steps in the synaptic transmission.

24S-Hydroxycholesterol enhances synaptic vesicle cycling in the mouse neuromuscular junction: Implication of glutamate NMDA receptors and nitric oxide.

24S-hydroxycholesterol (24S-HC) is a brain-derived product of lipid metabolism present in the systemic circulation, where its level can change significantly in response to physiological and pathophysiological conditions. Here, using electrophysiological and optical approaches, we have found a high sensitivity to 24S-HC of the synaptic vesicle cycle at the mouse neuromuscular junctions. Treatment with 24S-HC increased the end plate potential amplitude (EPP) in response to a single stimulus and attenuated the EPP amplitude rundown during high frequency (HF) activity but had no influence on miniature EPP amplitude or frequency. The effects on evoked responses were associated with enhanced FM1-43 dye loading and unloading by endo- and exocytosis. Comparison of electrophysiological and optical data revealed an increase in the rate of vesicular cycling. The impact of 24S-HC was abolished or potentiated by stimulation or inhibition of NMDA-receptors respectively. Moreover, 24S-HC, acting in the same manner as the endothelial NO synthase (eNOS) inhibitor cavtratin, suppressed an increase in NO-sensitive dye fluorescence during HF stimulation, while l-glutamate had the opposite effect. Inhibitors of NOS (l-NAME and cavtratin, but not the neuronal NOS inhibitor TRIM), a scavenger of extracellular NO and a protein kinase G blocker all had stimulatory effects, similar to those of 24S-HC, on exocytosis induced by HF activity and completely masked the effect of 24S-HC. The data suggest that 24S-HC enhances synaptic vesicle cycling due to an attenuation of retrograde NO signaling that depends on eNOS. In this regard, 24S-HC counteracts the effects of NMDA-receptor stimulation at mouse neuromuscular junctions.

[Capabilities of positron emission tomography to study mecha-nisms of multiple sclerosis: own data and literature]. / Vozmozhnosti pozitronno-emissionnoi tomografii dlya izucheniya mekhanizmov razvitiya rasseyannogo skleroza (literaturnye i sobstvennye dannye).

The article presents the literature data and results of our own researchon the use of positron emission tomography (PET) with different radiotracersin multiple sclerosis (MS). Informationon the operating principles of PET and PET studies with different radiotracers are considered. The results of PET studiesin different typesof MS, including determinationof the localization of neuronal damagein the corticalgray matter, assessmentof microglial activation, study of the relationship between glucose metabolismin the brain and the severity of cognitive impairmentin MS, can providenew information about the pathogenesis ofMS.

Brain Cholesterol Metabolism and Its Defects: Linkage to Neurodegenerative Diseases and Synaptic Dysfunction.

Cholesterol is an important constituent of cell membranes and plays a crucial role in the compartmentalization of the plasma membrane and signaling. Brain cholesterol accounts for a large proportion of the body's total cholesterol, existing in two pools: the plasma membranes of neurons and glial cells and the myelin membranes . Cholesterol has been recently shown to be important for synaptic transmission, and a link between cholesterol metabolism defects and neurodegenerative disorders is now recognized. Many neurodegenerative diseases are characterized by impaired cholesterol turnover in the brain. However, at which stage the cholesterol biosynthetic pathway is perturbed and how this contributes to pathogenesis remains unknown. Cognitive deficits and neurodegeneration may be associated with impaired synaptic transduction. Defects in cholesterol biosynthesis can trigger dysfunction of synaptic transmission. In this review, an overview of cholesterol turnover under physiological and pathological conditions is presented (Huntington's, Niemann-Pick type C diseases, Smith-Lemli-Opitz syndrome). We will discuss possible mechanisms by which cholesterol content in the plasma membrane influences synaptic processes. Changes in cholesterol metabolism in Alzheimer's disease, Parkinson's disease, and autistic disorders are beyond the scope of this review and will be summarized in our next paper.

Similar oxysterols may lead to opposite effects on synaptic transmission: Olesoxime versus 5α-cholestan-3-one at the frog neuromuscular junction.

Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle.

Effects of 5α-cholestan-3-one on the synaptic vesicle cycle at the mouse neuromuscular junction.

We have investigated the effects of 5α-cholesten-3-one (5Ch3, 200 nM) on synaptic transmission in mouse diaphragm. 5Ch3 had no impact on the amplitude or frequency of miniature endplate currents (MEPCs, spontaneous secretion), but decreased the amplitude of EPCs (evoked secretion) triggered by single action potentials. Treatment with 5Ch3 increased the depression of EPC amplitude and slowed the unloading of the dye FM1-43 from synaptic vesicles (exocytosis rate) during high-frequency stimulation. The estimated recycling time of vesicles did not change, suggesting that the decline of synaptic efficiency was due to the reduction in the size of the population of vesicles involved in release. The effects of 5Ch3 on synaptic transmission may be related to changes in the phase properties of the membrane. We have found that 5Ch3 reduces the staining of synaptic regions with the B-subunit of cholera toxin (a marker of lipid rafts) and increases the fluorescence of 22-NBD-cholesterol, indicating a phase change within the membrane. Manipulations of membrane cholesterol (saturation or depletion) strongly reduced the influence of 5Ch3 on both FM1-43 dye unloading and staining with the B-subunit of cholera toxin. Thus, 5Ch3 reduces the number of vesicles which are actively recruited during synaptic transmission and alters membrane properties. These effects of 5Ch3 depend on membrane cholesterol.

Role of cholesterol in the maintenance of endplate electrogenesis in rat diaphragm.

Methyl-ß-cyclodextrin (0.1 mM) reduced resting potential of muscle fibers and abolished local endplate membrane hyperpolarization in rat diaphragm. This effect was associated with selective reduction of electrogenic activity of α2-isoform of Na,K-ATPase without changes in the level of intracellular acetylcholine. Experiments with cholesterol marker filipin showed that methyl-ß-cyclodextrin in this dose induced cholesterol translocation from lipid rafts to liquid phase of the membrane without its release into extracellular space. This modification of lipid rafts by methyl-ß-cyclodextrin presumably impaired the mechanism maintaining electrogenesis in endplates mediated by modulation of Na,K-ATPase by non-quantum acetylcholine. Cholesterol can serve as a molecular component of this mechanism.

[The relationship between cognitive impairment and localization of white matter lesions in patients with multiple sclerosis]. / Sviaz' kognitivnykh narushenii s lokalizatsiei ochagov porazheniia belogo veshchestva golovnogo mozga u bol'nykh rasseiannym sklerozom.

Objective. To study a role of parenchymal white matter lesions and lesions of the white matter underlying the cortical grey matter in cognitive impairment in patients with multiple sclerosis (MS). Material and methods. We examined 33 patients with clinically definite RRMS in clinical remission. Cognitive performance was assessed with neuropsychological and psychometric tests. MRI (1.5 tesla) was used to assess volume and localization of focal demyelination in the cerebral cortex. Results. The volume of juxtacortical brain lesions in the white matter had a negative effect on the correct performance on neuropsychological tests. The volume of parenchymal white matter lesions had a negative effect on the speed of task performance. Conclusion. The results suggest that MS patients had multiple cognitive impairments.

[Cholesterol and lipid rafts in the biological membranes. Role in the release, reception and ion channel functions].

Traditionally, membrane protein molecules that form ion channels, transporters, pumps, signaling complexes, machine of exo- and endocytosis is assigned as the main players of the cellular processes. Recently, the findings that indicate the importance of lipids in regulating of cell physiology are accumulated. Attention is attracting to cholesterol molecule because it can directly interact with different proteins and together with sphingolipids to form membrane microdomains (lipid rafts). Many receptors (for neurotransmitters, hormones, growth factors), signaling proteins and proteins involved in vesicular and ion transport are concentrated in the lipid rafts. Changes in stability and structure of rafts cause dramatic cellular dysfunction. In the review the current views on lipid variants that make up the biological membrane, the distribution of cholesterol, the organization and the formation of lipid rafts and caveolae are described. Accent is made on researches that focus on the significance of lipid rafts in the extra- and intracellular signaling, neurotransmitters release, receptor and ion channels function at the excitable cells.

[Membrane cholesterol oxidation effects on synaptic vesicle cycle in frog (RANA ridibunda) motor nerve terminals].

In experiments on frog (Rana ridibunda) neuromuscular junction the influence of cholesterol oxidation on the presynaptic vesicular cycle was investigated. Application of cholesterol oxidase (1 u. a.) during 1/2 hour led to the oxidation of - 0.007 mg cholesterol per 1 g tissue and reduced stability of lipid rafts in the nerve terminals. Using electrophysiological techniques it was shown that the cholesterol oxidation decreases the evoked neurotransmitter release. In experiments with fluorescent FM-dyes the depression of the synaptic vesicles exo-endocytosis and the dispersion of synaptic vesicles clusters were revealed. Comparative analysis of electrophysiological and optical data, as well as experiments with water soluble quencher of FM-dye indicated the possibility of some neurotransmitter release by "kiss-and-run" pathway, when short-lived fusion pore is formed. It was concluded that cholesterol oxidation inhibit evoked exocytosis, and also synaptic vesicle delivery from reserve pool to cites of exocytosis probably by break of the clusterization. Perhaps the synaptic vesicles of recycling pool release the neurotransmitter using the kiss-and-run mechanism.

[Neuroimmunology: theoretical and clinical aspects].

The aspects of neuroimmune interactions (during cerebral ischemia and stroke--experimental and clinical data, demyelinating process in patients with multiple sclerosis,secondary immunodeficiency) are discussed in this article. Literature overview and authors' experience are presented as well.

[Teriflunomide is a new peroral disease-modifying drug for multiple sclerosis (a review)].

Teriflunomide is a once-daily, orally administered, disease-modifying drug. The review presents the data concerning the mechanism of action, pharmacokinetics as well as results of clinical studies. Teriflunomide has received the regulatory approval in the USA, Argentina, Australia, South Korea, Chile and EU. The submission of a regulatory dossier is performed in Russia.

Depolarization-induced calcium-independent synaptic vesicle exo- and endocytosis at frog motor nerve terminals.

The transmitter release and synaptic vesicle exo- and endocytosis induced by constant current depolarization of nerve terminals were studied by microelectode extracellular recording of miniature endplate currents and fluorescent microscopy (FM 1-43 styryl dye). Depolarization of the plasma membrane of nerve terminals in the control specimen was shown to significantly increase the MEPC frequency (quantal transmitter release) and exocytotic rate (FM 1-43 unloading from the synaptic vesicles preliminarily stained with the dye), which was caused by a rise in the intracellular Ca(2+) concentration due to opening of voltage-gated Ca channels. A slight increase in the MEPC frequency and in the rate of synaptic vesicle exocytosis was observed under depolarization in case of blockade of Ca channels and chelating of intracellular Ca(2+) ions (cooperative action of Cd(2+) and EGTA-AM). The processes of synaptic vesicle endocytosis (FM 1-43 loading) were proportional to the number of synaptic vesicles that had undergone exocytosis both in the control and in case of cooperative action of Cd(2+) and EGTA-AM. A hypothesis has been put forward that Ca-independent synaptic vesicle exo- and endocytosis that can be induced directly by depolarization of the membrane exists in the frog motor terminal in addition to the conventional Ca-dependent process.