Synaptopathy under conditions of altered gravity: Changes in synaptic vesicle fusion and glutamate release

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1 h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO (10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[¹⁴C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca²⁺ or Mg²⁺/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.     

Docking and fusion of synaptic vesicles in cell-free model system of exocytosis

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca²⁺ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca²⁺-independent step, termed docking and followed by fusion step that is triggered by Ca²⁺. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.     

The geometry of diphtheria toxoid CRM197 channel assessed by thiazolium salts and nonelectrolytes

The geometry of the channel formed by nontoxic derivative of diphtheria toxin CRM197 in lipid bilayer was determined using the dependence of single-channel conductance upon the hydrodynamic radii of different nonelectrolytes. It was found that the cis entrance of CRM197 channel on the side of membrane to which the toxoid was added at pH 4.8 and the trans entrance on the opposite side at pH 6.0 had effective radii of 3.90 and 3.48 Å, respectively. The 3-alkyloxycarbonylmethyl-5-(2-hydroxyethyl)-4-methyl-1,3-thiazolium salts reversibly reduced current via CRM197 channels. The potency of the blockers increased with increasing length of alkyl chain at symmetric pH 6.0 and remained high and stable at pH 4.8 on the cis side. Comparative analysis of CRM197 and amphotericin B pore size with the inhibitory action of thiazolium salts revealed a significant increase in CRM197 pore dimension at pH 6.0. Addition of thiazolium salt with nine carbons alkyl tail increased by ～30% the viability of human carcinoma cells A431 treated with diphtheria toxin.     

Study of channel-forming properties of latrotoxin in liposomes

Interaction of latrotoxin with phospholipid vesicles and bilayer lipid membranes is shown to proceed differently. Latrotoxin when interacting with liposomes is sorbed on the membrane surface forming no ionic channels in this case. Only latrotoxin fragments obtained due to the toxin hydrolysis by pronase or trypsin are able to form channels. These fragments being inserted into liposomes are coupled strongly with the membrane and are not subjected to the further splitting by proteinases. The electrophoretic spectrum of peptides bound with liposomes is presented by protein components with a molecular weight of 116, 100, 92, 67, 52 and 45 kDa, while zone typical of latrotoxin is absent in this spectrum. The method of small-angle X-rays scattering has shown that tryptic fragments of latrotoxin penetrate into the phospholipid bilayer of liposomes.     

Sterol composition of Palaemon adspersus

The sterol composition of Palaemon adspersus is studied. It is established that the nonsaponifiable fraction of Palaemon adspersus contains cholesterol, 7-dehydrocholesterol, desmosterol, substance "240" and inconsiderable amounts of other still nonidentified sterols.     

Some characteristics of fusogenic activity of cytoplasmic latrotoxin-like brain protein

We studied fusion of negatively charged artificial phospholipid vesicles (liposomes) in the presence of two electrophoretic fractions (molecular mass of about 90 and 50 kdalton) of latrotoxin-like (L) protein. It was shown that both fractions are capable of causing liposome fusion in acidic media. Treatment of native preparations of L protein with NEM depressed their fusogenic activity. Some common characteristics of L protein and well-known fusogenic proteins allow us to account for the possibility of participation of L protein in fusion of the membranes in the cell.     

Calcium-triggered fusion of synaptic vesicles and synaptosomal plasma membranesin vitro

We have developed a system, in which fusion of synaptic vesicles with synaptosomal plasma membranes in the presence of synaptic soluble proteins can be studiedin vitro. We found that in this system micromolar concentrations of Ca²⁺ trigger fusion. The extent of fusion is insensitive to Ca²⁺ in millimolar concentrations, but can be covered by addition of MgATP. Ultimately, characterization of such cell-free systems makes it possible to identify biochemical events, which mediate and regulate these membrane fusion eventsin vivo.     

Fusion of synaptic vesicles and plasma membrane in the presence of synaptosomal soluble proteins

Fusion between synaptic vesicles and plasma membranes isolated from rat brain synaptosomes is regarded as a model of neurosecretion. The main aim of current study is to investigate whether the synaptosomal soluble proteins are essential members of Ca(2+)-triggered fusion examined in this system. Fusion experiments were performed using fluorescent dye octadecylrhodamine B, which was incorporated into synaptic vesicle membranes at self-quenching concentration. The fusion of synaptic vesicles, containing marker octadecylrhodamine B, with plasma membranes was detected by dequenching of the probe fluorescence. Membrane fusion was not found in Ca(2+)-supplemented buffer solution, but was initiated by the addition of the synaptosomal soluble proteins. When soluble proteins were treated with trypsin, they lost completely the fusion activity. These experiments confirmed that soluble proteins of synaptosomes are sensitive to Ca(2+) signal and essential for membrane fusion. The experiments, in which members of fusion process were treated with monoclonal antibodies raised against synaptotagmin and synaptobrevin, have shown that antibodies only partially inhibited fusion of synaptic vesicles and plasma membranes in vitro. These results indicate that other additional component(s), which may or may not be related to synaptobrevin or synaptotagmin, mediate this process. It can be assumed that fusion of synaptic vesicles with plasma membranes in vitro depends upon the complex interaction of a large number of protein factors.