ROS-producing nanomaterial engineered from Cu(I) complexes with P2N2-ligands for cancer cells treating.

The work presents core-shell nanoparticles (NPs) built from the novel Cu(I) complexes with cyclic P2N2-ligands (1,5-diaza-3,7-diphosphacyclooctanes) that can visualize their entry into cancer and normal cells using a luminescent signal and treat cells by self-enhancing generation of reactive oxygen species (ROS). Variation of P- and N-substituents in the series of P2N2-ligands allows structure optimization of the Cu(I) complexes for the formation of the luminescent NPs with high chemical stability. The non-covalent modification of the NPs with triblock copolymer F-127 provides their high colloidal stability, followed by efficient cell internalization of the NPs visualized by their blue (⁓450 nm) luminescence. The cytotoxic effects of the NPs toward the normal and some of cancer cells are significantly lower than those of the corresponding molecular complexes, which correlates with the chemical stability of the NPs in the solutions. The ability of the NPs to self-enhanced and H2O2-induced ROS generation is demonstrated in solutions and intracellular space by means of the standard electron spin resonance (ESR) and fluorescence techniques correspondingly. The anticancer specificity of the NPs toward HuTu 80 cancer cells and the apoptotic cell death pathway correlate with the intracellular level of ROS, which agrees well with the self-enhancing ROS generation of the NPs. The enhanced level of ROS revealed in HuTu 80 cells incubated with the NPs can be associated with the significant level of their mitochondrial localization.

Activation of TRPV1 Channels Inhibits the Release of Acetylcholine and Improves Muscle Contractility in Mice.

TRPV1 represents a non-selective transient receptor potential cation channel found not only in sensory neurons, but also in motor nerve endings and in skeletal muscle fibers. However, the role of TRPV1 in the functioning of the neuromuscular junction has not yet been fully established. In this study, the Levator Auris Longus muscle preparations were used to assess the effect of pharmacological activation of TRPV1 channels on neuromuscular transmission. The presence of TRPV1 channels in the nerve terminal and in the muscle fiber was confirmed by immunohistochemistry. It was verified by electrophysiology that the TRPV1 channel agonist capsaicin inhibits the acetylcholine release, and this effect was completely absent after preliminary application of the TRPV1 channel blocker SB 366791. Nerve stimulation revealed an increase of amplitude of isometric tetanic contractions upon application of capsaicin which was also eliminated after preliminary application of SB 366791. Similar data were obtained during direct muscle stimulation. Thus, pharmacological activation of TRPV1 channels affects the functioning of both the pre- and postsynaptic compartment of the neuromuscular junction. A moderate decrease in the amount of acetylcholine released from the motor nerve allows to maintain a reserve pool of the mediator to ensure a longer signal transmission process, and an increase in the force of muscle contraction, in its turn, also implies more effective physiological muscle activity in response to prolonged stimulation. This assumption is supported by the fact that when muscle was indirect stimulated with a fatigue protocol, muscle fatigue was attenuated in the presence of capsaicin.

Early Alterations in Structural and Functional Properties in the Neuromuscular Junctions of Mutant FUS Mice.

Amyotrophic lateral sclerosis (ALS) is manifested as skeletal muscle denervation, loss of motor neurons and finally severe respiratory failure. Mutations of RNA-binding protein FUS are one of the common genetic reasons of ALS accompanied by a 'dying back' type of degeneration. Using fluorescent approaches and microelectrode recordings, the early structural and functional alterations in diaphragm neuromuscular junctions (NMJs) were studied in mutant FUS mice at the pre-onset stage. Lipid peroxidation and decreased staining with a lipid raft marker were found in the mutant mice. Despite the preservation of the end-plate structure, immunolabeling revealed an increase in levels of presynaptic proteins, SNAP-25 and synapsin 1. The latter can restrain Ca2+-dependent synaptic vesicle mobilization. Indeed, neurotransmitter release upon intense nerve stimulation and its recovery after tetanus and compensatory synaptic vesicle endocytosis were markedly depressed in FUS mice. There was a trend to attenuation of axonal [Ca2+]in increase upon nerve stimulation at 20 Hz. However, no changes in neurotransmitter release and the intraterminal Ca2+ transient in response to low frequency stimulation or in quantal content and the synchrony of neurotransmitter release at low levels of external Ca2+ were detected. At a later stage, shrinking and fragmentation of end plates together with a decrease in presynaptic protein expression and disturbance of the neurotransmitter release timing occurred. Overall, suppression of synaptic vesicle exo-endocytosis upon intense activity probably due to alterations in membrane properties, synapsin 1 levels and Ca2+ kinetics could be an early sign of nascent NMJ pathology, which leads to neuromuscular contact disorganization.

Presynaptic Acetylcholine Receptors Modulate the Time Course of Action Potential-Evoked Acetylcholine Quanta Secretion at Neuromuscular Junctions.

For effective transmission of excitation in neuromuscular junctions, the postsynaptic response amplitude must exceed a critical level of depolarization to trigger action potential spreading along the muscle-fiber membrane. At the presynaptic level, the end-plate potential amplitude depends not only on the acetylcholine quanta number released from the nerve terminals in response to the nerve impulse but also on a degree of synchronicity of quanta releases. The time course of stimulus-phasic synchronous quanta secretion is modulated by many extra- and intracellular factors. One of the pathways to regulate the neurosecretion kinetics of acetylcholine quanta is an activation of presynaptic autoreceptors. This review discusses the contribution of acetylcholine presynaptic receptors to the control of the kinetics of evoked acetylcholine release from nerve terminals at the neuromuscular junctions. The timing characteristics of neurotransmitter release is nowadays considered an essential factor determining the plasticity and efficacy of synaptic transmission.

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy.

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

Structure impact on photodynamic therapy and cellular contrasting functions of colloids constructed from dimeric Au(I) complex and hexamolybdenum clusters.

Electrostatically driven self-assembly of [Au2L2]2+ (L is cyclic PNNP ligand) with [{Mo6I8}(L')6]2- (L' = I-, CH3COO-) in aqueous solutions is introduced as facile route for combination of therapeutic and cellular contrasting functions within heterometallic colloids (Mo6-Au2). The nature of L' affects the size and aggregation behavior of crystalline Mo6-Au2 aggregates, which in turn affect the luminescence of the cluster units incorporated into Mo6-Au2 colloids. The spin trap facilitated electron spin resonance spectroscopy technique indicates that the level of ROS generated by Mo6-Au2 colloids is also affected by their size. Both (L' = I-, CH3COO-) Mo6-Au2 colloids undergo cell internalization, which is enhanced by their assembly with poly-DL-lysine (PL) for L' = CH3COO-, but remains unchanged for L' = I-. The colloids PL-Mo6-Au2 (L' = CH3COO-) are visualized as huge crystalline aggregates both outside and inside the cell cytoplasm by confocal microscopy imaging of the incubated cells, while the smaller sized (30-50 nm) PL-Mo6-Au2 (L' = I-) efficiently stain the cell nuclei. Quantitative colocalization analysis of PL-Mo6-Au2 (L' = CH3COO-) in lysosomal compartments points to the fast endo-lysosomal escape of the colloids followed by their intracellular aggregation. The cytotoxicity of PL-Mo6-Au2 differs from that of Mo6 and Au2 blocks, predominantly acting through apoptotic pathway. The photodynamic therapeutic effect of the PL-Mo6-Au2 colloids on the cancer cells correlates with their intracellular trafficking and aggregation.

Mitochondria-targeted mesoporous silica nanoparticles noncovalently modified with triphenylphosphonium cation: Physicochemical characteristics, cytotoxicity and intracellular uptake.

Novel nanocomposite system based on mesoporous silica nanoparticles (MSNs) noncovalently modified with hexadecyltriphenylphosphonium bromide (HTPPB) has been prepared, thoroughly characterized and used for encapsulation of model cargo Rhodamine B (RhB). The high encapsulation efficacy of this dye by HTPPB-modified mesoporous particles was demonstrated by spectrophotometry and thermography techniques. The bioavailability of MSN@HTPPB was testified. Cytotoxicity assay revealed that a marked suppression of M-HeLa cancer cells (epithelioid carcinoma of the cervix) occurs at concentration of 0.06 µg/mL, while the higher viability of Chang liver normal cell line was preserved in the concentration range of 0.98-0.06 µg/mL. Hemolysis assay demonstrated that only 2% of red blood cells are destructed at ~ 30 µg/mL concentration. This allows us to select the most harmless compositions based on MSN@HTPPB with minimal side effects toward normal cells and recommend them for the development of antitumor formulations. Fluorescence microscopy technique testified satisfactory penetration of HTPPB-modified carriers into M-HeLa cells. Importantly, modification of the MSN with HTPPB is shown to promote efficient delivery to mitochondria. To the best of our knowledge, it is one of the first successful examples of noncovalent surface modification of the MSNs with lipophilic phosphonium cation that improves targeted delivery of loads to mitochondria.

Enhancement of the Temporal Resolution of Fluorescent Signals Acquired by the Confocal Microscope.

Here, we describe a method of acquisition of fast fluorescent signals with the help of the laser scanning confocal microscope (LSCM). Our method permits an increase in the temporal resolution of acquired signals. The method is based on LSCM recordings of fast fluorescent signals with the shortest achievable time sweep, which are performed with the help of a proprietary algorithm. A series of recordings is made in multiple steps; at each step, the fluorescent signal is incremented by a time interval smaller than the time sweep of the frame of LSCM. The size of the increment determines the achievable time resolution. The convolution of the recorded images results in a signal with the temporal resolution determined by the chosen time increment. This method was applied to register the change in fluorescence (calcium transient) of calcium dye preloaded into peripheral nerve endings by electrical stimulation of the motor nerve. Calculated parameters of the calcium transient were identical to the parameters obtained earlier with the help of a high-speed camera and photodiode. We conclude that the method described here can be applied for the registration of fast fluorescent signals by LSCM with a high spatial and temporal resolution.

CLARITY analysis of the Cl/pH sensor expression in the brain of transgenic mice.

Genetically encoded biosensors are widely used in cell biology for the non-invasive imaging of concentrations of ions or the activity of enzymes, to evaluate the distribution of small molecules, proteins and organelles, and to image protein interactions in living cells. These fluorescent molecules can be used either by transient expression in cultured cells or in entire organisms or through stable expression by producing transgenic animals characterized by genetically encoded and heritable biosensors. Using the mouse Thy1 mini-promoter, we generated a line of transgenic mice expressing a genetically encoded sensor for the simultaneous measurements of intracellular Cl- and pH. This construct, called ClopHensor, consists of a H+- and Cl--sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a red fluorescent protein (DsRedm). Stimulation of hippocampal Schaffer collaterals proved that the sensor is functionally active. To reveal the expression pattern of ClopHensor across the brain of Thy1::ClopHensor mice, we obtained transparent brain samples using the CLARITY method and imaged them with confocal and light-sheet microscopy. We then developed a semi-quantitative approach to identify brain structures with high intrinsic sensor fluorescence. This approach allowed us to assess cell morphology and track axonal projection, as well as to confirm E2GFP and DsRedm fluorescence colocalization. This analysis also provides a map of the brain areas suitable for non-invasive monitoring of intracellular Cl-/pH in normal and pathological conditions. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Mitochondria-targeted cationic liposomes modified with alkyltriphenylphosphonium bromides loaded with hydrophilic drugs: preparation, cytotoxicity and colocalization assay.

The purpose of this work was to obtain cationic liposomes based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine noncovalently modified using alkyltriphenylphosphonium bromides (TPPB-n) with different lengths of hydrocarbon tail for targeted delivery to mitochondria. The hydrodynamic diameter and electrokinetic potential of hybrid liposomes depending on the lipid/surfactant ratio were monitored in time with the aim to optimize the composition with sufficient stability and positive charge for mitochondria-targeted delivery. It was found that increasing the alkyl tail length of the surfactant (up to TPPB-14) leads to an increase in the positive charge of the liposomes. The most optimal results of stability were obtained for hybrid liposomes based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and TPPB-12, TPPB-14. The obtained modified liposomes were loaded with hydrophilic substrates (a model probe Rhodamine B and medicines metronidazole and doxorubicin). This is one of the first examples of fabrication of liposomes noncovalently modified using an amphiphilic TPP cation, with the alkyl tail length of surfactant and TPP/lipid ratio optimized in terms of stability of the liposomes and the binding/release behavior of hydrophilic probes. Using the confocal microscopy method, it was shown that modification of liposomes with a triphenylphosphonium cation results in targeted delivery of encapsulated compounds to mitochondria.

Reorganization of Septins Modulates Synaptic Transmission at Neuromuscular Junctions.

Septins (Sept) are highly conserved Guanosine-5'-triphosphate (GTP)-binding cytoskeletal proteins involved in neuronal signaling in the central nervous system but their involvement in signal transmission in peripheral synapses remains unclear. Sept5 and Sept9 proteins were detected in mouse peripheral neuromuscular junctions by immunofluorescence with a greater degree of co-localization with presynaptic than postsynaptic membranes. Preincubation of neuromuscular junction preparations with the inhibitor of Sept dynamics, forchlorfenuron (FCF), decreased co-localization of Sept with presynaptic membranes. FCF introduced ex vivo or in vivo had no effect on the amplitude of the spontaneous endplate currents (EPCs), indicating the absence of postsynaptic effects of FCF. However, FCF decreased acetylcholine (ACh) quantal release in response to nerve stimulation, reduced the amplitude of evoked quantal currents and decreased the number of quanta with long synaptic delays, demonstrating the presynaptic action of FCF. Nevertheless, FCF had no effect on the amplitude of calcium transient in nerve terminals, as detected by calcium-sensitive dye, and slightly decreased the ratio of the second response amplitude to the first one in paired-pulse experiments. These results suggest that FCF-induced decrease in ACh quantal secretion is not due to a decrease in Ca2+ influx but is likely related to the impairment of later stages occurring after Ca2+ entry, such as trafficking, docking or membrane fusion of synaptic vesicles. Therefore, Sept9 and Sept5 are abundantly expressed in presynaptic membranes, and disruption of Sept dynamics suppresses the evoked synchronous and delayed asynchronous quantal release of ACh, strongly suggesting an important role of Sept in the regulation of neurotransmission in peripheral synapses.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers.

The local calcium concentration in the active zone of secretion determines the number and kinetics of neurotransmitter quanta released after the arrival of a nerve action potential in chemical synapses. The small size of mammalian neuromuscular junctions does not allow direct measurement of the correlation between calcium influx, the state of endogenous calcium buffers determining the local concentration of calcium and the time course of quanta exocytosis. In this work, we used computer modeling of quanta release kinetics with various levels of calcium influx and in the presence of endogenous calcium buffers with varying mobilities. The results of this modeling revealed the desynchronization of quanta release under low calcium influx in the presence of an endogenous fixed calcium buffer, with a diffusion coefficient much smaller than that of free Ca(2+), and synchronization occurred upon adding a mobile buffer. This corresponds to changes in secretion time course parameters found experimentally (Samigullin et al., Physiol Res 54:129-132, 2005; Bukharaeva et al., J Neurochem 100:939-949, 2007).