Optogenetics: Perspectives in biomedical research

© 2016, Nizhny Novgorod State Medical Academy. All rights reserved.Optogenetic tools, photochromic switches and genetically encoded biosensors revolutionized contemporary neuroscience research. These approaches provided unprecedented opportunities for monitoring and modulating the function of specific neurons and have literally shed light on the mechanisms of neuronal networks function in the brain. A number of light-sensitive biosensors for non-invasive monitoring of ions and enzymes have been developed. These molecular designs expand extremely rapidly and a number of new approaches for image analysis of various proteins in living cells have being proposed. In this review we discuss new tools for molecular imaging and remote activation of receptors, ionic channels and synaptic networks, as well as its potential for biomedical research

Optogenetics and photopharmacology - effective tools for managing cell activity using light

Contemporary research has been enriched by the new directions in which the light plays a key role as a tool for modulation of cellular activity and invasive monitoring of intracellular ions and other components. The main advantages of these approaches are the possibilities to precisely control the intensity, spectral characteristics and durations of light signals in space and time. This review summarizes the key areas, optogenetics and photopharmacology, - directions that allow to control cellular activity with light. Optogenetics is the use of light-sensitive transmembrane proteins capable of exciting or inhibiting cellular activity under illumination by different wavelengths. In 2003 a light-sensitive protein canalo-rodopsine was isolated and cloned which is capable of inducing ion currents and changing cellular rest potential with its excitation under the blue light when embedded into the neurons or other cell types. Inhibition of cellular activity is caused by expression of other lightsensitive proteins - chloride or hydrogen pumps, or anion-selective ion channels. These principles turns out to be efficacious for the study of the functions of solitary cells and neural nets as well as for the control of living organisms behavior but their use in medicine is complicated because of necessary genetic manipulations. Photopharmacology is based on creating and using of chemical compounds changing conformations and/or activity under the light. Photochromic compounds with the use of photosensitive switches are capable of selective activation or inhibition of the activity of functionally important proteins - receptors, ion channels, enzymes, etc. The principles and the potential use of optogenetics and photopharmacology in the analysis of the neuronal functions and the perspectives for new approaches to treat some diseases of the nervous system are discussed

Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo. © 2013 Batti, Mukhtarov, Audero, Ivanov, Paolicelli, Zurborg, Gross, Bregestovski and Heppenstall

Calibration and functional analysis of three genetically encoded Cl-/pH sensors

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

Reactive oxygen species initiate a metabolic collapse in hippocampal slices: Potential trigger of cortical spreading depression

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

ANO1 amplification and expression in HNSCC with a high propensity for future distant metastasis and its functions in HNSCC cell lines

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is associated with poor survival. To identify prognostic and diagnostic markers and therapeutic targets, we studied ANO1, a recently identified calcium-activated chloride channel (CaCC). METHODS: High-resolution genomic and transcriptomic microarray analysis and functional studies using HNSCC cell line and CaCC inhibitors. RESULTS: Amplification and overexpression of genes within the 11q13 amplicon are associated with the propensity for future distance metastasis of HPV-negative HNSCC. ANO1 was selected for functional studies based on high correlations, cell surface expression and CaCC activity. ANO1 overexpression in cells that express low endogenous levels stimulates cell movement, whereas downregulation in cells with high endogenous levels has the opposite effect. ANO1 overexpression also stimulates attachment, spreading, detachment and invasion, which could account for its effects on migration. CaCC inhibitors decrease movement, suggesting that channel activity is required for the effects of ANO1. In contrast, ANO1 overexpression does not affect cell proliferation. INTERPRETATION: ANO1 amplification and expression could be markers for distant metastasis in HNSCC. ANO1 overexpression affects cell properties linked to metastasis. Inhibitors of CaCCs could be used to inhibit the tumourigenic properties of ANO1, whereas activators developed to increase CaCC activity could have adverse effects

Elie Metchnikoff: Father of phagocytosis theory and pioneer of experiments in vivo

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Ricardo Miledi - an outstanding neurophysiologist of 20th-21st centuries (1927-2017)

Ricardo Miledi (16.09.1927-18.12.2017) is an outstanding neurophysiologist and biophysicist who made a great contribution to the study of synaptic transmission functions. He proved the key role of сalcium ions in the release of neuromediators, developed methods of receptor expression and membrane fragments integration into large oocytes that provided huge possibilities for thousands of researchers to study subtle mechanisms of transmembrane proteins function in norm and pathology. Ricardo Miledi received his MD degree in the National Autonomous University of Mexico and in 1954 he defensed his dissertation on the study of electrical nature of cardiac fibrillation in the National Institute of Cardiology (Mexico). In 1956-1958 he underwent training in Canberra Health Research Institute (Australia) in the laboratory headed by John Eccles (Nobel Prize 1963). In 1958 R. Miledi was invited to the Department of biophysics of University College London where in cooperation with Bernard Katz (Nobel Prize 1970) made a number of important discoveries in the analysis of acetylcholine receptor expression in denervated mucle; determination of the role of calcium in neuromediators release; analysis of membrane noise on neuromediator application to neuromuscular synapses; study of the effect of antibodies from patients with myasthenia gravis on neuromuscular transmission. In the early 1980s Ricardo Miledi implemented the method of functional expression in Xenopus frog oocytes of receptors and ion channels from messenger ribonucleic acid (mRNA). His heritage running the gamut is presented in more than 500 articles

Photochromic Modulation of Cys-loop Ligand-gated Ion Channels

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Optogenetics: Perspectives in biomedical research

© 2016, Nizhny Novgorod State Medical Academy. All rights reserved.Optogenetic tools, photochromic switches and genetically encoded biosensors revolutionized contemporary neuroscience research. These approaches provided unprecedented opportunities for monitoring and modulating the function of specific neurons and have literally shed light on the mechanisms of neuronal networks function in the brain. A number of light-sensitive biosensors for non-invasive monitoring of ions and enzymes have been developed. These molecular designs expand extremely rapidly and a number of new approaches for image analysis of various proteins in living cells have being proposed. In this review we discuss new tools for molecular imaging and remote activation of receptors, ionic channels and synaptic networks, as well as its potential for biomedical research