RESUMO
Animal models of human neurological disorders provide valuable experimental tools which enable us to study various aspects of disorder pathogeneses, ranging from structural abnormalities and disrupted metabolism and signaling to motor and mental deficits, and allow us to test novel therapies in preclinical studies. To be valid, these animal models should recapitulate complex pathological features at the molecular, cellular, tissue, and behavioral levels as closely as possible to those observed in human subjects. Pathological states resembling known human neurological disorders can be induced in animal species by toxins, genetic factors, lesioning, or exposure to extreme conditions. In recent years, novel animal models recapitulating neuropathologies in humans have been introduced. These animal models are based on synthetic biology approaches: opto- and chemogenetics. In this paper, we review recent opto- and chemogenetics-based animal models of human neurological disorders. These models allow for the creation of pathological states by disrupting specific processes at the cellular level. The artificial pathological states mimic a range of human neurological disorders, such as aging-related dementia, Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, epilepsy, and ataxias. Opto- and chemogenetics provide new opportunities unavailable with other animal models of human neurological disorders. These techniques enable researchers to induce neuropathological states varying in severity and ranging from acute to chronic. We also discuss future directions for the development and application of synthetic biology approaches for modeling neurological disorders.
Assuntos
Epilepsia , Doença de Parkinson , Animais , Humanos , Optogenética/métodos , Modelos Animais , NeuropatologiaRESUMO
The objective of the current review is to summarize the current state of optical methods in redox biology. It consists of two parts, the first is dedicated to genetically encoded fluorescent indicators and the second to Raman spectroscopy. In the first part, we provide a detailed classification of the currently available redox biosensors based on their target analytes. We thoroughly discuss the main architecture types of these proteins, the underlying engineering strategies for their development, the biochemical properties of existing tools and their advantages and disadvantages from a practical point of view. Particular attention is paid to fluorescence lifetime imaging microscopy as a possible readout technique, since it is less prone to certain artifacts than traditional intensiometric measurements. In the second part, the characteristic Raman peaks of the most important redox intermediates are listed, and examples of how this knowledge can be implemented in biological studies are given. This part covers such fields as estimation of the redox states and concentrations of Fe-S clusters, cytochromes, other heme-containing proteins, oxidative derivatives of thiols, lipids, and nucleotides. Finally, we touch on the issue of multiparameter imaging, in which biosensors are combined with other visualization methods for simultaneous assessment of several cellular parameters.
Assuntos
Técnicas Biossensoriais , Análise Espectral Raman , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Técnicas Biossensoriais/métodos , Oxirredução , BiologiaRESUMO
Reactive oxygen species (ROS) are considered a primary source of damage during ischemic stroke. However, the precise timing of ROS production (during hypoxia or reperfusion) remains unclear. Cellular 3D spheroids are often proposed as an optimal alternative to both 2D cell cultures and animal models in modeling disease conditions. Here we report live imaging of hydrogen peroxide dynamics during the acute phase of hypoxia and reperfusion in human iPSC-derived neural spheroids, stably expressing fluorescent biosensor HyPer7. Contrary to previous reports, we did not observe a hydrogen peroxide production burst neither during hypoxia nor in course of reperfusion. Our data suggest either lack of oxidative stress during ischemia-reperfusion in spheroids or existence of different mechanisms of oxidative damage.
Assuntos
Traumatismo por Reperfusão Miocárdica , Traumatismo por Reperfusão , Animais , Humanos , Espécies Reativas de Oxigênio , Peróxido de Hidrogênio , Estresse Oxidativo , Isquemia , Reperfusão , HipóxiaRESUMO
Feed-forward inhibition is vital in the transfer and processing of synaptic information within the hippocampal-entorhinal loop by controlling the strength and direction of excitation flow between different neuronal populations and individual neurons. While the cellular targets in the hippocampus that receive excitatory inputs from the entorhinal cortex have been well studied, and the role of feedforward inhibitory neurons has been attributed to neurogliafom cells, the cortical interneurons providing feed-forward control over receiving layer V in the entorhinal cortex remain unknown. We used sharp-wave ripple oscillations as a natural excitatory stimulus of the entorhinal cortex, driven by the hippocampus, to study the function of synaptic interactions between neurons in the deep layers of the entorhinal cortex. We discovered that CB1R-expressing interneurons in the deep layers of the entorhinal cortex constitute the major relay station that translates hippocampal excitation into efficient inhibition of cortical pyramidal cells. The impact of inhibition provided by these interneurons is under strong endocannabinoid control and can be drastically reduced either by enhanced activity of postsynaptic targets or by stress-induced elevation of cannabinoids.
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Oscillation of intracellular calcium concentration is a stable phenomenon that affects cellular function throughout the lifetime of both electrically excitable and non-excitable cells. Nitric oxide, a gaseous secondary messenger and the product of nitric oxide synthase (NOS), affects intracellular calcium dynamics. Using mouse hippocampal primary cultures, we recorded the effect of NOS blockade on neuronal spontaneous calcium activity. There was a correlation between the amplitude of spontaneous calcium events and the number of action potentials (APs) (Spearman R = 0.94). There was a linear rise of DAF-FM fluorescent emission showing an increase in NO concentration with time in neurons (11.9 ± 1.0%). There is correlation between the integral of the signal from DAF-FM and the integral of the spontaneous calcium event signal from Oregon Green 488 (Spearman R = 0.58). Blockade of NOS affected the parameters of the spontaneous calcium events studied (amplitude, frequency, integral, rise slope and decay slope). NOS blockade by Nw-Nitro-L-arginine suppressed the amplitude and frequency of spontaneous calcium events. The NOS blocker 3-Bromo-7-Nitroindazole reduced the frequency but not the amplitude of spontaneous calcium activity. Blockade of the well-known regulator of NOS, calcineurin with cyclosporine A reduced the integral of calcium activity in neurons. The differences and similarities in the effects on the parameters of spontaneous calcium effects caused by different blockades of NO production help to improve understanding of how NO synthesis affects calcium dynamics in neurons.
Assuntos
Cálcio , Óxido Nítrico Sintase , Camundongos , Animais , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico , Doadores de Óxido Nítrico , Cálcio da Dieta , Hipocampo/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismoRESUMO
Oxidative stress, a state of disrupted redox signaling, reactive oxygen species (ROS) overproduction, and oxidative cell damage, accompanies numerous brain pathologies, including aging-related dementia and Alzheimer's disease, the most common neurodegenerative disorder of the elderly population. However, a causative role of neuronal oxidative stress in the development of aging-related cognitive decline and neurodegeneration remains elusive because of the lack of approaches for modeling isolated oxidative injury in the brain. Here, we present a chemogenetic approach based on the yeast flavoprotein d-amino acid oxidase (DAAO) for the generation of intraneuronal hydrogen peroxide (H2O2). To validate this chemogenetic tool, DAAO and HyPer7, an ultrasensitive genetically encoded H2O2 biosensor, were targeted to neurons. Changes in the fluorescence of HyPer7 upon treatment of neurons expressing DAAO with d-norvaline (D-Nva), a DAAO substrate, confirmed chemogenetically induced production of intraneuornal H2O2. Then, using the verified chemogenetic tool, we emulated isolated intraneuronal oxidative stress in acute brain slices and, using electrophysiological recordings, revealed that it does not alter basal synaptic transmission and the probability of neurotransmitter release from presynaptic terminals but reduces long-term potentiation (LTP). Moreover, treating neurons expressing DAAO with D-Nva via the patch pipette also decreases LTP. This observation indicates that isolated oxidative stress affects synaptic plasticity at single cell level. Our results broaden the toolset for studying normal redox regulation in the brain and elucidating the role of oxidative stress to the pathogenesis of cognitive aging and the early stages of aging-related neurodegenerative diseases. The proposed approach is useful for identification of early markers of neuronal oxidative stress and may be used in screens of potential antioxidants effective against neuronal oxidative injury.
Assuntos
Peróxido de Hidrogênio , Estresse Oxidativo , Humanos , Idoso , Peróxido de Hidrogênio/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Antioxidantes/farmacologia , Plasticidade Neuronal/fisiologiaRESUMO
A reduced recombinant system provides a unique opportunity to study the biophysical properties of NMDAR channels with known subunit compositions, by using a point mutation approach to analyze the structural determinants of receptor function (Wollmuth and Sobolevsky, Trends Neurosci 27:321-328, 2004). However, in addition to the well-developed repertoire of molecular biological techniques, these types of studies also require electrophysiological methods that allow a wide range of receptor activation protocols that can adequately assess desensitization, inactivation, ion permeability, and other properties of the channels. Currently, one of the most well-developed techniques suitable for addressing these issues is use of the fast agonist application system for rapid activation of ligand gated ion-channels (Colquhoun et al., J Physiol 458:261-287, 1992; Jonas and Sakmann, J Physiol 455:143-171, 1992).