Modern optical approaches in redox biology: Genetically encoded sensors and Raman spectroscopy.

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.

Redox differences between rat neonatal and adult cardiomyocytes under hypoxia.

It is generally accepted that oxidative stress plays a key role in the development of ischemia-reperfusion injury in ischemic heart disease. However, the mechanisms how reactive oxygen species trigger cellular damage are not fully understood. Our study investigates redox state and highly reactive substances within neonatal and adult cardiomyocytes under hypoxia conditions. We have found that hypoxia induced an increase in H2O2 production in adult cardiomyocytes, while neonatal cardiomyocytes experienced a decrease in H2O2 levels. This finding correlates with our observation of the difference between the electron transport chain (ETC) properties and mitochondria amount in adult and neonatal cells. We demonstrated that in adult cardiomyocytes hypoxia caused the significant increase in the ETC loading with electrons compared to normoxia. On the contrary, in neonatal cardiomyocytes ETC loading with electrons was similar under both normoxic and hypoxic conditions that could be due to ETC non-functional state and the absence of the electrons transfer to O2 under normoxia. In addition to the variations in H2O2 production, we also noted consistent pH dynamics under hypoxic conditions. Notably, the pH levels exhibited a similar decrease in both cell types, thus, acidosis is a more universal cellular response to hypoxia. We also demonstrated that the amount of mitochondria and the levels of cardiac isoforms of troponin I, troponin T, myoglobin and GAPDH were significantly higher in adult cardiomyocytes compared to neonatal ones. Remarkably, we found out that under hypoxia, the levels of cardiac isoforms of troponin T, myoglobin, and GAPDH were elevated in adult cardiomyocytes, while their level in neonatal cells remained unchanged. Obtained data contribute to the understanding of the mechanisms of neonatal cardiomyocytes' resistance to hypoxia and the ability to maintain the metabolic homeostasis in contrast to adult ones.

Astrocytes produce nitric oxide via nitrite reduction in mitochondria to regulate cerebral blood flow during brain hypoxia.

During hypoxia, increases in cerebral blood flow maintain brain oxygen delivery. Here, we describe a mechanism of brain oxygen sensing that mediates the dilation of intraparenchymal cerebral blood vessels in response to reductions in oxygen supply. In vitro and in vivo experiments conducted in rodent models show that during hypoxia, cortical astrocytes produce the potent vasodilator nitric oxide (NO) via nitrite reduction in mitochondria. Inhibition of mitochondrial respiration mimics, but also occludes, the effect of hypoxia on NO production in astrocytes. Astrocytes display high expression of the molybdenum-cofactor-containing mitochondrial enzyme sulfite oxidase, which can catalyze nitrite reduction in hypoxia. Replacement of molybdenum with tungsten or knockdown of sulfite oxidase expression in astrocytes blocks hypoxia-induced NO production by these glial cells and reduces the cerebrovascular response to hypoxia. These data identify astrocyte mitochondria as brain oxygen sensors that regulate cerebral blood flow during hypoxia via release of nitric oxide.

Mitochondrial malfunction and atrophy of astrocytes in the aged human cerebral cortex.

How aging affects cells of the human brain active milieu remains largely unknown. Here, we analyze astrocytes and neurons in the neocortical tissue of younger (22-50 years) and older (51-72 years) adults. Aging decreases the amount of reduced mitochondrial cytochromes in astrocytes but not neurons. The protein-to-lipid ratio decreases in astrocytes and increases in neurons. Aged astrocytes show morphological atrophy quantified by the decreased length of branches, decreased volume fraction of leaflets, and shrinkage of the anatomical domain. Atrophy correlates with the loss of gap junction coupling between astrocytes and increased input resistance. Aging is accompanied by the upregulation of glial fibrillary acidic protein (GFAP) and downregulation of membrane-cytoskeleton linker ezrin associated with leaflets. No significant changes in neuronal excitability or spontaneous inhibitory postsynaptic signaling is observed. Thus, brain aging is associated with the impaired morphological presence and mitochondrial malfunction of cortical astrocytes, but not neurons.

Astrocytes in human central nervous system diseases: a frontier for new therapies.

Astroglia are a broad class of neural parenchymal cells primarily dedicated to homoeostasis and defence of the central nervous system (CNS). Astroglia contribute to the pathophysiology of all neurological and neuropsychiatric disorders in ways that can be either beneficial or detrimental to disorder outcome. Pathophysiological changes in astroglia can be primary or secondary and can result in gain or loss of functions. Astroglia respond to external, non-cell autonomous signals associated with any form of CNS pathology by undergoing complex and variable changes in their structure, molecular expression, and function. In addition, internally driven, cell autonomous changes of astroglial innate properties can lead to CNS pathologies. Astroglial pathophysiology is complex, with different pathophysiological cell states and cell phenotypes that are context-specific and vary with disorder, disorder-stage, comorbidities, age, and sex. Here, we classify astroglial pathophysiology into (i) reactive astrogliosis, (ii) astroglial atrophy with loss of function, (iii) astroglial degeneration and death, and (iv) astrocytopathies characterised by aberrant forms that drive disease. We review astroglial pathophysiology across the spectrum of human CNS diseases and disorders, including neurotrauma, stroke, neuroinfection, autoimmune attack and epilepsy, as well as neurodevelopmental, neurodegenerative, metabolic and neuropsychiatric disorders. Characterising cellular and molecular mechanisms of astroglial pathophysiology represents a new frontier to identify novel therapeutic strategies.

Hyperglycemia exacerbates ischemic stroke not through increased generation of hydrogen peroxide.

Diabetes is one of the significant risk factors for ischemic stroke. Hyperglycemia exacerbates the pathogenesis of stroke, leading to more extensive cerebral damage and, as a result, to more severe consequences. However, the mechanism whereby the hyperglycemic status in diabetes affects biochemical processes during the development of ischemic injury is still not fully understood. In the present work, we record for the first time the real-time dynamics of H2O2 in the matrix of neuronal mitochondria in vitro in culture and in vivo in the brain tissues of rats during development of ischemic stroke under conditions of hyperglycemia and normal glucose levels. To accomplish this, we used a highly sensitive HyPer7 biosensor and a fiber-optic interface technology. We demonstrated that a high glycemic status does not affect the generation of H2O2 in the tissues of the ischemic core, while significantly exacerbating the consequences of pathogenesis. For the first time using Raman microspectroscopy approach, we have shown how a sharp increase in the blood glucose level increases the relative amount of reduced cytochromes in the mitochondrial electron transport chain in neurons under normal conditions in awake mice.

KCC2 reverse mode helps to clear postsynaptically released potassium at glutamatergic synapses.

Extracellular potassium [K+]o elevation during synaptic activity retrogradely modifies presynaptic release and astrocytic uptake of glutamate. Hence, local K+ clearance and replenishment mechanisms are crucial regulators of glutamatergic transmission and plasticity. Based on recordings of astrocytic inward rectifier potassium current IKir and K+-sensitive electrodes as sensors of [K+]o as well as on in silico modeling, we demonstrate that the neuronal K+-Cl- co-transporter KCC2 clears local perisynaptic [K+]o during synaptic excitation by operating in an activity-dependent reversed mode. In reverse mode, KCC2 replenishes K+ in dendritic spines and complements clearance of [K+]o, therewith attenuating presynaptic glutamate release and shortening LTP. We thus demonstrate a physiological role of KCC2 in neuron-glial interactions and regulation of synaptic signaling and plasticity through the uptake of postsynaptically released K+.

Dissociation Between Neuronal and Astrocytic Calcium Activity in Response to Locomotion in Mice.

Locomotion triggers a coordinated response of both neurons and astrocytes in the brain. Here we performed calcium (Ca2+) imaging of these two cell types in the somatosensory cortex in head-fixed mice moving on the airlifted platform. Ca2+ activity in astrocytes significantly increased during locomotion from a low quiescence level. Ca2+ signals first appeared in the distal processes and then propagated to astrocytic somata, where it became significantly larger and exhibited oscillatory behaviour. Thus, astrocytic soma operates as both integrator and amplifier of Ca2+ signal. In neurons, Ca2+ activity was pronounced in quiescent periods and further increased during locomotion. Neuronal Ca2+ concentration ([Ca2+]i) rose almost immediately following the onset of locomotion, whereas astrocytic Ca2+ signals lagged by several seconds. Such a long lag suggests that astrocytic [Ca2+]i elevations are unlikely to be triggered by the activity of synapses among local neurons. Ca2+ responses to pairs of consecutive episodes of locomotion did not significantly differ in neurons, while were significantly diminished in response to the second locomotion in astrocytes. Such astrocytic refractoriness may arise from distinct mechanisms underlying Ca2+ signal generation. In neurons, the bulk of Ca2+ enters through the Ca2+ channels in the plasma membrane allowing for steady-level Ca2+ elevations in repetitive runs. Astrocytic Ca2+ responses originate from the intracellular stores, the depletion of which affects subsequent Ca2+ signals. Functionally, neuronal Ca2+ response reflects sensory input processed by neurons. Astrocytic Ca2+ dynamics is likely to provide metabolic and homeostatic support within the brain active milieu.

Decline of astrocyte Ca2+ signalling in Alzheimer's disease: STIM1 to the rescue!

Lia et al. [1] discovered the critical role of STIM1 ER Ca2+ sensor in the functional decline of astrocytes in the AD-like pathology in PS2APP mice. Profound downregulation of STIM1 expression in astrocytes in the disease results in the decreased ER Ca2+ content and severely impaired evoked as well as spontaneous astrocytic Ca2+ signalling. Aberrant astrocytic Ca2+ signalling translated into impaired synaptic plasticity and memory. Astrocyte-specific overexpression of STIM1 restored Ca2+ excitability and rectified synaptic and memory deficits.

Electroacupuncture prevents astrocyte atrophy to alleviate depression.

Astrocyte atrophy is the main histopathological hallmark of major depressive disorder (MDD) in humans and in animal models of depression. Here we show that electroacupuncture prevents astrocyte atrophy in the prefrontal cortex and alleviates depressive-like behaviour in mice subjected to chronic unpredictable mild stress (CUMS). Treatment of mice with CUMS induced depressive-like phenotypes as confirmed by sucrose preference test, tail suspension test, and forced swimming test. These behavioural changes were paralleled with morphological atrophy of astrocytes in the prefrontal cortex, revealed by analysis of 3D reconstructions of confocal Z-stack images of mCherry expressing astrocytes. This morphological atrophy was accompanied by a decrease in the expression of cytoskeletal linker Ezrin, associated with formation of astrocytic leaflets, which form astroglial synaptic cradle. Electroacupuncture at the acupoint ST36, as well as treatment with anti-depressant fluoxetine, prevented depressive-like behaviours, astrocytic atrophy, and down-regulation of astrocytic ezrin. In conclusion, our data further strengthen the notion of a primary role of astrocytic atrophy in depression and reveal astrocytes as cellular target for electroacupuncture in treatment of depressive disorders.

Astrocytes in Ageing.

Ageing is associated with a morphological and functional decline of astrocytes with a prevalence of morphological atrophy and loss of function. In particular, ageing is manifested by the shrinkage of astrocytic processes: branches and leaflets, which decreases synaptic coverage. Astrocytic dystrophy affects multiple functions astrocytes play in the brain active milieu. In particular, and in combination with an age-dependent decline in the expression of glutamate transporters, astrocytic atrophy translates into deficient glutamate clearance and K+ buffering. Decreased astrocyte presence may contribute to age-dependent remodelling of brain extracellular space, hence affecting extrasynaptic signalling. Old astrocytes lose endfeet polarisation of AQP4 water channels, thus limiting the operation of the glymphatic system. In ageing, astrocytes down-regulate their antioxidant capacity leading to decreased neuroprotection. All these changes may contribute to an age-dependent cognitive decline.

Delayed Impairment of Hippocampal Synaptic Plasticity after Pentylenetetrazole-Induced Seizures in Young Rats.

Data on the long-term consequences of a single episode of generalized seizures in infants are inconsistent. In this study, we examined the effects of pentylenetetrazole-induced generalized seizures in three-week-old rats. One month after the seizures, we detected a moderate neuronal loss in several hippocampal regions: CA1, CA3, and hilus, but not in the dentate gyrus. In addition, long-term synaptic potentiation (LTP) was impaired. We also found that the mechanism of plasticity induction was altered: additional activation of metabotropic glutamate receptors (mGluR1) is required for LTP induction in experimental rats. This disturbance of the plasticity induction mechanism is likely due to the greater involvement of perisynaptic NMDA receptors compared to receptors located in the core part of the postsynaptic density. This hypothesis is supported by experiments with selective blockades of core-located NMDA receptors by the use-dependent blocker MK-801. MK-801 had no effect on LTP induction in experimental rats and suppressed LTP in control animals. The weakening of the function of core-located NMDA receptors may be due to the disturbed clearance of glutamate from the synaptic cleft since the distribution of the astrocytic glutamate transporter EAAT2 in experimental animals was found to be altered.

A high-fat diet changes astrocytic metabolism to promote synaptic plasticity and behavior.

AIM: A high-fat diet (HFD) is generally considered to negatively influence the body, the brain, and cognition. Nonetheless, fat and fatty acids are essential for nourishing and constructing brain tissue. Astrocytes are central for lipolysis and fatty acids metabolism. We tested how HFD affects astrocyte metabolism, morphology, and physiology. METHODS: We used Raman microspectroscopy to assess the redox state of mitochondria and lipid content in astrocytes and neurons in hippocampal slices of mice subjected to HFD. Astrocytes were loaded with fluorescent dye through patch pipette for morphological analysis. Whole-cell voltage-clamp recordings were performed to measure transporter and potassium currents. Western blot analysis quantified the expression of astrocyte-specific proteins. Field potential recordings measured the magnitude of long-term potentiation (LTP). Open filed test was performed to evaluate the effect of HFD on animal behavior. RESULTS: We found that exposure of young mice to 1 month of HFD increases lipid content and relative amount of reduced cytochromes in astrocytes but not in neurons. Metabolic changes were paralleled with an enlargement of astrocytic territorial domains due to an increased outgrowth of branches and leaflets. Astrocyte remodeling was associated with an increase in expression of ezrin and with no changes in glial fibrillary acidic protein (GFAP), glutamate transporter-1 (GLT-1), and glutamine synthetase (GS). Such physiological (non-reactive) enlargement of astrocytes in the brain active milieu promoted glutamate clearance and LTP and translated into behavioral changes. CONCLUSION: Dietary fat intake is not invariably harmful and might exert beneficial effects depending on the biological context.

K+ efflux through postsynaptic NMDA receptors suppresses local astrocytic glutamate uptake.

Glutamatergic transmission prompts K+ efflux through postsynaptic NMDA receptors. The ensuing hotspot of extracellular K+ elevation depolarizes presynaptic terminal, boosting glutamate release, but whether this also affects glutamate uptake in local astroglia has remained an intriguing question. Here, we find that the pharmacological blockade, or conditional knockout, of postsynaptic NMDA receptors suppresses use-dependent increase in the amplitude and duration of the astrocytic glutamate transporter current (IGluT ), whereas blocking astrocytic K+ channels prevents the duration increase only. Glutamate spot-uncaging reveals that astrocyte depolarization, rather than extracellular K+ rises per se, is required to reduce the amplitude and duration of IGluT . Biophysical simulations confirm that local transient elevations of extracellular K+ can inhibit local glutamate uptake in fine astrocytic processes. Optical glutamate sensor imaging and a two-pathway test relate postsynaptic K+ efflux to enhanced extrasynaptic glutamate signaling. Thus, repetitive glutamatergic transmission triggers a feedback loop in which postsynaptic K+ efflux can transiently facilitate presynaptic release while reducing local glutamate uptake.

Astrocytic processes: from tripartite synapses to the active milieu.

We define a new concept of 'active milieu' that unifies all components of nervous tissue (neuronal and glial compartments, extracellular space, extracellular matrix, and vasculature) into a dynamic information processing system. Within this framework, we focus on the role of astrocytic processes, classified into organelle-containing branches and organelle-free leaflets. We argue that astrocytic branches with emanating leaflets are homologous to dendritic shafts with spines. Within the active milieu, astrocytic processes are engaged in reciprocal interactions with neuronal compartments and communication with other cellular and non-cellular elements of the nervous tissue.