Astrocyte Regulation of Cerebral Blood Flow in Health and Disease.

Astrocytes play an important role in controlling microvascular diameter and regulating local cerebral blood flow (CBF) in several physiological and pathological scenarios. Neurotransmitters released from active neurons evoke Ca2+ increases in astrocytes, leading to the release of vasoactive metabolites of arachidonic acid (AA) from astrocyte endfeet. Synthesis of prostaglandin E2 (PGE2) and epoxyeicosatrienoic acids (EETs) dilate blood vessels while 20-hydroxyeicosatetraenoic acid (20-HETE) constricts vessels. The release of K+ from astrocyte endfeet also contributes to vasodilation or constriction in a concentration-dependent manner. Whether astrocytes exert a vasodilation or vasoconstriction depends on the local microenvironment, including the metabolic status, the concentration of Ca2+ reached in the endfoot, and the resting vascular tone. Astrocytes also contribute to the generation of steady-state vascular tone. Tonic release of both 20-HETE and ATP from astrocytes constricts vascular smooth muscle cells, generating vessel tone, whereas tone-dependent elevations in endfoot Ca2+ produce tonic prostaglandin dilators to limit the degree of constriction. Under pathological conditions, including Alzheimer's disease, epilepsy, stroke, and diabetes, disruption of normal astrocyte physiology can compromise the regulation of blood flow, with negative consequences for neurological function.

Chronic pregabalin treatment protects against spreading depolarization and alters hippocampal synaptic characteristics in a model of familial hemiplegic migraine-type 1.

Familial hemiplegic migraine type-1 (FHM-1) is a form of migraine with aura caused by mutations in the P/Q-type (Cav2.1) voltage-gated calcium channel. Pregabalin, used clinically in the treatment of chronic pain and epilepsy, inhibits P/Q-type calcium channel activity and recent studies suggest that it may have potential for the treatment of migraine. Spreading Depolarization (SD) is a neurophysiological phenomenon that can occur during migraine with aura by propagating a wave of silenced neuronal function through cortex and sometimes subcortical brain structures. Here, utilizing an optogenetic stimulation technique optimized to allow for non-invasive initiation of cortical SD, we demonstrate that chronic pregabalin administration [12 mg/kg/day (s.c.)] in vivo increased the threshold for cortical spreading depolarization in transgenic mice harboring the clinically-relevant Cav2.1S218L mutation (S218L). In addition, chronic pregabalin treatment limited subcortical propagation of recurrent spreading depolarization events to the striatum and hippocampus in both wild-type and S218L mice. To examine contributing underlying mechanisms of action of chronic pregabalin, we performed whole-cell patch-clamp electrophysiology in CA1 neurons in ex vivo brain slices from mice treated with chronic pregabalin vs vehicle. In WT mice, chronic pregabalin produced a decrease in spontaneous excitatory postsynaptic current (sEPSC) amplitude with no effect on frequency. In contrast, in S218L mice chronic pregabalin produced an increase in sEPSC amplitude and decreased frequency. These electrophysiological findings suggest that in FHM-1 mice chronic pregabalin acts through both pre- and post-synaptic mechanisms in CA1 hippocampal neurons to elicit FHM-1 genotype-specific inhibitory action. The results highlight the potential of chronic pregabalin to limit recurrent SD to subcortical brain structures during pathophysiological events in both the genetically-normal and FHM-1 brain. The work further provides insights into FHM-1 pathophysiology and the potential for chronic pregabalin treatment to prevent SD in migraineurs.

Pannexin-1 opening in neuronal edema causes cell death but also leads to protection via increased microglia contacts.

Neuronal swelling during cytotoxic edema is triggered by Na+ and Cl- entry and is Ca2+ independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema. Panx1 channel inhibitors reduce and delay neuronal death in swelling triggered by voltage-gated Na+ entry with veratridine. Neuronal swelling causes downstream production of reactive oxygen species (ROS) that opens Panx1 channels. We confirm that ROS activates Panx1 currents with whole-cell electrophysiology and find scavenging ROS is neuroprotective. Panx1 opening and subsequent ATP release attract microglial processes to contact swelling neurons. Depleting microglia using the CSF1 receptor antagonist PLX3397 or blocking P2Y12 receptors exacerbates neuronal death, suggesting that the Panx1-ATP-dependent microglia contacts are neuroprotective. We conclude that cytotoxic edema triggers oxidative stress in neurons that opens Panx1 to trigger death but also initiates neuroprotective feedback mediated by microglia contacts.

Modelling Microglial Innate Immune Memory In Vitro: Understanding the Role of Aerobic Glycolysis in Innate Immune Memory.

Microglia, the resident macrophages of the central nervous system, play important roles in maintaining brain homeostasis and facilitating the brain's innate immune responses. Following immune challenges microglia also retain immune memories, which can alter responses to secondary inflammatory challenges. Microglia have two main memory states, training and tolerance, which are associated with increased and attenuated expression of inflammatory cytokines, respectively. However, the mechanisms differentiating these two distinct states are not well understood. We investigated mechanisms underlying training versus tolerance memory paradigms in vitro in BV2 cells using B-cell-activating factor (BAFF) or bacterial lipopolysaccharide (LPS) as a priming stimulus followed by LPS as a second stimulus. BAFF followed by LPS showed enhanced responses indicative of priming, whereas LPS followed by LPS as the second stimulus caused reduced responses suggestive of tolerance. The main difference between the BAFF versus the LPS stimulus was the induction of aerobic glycolysis by LPS. Inhibiting aerobic glycolysis during the priming stimulus using sodium oxamate prevented the establishment of the tolerized memory state. In addition, tolerized microglia were unable to induce aerobic glycolysis upon LPS restimulus. Therefore, we conclude that aerobic glycolysis triggered by the first LPS stimulus was a critical step in the induction of innate immune tolerance.

hiPSC-derived GRN-deficient astrocytes delay spiking activity of developing neurons.

Frontotemporal dementia (FTD) refers to a group of neurodegenerative disorders that are characterized by pathology predominantly localized to the frontal and temporal lobes. Approximately 40% of FTD cases are familial, and up to 20% of these are caused by heterozygous loss of function mutations in the gene encoding for progranulin (PGRN), GRN. The mechanisms by which loss of PGRN leads to FTD remain incompletely understood. While astrocytes and microglia have long been linked to the neuropathology of FTD due to mutations in GRN (FTD-GRN), a primary mechanistic role of these supporting cells have not been thoroughly addressed. In contrast, mutations in MAPT, another leading cause of familial FTD, greatly alters astrocyte gene expression leading to subsequent non-cell autonomous effects on neurons, suggesting similar mechanisms may be present in FTD-GRN. Here, we utilized human induced pluripotent stem cell (hiPSC)-derived neural tissue carrying a homozygous GRN R493X-/- knock-in mutation to investigate in vitro whether GRN mutant astrocytes have a non-cell autonomous effect on neurons. Using microelectrode array (MEA) analysis, we demonstrate that the development of spiking activity of neurons cultured with GRN R493X-/- astrocytes was significantly delayed compared to cultures with WT astrocytes. Histological analysis of synaptic markers in these cultures showed an increase in GABAergic synaptic markers and a decrease in glutamatergic synaptic markers during this period when activity was delayed. We also demonstrate that this effect may be due in-part to soluble factors. Overall, this work represents one of the first studies investigating astrocyte-induced neuronal pathology in GRN mutant hiPSCs, and supports the hypothesis of astrocyte involvement in the early pathophysiology of FTD.

KCC2 drives chloride microdomain formation in dendritic blebbing.

Intracellular chloride ion concentration ([Cl-]i) homeostasis is critical for excitatory/inhibitory balance and volume regulation in neurons. We quantitatively map spatiotemporal dendritic [Cl-]i dynamics during N-methyl-d-aspartate (NMDA) excitotoxicity to determine how Cl- changes contribute to localized dendritic swelling (blebbing) in stroke-like conditions. Whole-cell patch clamp electrophysiology combined with simultaneous fluorescence lifetime imaging (FLIM) of the Cl- dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE; MQAE-FLIM) reliably report resting and dynamic [Cl-]i shifts in dendrites. NMDA application generates spatially restricted and persistent high [Cl-]i subdomains at dendritic blebs in a process that requires Ca2+ influx and the subsequent opening of small-conductance Ca2+-activated K+ (SK) channels. We propose sustained and localized K+ efflux increased extracellular K+ concentrations ([K+]o) sufficiently at discrete regions to reverse K+-Cl- cotransporter (KCC2) transport and trigger synaptic swelling. Together, our data establish a mechanism for KCC2 to generate pathological [Cl-]i microdomains in blebbing with relevance for multiple neurological disorders.

Mitochondrial Ca2+ uptake by the MCU facilitates pyramidal neuron excitability and metabolism during action potential firing.

Neuronal activation is fundamental to information processing by the brain and requires mitochondrial energy metabolism. Mitochondrial Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) has long been implicated in the control of energy metabolism and intracellular Ca2+ signalling, but its importance to neuronal function in the brain remains unclear. Here, we used in situ electrophysiology and two-photon imaging of mitochondrial Ca2+, cytosolic Ca2+, and NAD(P)H to test the relevance of MCU activation to pyramidal neuron Ca2+ signalling and energy metabolism during action potential firing. We demonstrate that mitochondrial Ca2+ uptake by the MCU is tuned to enhanced firing rate and the strength of this relationship varied between neurons of discrete brain regions. MCU activation promoted electron transport chain activity and chemical reduction of NAD+ to NADH. Moreover, Ca2+ buffering by mitochondria attenuated cytosolic Ca2+ signals and thereby reduced the coupling between activity and the slow afterhyperpolarization, a ubiquitous regulator of excitability. Collectively, we demonstrate that the MCU is engaged by accelerated spike frequency to facilitate neuronal activity through simultaneous control of energy metabolism and excitability. As such, the MCU is situated to promote brain functions associated with high frequency signalling and may represent a target for controlling excessive neuronal activity.

Simultaneous imaging of redox states in dystrophic neurites and microglia at Aß plaques indicate lysosome accumulation not microglia correlate with increased oxidative stress.

The inter-relationship between microglia dynamics and oxidative stress (Ox-stress) in dystrophic neurites (DNs) at Alzheimer's Disease (AD) plaques may contribute to the pathological changes in neurons. We developed new in vivo imaging strategies to combine EGFP expression in microglia with neuronal expression of genetically encoded ratiometric redox sensors (rogRFP2 or roGFP1), and immunohistochemistry to investigate how microglia influence Ox-stress at amyloid plaques in 5xFAD AD mice. By simultaneously imaging microglia morphology and neuronal Ox-stress over time in vivo and in fixed brains we found that microglia preferentially enwrapped DNs exhibiting the greatest degree of Ox-stress. After microglia were partially depleted with the CSF1 receptor antagonist PLX3397, Ox-stress in DNs increased in a manner that was inversely correlated to the extent of coverage of the adjacent Aß plaques by the remaining microglia. These data suggest that microglia do not create Ox-stress at Aß plaques but instead create protective barriers around Aß plaques possibly reducing the spread of Aß. Intracranial injection of Aß was sufficient to induce neuronal Ox-stress suggesting it to be the initial trigger of Ox-stress generation. Although Ox-stress is increased in DNs, neuronal survival is enhanced following microglia depletion indicating complex and multifactorial roles of microglia with both neurotoxic and neuroprotective components. Increased Ox-stress of DNs was correlated with higher LAMP1 and ubiquitin immunoreactivity supporting proposed mechanistic links between lysosomal accumulation in DNs and their intrinsic generation of Ox-stress. Our results suggest protective as well as neurotoxic roles for microglia at plaques and that the generation of Ox-stress of DNs could intrinsically be generated via lysosomal disruption rather than by microglia. In Brief: Simultaneous imaging of microglia and neuronal Ox-stress revealed a double-edged role for microglia in 5xFAD mice. Plaque associated microglia were attracted to and enwrapped Aß plaques as well as the most highly oxidized DNs. After partial depletion of microglia, DNs were larger with greater levels of Ox-stress. Despite increased Ox-stress after microglia removal neuronal survival improved. Greater Ox-stress was correlated with increased levels of LAMP1 and ubiquitin thereby linking lysosome accumulation and Ox-stress in DNs.

Age-dependent gray matter demyelination is associated with leptomeningeal neutrophil accumulation.

People living with multiple sclerosis (MS) experience episodic CNS white matter lesions instigated by autoreactive T cells. With age, patients with MS show evidence of gray matter demyelination and experience devastating nonremitting symptomology. What drives progression is unclear and studying this has been hampered by the lack of suitable animal models. Here, we show that passive experimental autoimmune encephalomyelitis (EAE) induced by an adoptive transfer of young Th17 cells induced a nonremitting clinical phenotype that was associated with persistent leptomeningeal inflammation and cortical pathology in old, but not young, SJL/J mice. Although the quantity and quality of T cells did not differ in the brains of old versus young EAE mice, an increase in neutrophils and a decrease in B cells were observed in the brains of old mice. Neutrophils were also found in the leptomeninges of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and subpial cortical demyelination. Taken together, our data show that while Th17 cells initiate CNS inflammation, subsequent clinical symptoms and gray matter pathology are dictated by age and associated with other immune cells, such as neutrophils.

Hyperexcitable superior colliculus and fatal brainstem spreading depolarization in a model of Sudden Unexpected Death in Epilepsy.

Cardiorespiratory arrest and death in mouse models of sudden unexpected death in epilepsy occur when spreading depolarization is triggered by cortical seizures and then propagates to the brainstem. However, the critical brain regions and the specific changes required to allow spreading depolarization to propagate to the brainstem under the relatively rare circumstances leading to a fatal seizure are unknown. We previously found that following cortical seizure-inducing electrical stimulation, spreading depolarization could occur in both the superior and inferior colliculi in Cacna1aS218L mice, but was never observed in wild-type animals or following non-seizure-inducing stimuli in Cacna1aS218L mice. Here, we show that optogenetic stimulation of the superior/inferior colliculi in Cacna1aS218L mice induces severe seizures, and resulting spreading depolarization in the superior/inferior colliculi that propagates to the brainstem and correlates with the respiratory arrest followed by cardiac arrest. Further, we show that neurons of the superior colliculus in Cacna1aS218L mice exhibit hyperexcitable properties that we propose underlie a distinct susceptibility to spreading depolarization. Our data suggest that the susceptibility of the superior colliculus to elicit fatal spreading depolarization is a result of either genetic or seizure-related alterations within the superior colliculus that may involve changes to structure, connectivity and/or excitability.

The Oral and Fecal Microbiota in a Canadian Cohort of Alzheimer's Disease.

BACKGROUND: Despite decades of research, our understanding of Alzheimer's disease (AD) etiology remains incomplete. In recent years, appreciation has grown for potential roles for the microbiota in shaping neurological health. OBJECTIVE: This study aimed to examine associations between the microbiota and AD in a human cross-sectional cohort. METHODS: Forty-five AD patients and 54 matched controls were recruited in Vancouver, Canada. Fecal and oral samples underwent 16S microbiota sequencing. A wide array of demographic and clinical data were collected. Differences between participant groups were assessed, and associations between microbes and clinical variables were examined within the AD population. RESULTS: The gut microbiota of AD patients displayed lower diversity relative to controls, although taxonomic differences were sparse. In contrast, the AD oral microbiota displayed higher diversity, with several taxonomic differences relative to controls, including a lower abundance of the families Streptococcaceae and Actinomycetaceae, and a higher abundance of Weeksellaceae, among others. The periodontitis-associated oral microbe Porphyromonas gingivalis was 5 times more prevalent among patients. No significant associations between gut or oral microbes and cognition were detected, but several correlations existed between microbes and mood disorders and BMI among patients, including a strong positive correlation between Alphaproteobacteria and depression score. CONCLUSION: The gut microbiota of AD patients was not overtly different from controls, although it displayed lower diversity, an overall marker of microbiota health. The oral microbiota did display marked differences. Cognition was not associated with a microbial signature, but other relevant AD factors including mood and BMI did demonstrate an association.

Gut microbes shape microglia and cognitive function during malnutrition.

Fecal-oral contamination promotes malnutrition pathology. Lasting consequences of early life malnutrition include cognitive impairment, but the underlying pathology and influence of gut microbes remain largely unknown. Here, we utilize an established murine model combining malnutrition and iterative exposure to fecal commensals (MAL-BG). The MAL-BG model was analyzed in comparison to malnourished (MAL mice) and healthy (CON mice) controls. Malnourished mice display poor spatial memory and learning plasticity, as well as altered microglia, non-neuronal CNS cells that regulate neuroimmune responses and brain plasticity. Chronic fecal-oral exposures shaped microglial morphology and transcriptional profile, promoting phagocytic features in MAL-BG mice. Unexpectedly, these changes occurred independently from significant cytokine-induced inflammation or blood-brain barrier (BBB) disruption, key gut-brain pathways. Metabolomic profiling of the MAL-BG cortex revealed altered polyunsaturated fatty acid (PUFA) profiles and systemic lipoxidative stress. In contrast, supplementation with an ω3 PUFA/antioxidant-associated diet (PAO) mitigated cognitive deficits within the MAL-BG model. These findings provide valued insight into the malnourished gut microbiota-brain axis, highlighting PUFA metabolism as a potential therapeutic target.

Reactive astrocyte nomenclature, definitions, and future directions.

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions.

Gamma frequency activation of inhibitory neurons in the acute phase after stroke attenuates vascular and behavioral dysfunction.

Alterations in gamma oscillations occur in several neurological disorders, and the entrainment of gamma oscillations has been recently proposed as a treatment for neurodegenerative disease. Optogenetic stimulation enhances recovery in models of stroke when applied weeks after injury; however, the benefits of acute brain stimulation have not been investigated. Here, we report beneficial effects of gamma-frequency modulation in the acute phase, within 1 h, after stroke. Transgenic VGAT-ChR2 mice are subject to awake photothrombotic stroke in an area encompassing the forelimb sensory and motor cortex. Optogenetic stimulation at 40 Hz in the peri-infarct zone recovers neuronal activity 24 h after stroke in motor and parietal association areas, as well as blood flow over the first week after stroke. Stimulation significantly reduces lesion volume and improves motor function. Our results suggest that acute-phase modulation of cortical oscillatory dynamics may serve as a target for neuroprotection against stroke.

The Laminin-Induced Phosphorylation of PKCδ Regulates AQP4 Distribution and Water Permeability in Rat Astrocytes.

In astrocytes, the water-permeable channel aquaporin-4 (AQP4) is concentrated at the endfeet that abut the blood vessels of the brain. The asymmetric distribution of this channel is dependent on the function of dystroglycan (DG), a co-expressed laminin receptor, and its associated protein complex. We have demonstrated that the addition of laminin to astrocytes in culture causes the clustering of AQP4, DG, and lipid rafts. The last, in particular, have been associated with the initiation of cell signaling. As laminin binding to DG in muscle cells can induce the tyrosine phosphorylation of syntrophin and laminin requires tyrosine kinases for acetylcholine receptor clustering in myotubes, we asked if signal transduction might also be involved in AQP4 clustering in astrocytes. We analyzed the timecourse of AQP4, DG, and monosialotetrahexosylganglioside (GM1) clustering in primary cultures of rat astrocytes following the addition of laminin, and determined that the clustering of DG precedes that of AQP4 and GM1. We also showed that laminin induces the formation of phosphotyrosine-rich clusters and that the tyrosine kinase inhibitor, genistein, disrupts the laminin-induced clustering of both ß-DG and AQP4. Using the Kinexus antibody microarray chip, we then identified protein-serine kinase C delta (PKCδ) as one of the main proteins exhibiting high levels of tyrosine phosphorylation upon laminin treatment. Selective inhibitors of PKC and siRNA against PKCδ disrupted ß-DG and AQP4 clustering, and also caused water transport to increase in astrocytes treated with laminin. Our results demonstrate that the effects of laminin on AQP4 localization and function are relayed, at least in part, through PKC signaling.

Neuroinflammatory inhibition of synaptic long-term potentiation requires immunometabolic reprogramming of microglia.

Immunometabolism refers to the rearrangement of metabolic pathways in response to immune stimulation, and the ability of these metabolic pathways themselves to control immune functions. Many aspects of immunometabolism have been revealed through studies of peripheral immune cells. However, immunometabolic reprogramming of microglia, the resident immune cell of the central nervous system, and the consequential outcome on neuronal activity have remained difficult to unravel. Microglia are highly sensitive to subtle changes in their environment, limiting the techniques available to study their metabolic and inflammatory profiles. Here, using fluorescence lifetime imaging of endogenous NAD(P)H, we measure the metabolic activity of individual microglia within acute hippocampal slices. We observed an LPS-induced increase in aerobic glycolysis, which was blocked by the addition of 5 mM 2-deoxyglucose (2DG). This LPS-induced glycolysis in microglia was necessary for the stabilization of hypoxia inducible factor-1α (HIF-1α) and production of the proinflammatory cytokine, interleukin-1ß (IL-1ß). Upon release, IL-1ß acted via the neuronal interleukin-1 receptor to inhibit the formation of synaptic long-term potentiation (LTP) following high frequency stimulation. Remarkably, the addition of 2DG to blunt the microglial glycolytic increase also inhibited HIF-1α accumulation and IL-1ß production, and therefore rescued LTP in LPS-stimulated slices. Overall, these data reveal the importance of metabolic reprogramming in regulating microglial immune functions, with appreciable outcomes on cytokine release and neuronal activity.

An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases.

INTRODUCTION: The neurovascular unit (NVU) - the interaction between the neurons and the cerebrovasculature - is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU. METHOD: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. RESULTS: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer's disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model. CONCLUSION: This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

Immunometabolism in the Brain: How Metabolism Shapes Microglial Function.

Immune cells react to their environment by flexibly reprogramming intracellular metabolic pathways that subsequently alter immune function, in a process called immunometabolism. However, in the CNS, the impact of metabolic reprogramming on microglia, neuroinflammation, and subsequently on brain function is poorly understood. As brain-resident macrophages, microglia are the CNS immune effectors and share similarities with peripheral immune cells. New tools for studying immunometabolism now allow the analysis of bioenergetic regulation with cellular resolution and, as a result, have uncovered previously unappreciated roles for microglial immunometabolism in shaping neuroinflammation. This review highlights evidence that microglia metabolism adapts to changes in brain energy homeostasis and that metabolic reprogramming regulates microglial polarization, thereby impacting pathological inflammatory responses in the brain.

Neuron Activity Dependent Redox Compartmentation Revealed with a Second Generation Red-Shifted Ratiometric Sensor.

Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. We previously engineered genetically encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of cocultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric one- and two-photon redox imaging in rat brain slices and Drosophila retinas. Overall, the proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms.

PANX1 in inflammation heats up: New mechanistic insights with implications for injury and infection.

A new study by Yang and colleagues has revealed that TNF-alpha regulates PANX1 levels through an NF-kB-dependent mechanism in human endothelial cells. PANX1 modulates Ca2+ influx contributing to IL-1beta production independent of purinergic signaling. These novel findings expand our understanding of TNF-alpha-mediated upregulation of IL-1beta with implications for responses to tissue injury and infection.