Ultra-fast genetically encoded sensor for precise real-time monitoring of physiological and pathophysiological peroxide dynamics.

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity and response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse brain ex vivo and in vivo. These applications demonstrate oROS's capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aß-putriscine-MAOB axis, highlighting the sensor's relevance in validating neurodegenerative disease models. Lastly, we demonstrated acute opioid-induced generation of H2O2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for understanding diseases associated with oxidative stress, such as cancer, neurodegenerative, and cardiovascular diseases.

Rejuvenating aged microglia by p16ink4a-siRNA-loaded nanoparticles increases amyloid-ß clearance in animal models of Alzheimer's disease.

Age-dependent accumulation of amyloid plaques in patients with sporadic Alzheimer's disease (AD) is associated with reduced amyloid clearance. Older microglia have a reduced ability to phagocytose amyloid, so phagocytosis of amyloid plaques by microglia could be regulated to prevent amyloid accumulation. Furthermore, considering the aging-related disruption of cell cycle machinery in old microglia, we hypothesize that regulating their cell cycle could rejuvenate them and enhance their ability to promote more efficient amyloid clearance. First, we used gene ontology analysis of microglia from young and old mice to identify differential expression of cyclin-dependent kinase inhibitor 2A (p16ink4a), a cell cycle factor related to aging. We found that p16ink4a expression was increased in microglia near amyloid plaques in brain tissue from patients with AD and 5XFAD mice, a model of AD. In BV2 microglia, small interfering RNA (siRNA)-mediated p16ink4a downregulation transformed microglia with enhanced amyloid phagocytic capacity through regulated the cell cycle and increased cell proliferation. To regulate microglial phagocytosis by gene transduction, we used poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, which predominantly target microglia, to deliver the siRNA and to control microglial reactivity. Nanoparticle-based delivery of p16ink4a siRNA reduced amyloid plaque formation and the number of aged microglia surrounding the plaque and reversed learning deterioration and spatial memory deficits. We propose that downregulation of p16ink4a in microglia is a promising strategy for the treatment of Alzheimer's disease.

Tonic NMDAR Currents in the Brain: Regulation and Cognitive Functions.

Synaptically localized N-methyl-D-aspartate receptors (NMDARs) play a crucial role in important cognitive functions by mediating synaptic transmission and plasticity. In contrast, a tonic NMDAR current exists, thought to be mediated by extrasynaptic NMDARs, with a less clear function. This review provides a comprehensive overview of tonic NMDAR currents, focusing on their roles in synaptic transmission/plasticity and their impact on cognitive functions and psychiatric disorders. We discuss the roles of three endogenous ligands (i.e. glutamate, glycine, and D-serine) and receptors in mediating tonic NMDAR currents and explore the diverse mechanisms that regulate tonic NMDAR currents. In light of recent controversies surrounding the source of D-serine, we highlight the recent findings suggesting that astrocytes release D-serine to modulate tonic NMDAR currents and control cognitive flexibility. Furthermore, we propose the distinct roles of neuronal and astrocytic D-serine in different locations and their implications on synaptic regulation and cognitive functions. The potential roles of tonic NMDAR currents in various psychiatric disorders, such as schizophrenia and autism spectrum disorders (ASD), are discussed in the context of the NMDAR hypofunction hypothesis. By presenting the mechanisms in which various cells, particularly astrocytes, regulate tonic NMDAR currents, we aim to stimulate future research in NMDAR hypofunction- or hyperfunction-related psychiatric disorders. This review will not only provide a better understanding of the complex interplay between tonic NMDAR currents and cognitive functions but also shed light on its potential therapeutic target for the treatment of various psychiatric disorders.

Neurotoxic Microglial Activation via IFNγ-Induced Nrf2 Reduction Exacerbating Alzheimer's Disease.

Microglial neuroinflammation appears to be neuroprotective in the early pathological stage, yet neurotoxic, which often precedes neurodegeneration in Alzheimer's disease (AD). However, it remains unclear how the microglial activities transit to the neurotoxic state during AD progression, due to complex neuron-glia interactions. Here, the mechanism of detrimental microgliosis in AD by employing 3D human AD mini-brains, brain tissues of AD patients, and 5XFAD mice is explored. In the human and animal AD models, amyloid-beta (Aß)-overexpressing neurons and reactive astrocytes produce interferon-gamma (IFNγ) and excessive oxidative stress. IFNγ results in the downregulation of mitogen-activated protein kinase (MAPK) and the upregulation of Kelch-like ECH-associated Protein 1 (Keap1) in microglia, which inactivate nuclear factor erythroid-2-related factor 2 (Nrf2) and sensitize microglia to the oxidative stress and induces a proinflammatory microglia via nuclear factor kappa B (NFκB)-axis. The proinflammatory microglia in turn produce neurotoxic nitric oxide and proinflammatory mediators exacerbating synaptic impairment, phosphorylated-tau accumulation, and discernable neuronal loss. Interestingly, recovering Nrf2 in the microglia prevents the activation of proinflammatory microglia and significantly blocks the tauopathy in AD minibrains. Taken together, it is envisioned that IFNγ-driven Nrf2 downregulation in microglia as a key target to ameliorate AD pathology.

Transmembrane proteins with unknown function (TMEMs) as ion channels: electrophysiological properties, structure, and pathophysiological roles.

A transmembrane (TMEM) protein with an unknown function is a type of membrane-spanning protein expressed in the plasma membrane or the membranes of intracellular organelles. Recently, several TMEM proteins have been identified as functional ion channels. The structures and functions of these proteins have been extensively studied over the last two decades, starting with TMEM16A (ANO1). In this review, we provide a summary of the electrophysiological properties of known TMEM proteins that function as ion channels, such as TMEM175 (KEL), TMEM206 (PAC), TMEM38 (TRIC), TMEM87A (GolpHCat), TMEM120A (TACAN), TMEM63 (OSCA), TMEM150C (Tentonin3), and TMEM43 (Gapjinc). Additionally, we examine the unique structural features of these channels compared to those of other well-known ion channels. Furthermore, we discuss the diverse physiological roles of these proteins in lysosomal/endosomal/Golgi pH regulation, intracellular Ca2+ regulation, spatial memory, cell migration, adipocyte differentiation, and mechanical pain, as well as their pathophysiological roles in Parkinson's disease, cancer, osteogenesis imperfecta, infantile hypomyelination, cardiomyopathy, and auditory neuropathy spectrum disorder. This review highlights the potential for the discovery of novel ion channels within the TMEM protein family and the development of new therapeutic targets for related channelopathies.

Hypothalamic astrocyte NAD+ salvage pathway mediates the coupling of dietary fat overconsumption in a mouse model of obesity.

Nicotinamide adenine dinucleotide (NAD)+ serves as a crucial coenzyme in numerous essential biological reactions, and its cellular availability relies on the activity of the nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed salvage pathway. Here we show that treatment with saturated fatty acids activates the NAD+ salvage pathway in hypothalamic astrocytes. Furthermore, inhibition of this pathway mitigates hypothalamic inflammation and attenuates the development of obesity in male mice fed a high-fat diet (HFD). Mechanistically, CD38 functions downstream of the NAD+ salvage pathway in hypothalamic astrocytes burdened with excess fat. The activation of the astrocytic NAMPT-NAD+-CD38 axis in response to fat overload induces proinflammatory responses in the hypothalamus. It also leads to aberrantly activated basal Ca2+ signals and compromised Ca2+ responses to metabolic hormones such as insulin, leptin, and glucagon-like peptide 1, ultimately resulting in dysfunctional hypothalamic astrocytes. Our findings highlight the significant contribution of the hypothalamic astrocytic NAD+ salvage pathway, along with its downstream CD38, to HFD-induced obesity.

Long-lasting forms of plasticity through patterned ultrasound-induced brainwave entrainment.

Achieving long-lasting neuronal modulation with low-intensity, low-frequency ultrasound is challenging. Here, we devised theta burst ultrasound stimulation (TBUS) with gamma bursts for brain entrainment and modulation of neuronal plasticity in the mouse motor cortex. We demonstrate that two types of TBUS, intermittent and continuous TBUS, induce bidirectional long-term potentiation or depression-like plasticity, respectively, as evidenced by changes in motor-evoked potentials. These effects depended on molecular pathways associated with long-term plasticity, including N-methyl-d-aspartate receptor and brain-derived neurotrophic factor/tropomyosin receptor kinase B activation, as well as de novo protein synthesis. Notably, bestrophin-1 and transient receptor potential ankyrin 1 play important roles in these enduring effects. Moreover, pretraining TBUS enhances the acquisition of previously unidentified motor skills. Our study unveils a promising protocol for ultrasound neuromodulation, enabling noninvasive and sustained modulation of brain function.

Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity or response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse neurons and astrocytes in ex vivo brain slices. These applications demonstrate oROS's capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress, G-protein coupled receptor (GPCR)-induced cell signaling, and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aß-putriscine-MAOB axis, highlighting the sensor's relevance in validating neurodegenerative disease models. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for diseases associated with oxidative stress, such as cancer, neurodegenerative disorders, and cardiovascular diseases.

KDS2010, a reversible MAO-B inhibitor, extends the lifetime of neural probes by preventing glial scar formation.

Implantable neural probes have been extensively utilized in the fields of neurocircuitry, systems neuroscience, and brain-computer interface. However, the long-term functionality of these devices is hampered by the formation of glial scar and astrogliosis at the surface of electrodes. In this study, we administered KDS2010, a recently developed reversible MAO-B inhibitor, to mice through ad libitum drinking in order to prevent glial scar formation and astrogliosis. The administration of KDS2010 allowed long-term recordings of neural signals with implantable devices, which remained stable over a period of 6 months and even restored diminished neural signals after probe implantation. KDS2010 effectively prevented the formation of glial scar, which consists of reactive astrocytes and activated microglia around the implant. Furthermore, it restored neural activity by disinhibiting astrocytic MAO-B dependent tonic GABA inhibition induced by astrogliosis. We suggest that the use of KDS2010 is a promising approach to prevent glial scar formation around the implant, thereby enabling long-term functionality of neural devices.

Long-term inhibition of ODC1 in APP/PS1 mice rescues amyloid pathology and switches astrocytes from a reactive to active state.

Alzheimer's disease (AD) is characterized by the loss of memory due to aggregation of misphosphorylated tau and amyloid beta (Aß) plaques in the brain, elevated release of inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and reactive oxygen species from astrocytes, and subsequent neurodegeneration. Recently, it was found that enzyme Ornithine Decarboxylase 1 (ODC1) acts as a bridge between the astrocytic urea cycle and the putrescine-to-GABA conversion pathway in the brain of AD mouse models as well as human patients. In this study, we show that the long-term knockdown of astrocytic Odc1 in APP/PS1 animals was sufficient to completely clear Aß plaques in the hippocampus while simultaneously switching the astrocytes from a detrimental reactive state to a regenerative active state, characterized by proBDNF expression. Our experiments also reveal an effect of astrocytic ODC1 inhibition on the expression of genes involved in synapse pruning and organization, histone modification, apoptotic signaling and protein processing. These genes are previously known to be associated with astrocytic activation and together create a neuroregeneration-supportive environment in the brain. By inhibiting ODC1 for a long period of 3 months in AD mice, we demonstrate that the beneficial amyloid-clearing process of astrocytes can be completely segregated from the systemically harmful astrocytic response to insult. Our study reports an almost complete clearance of Aß plaques by controlling an endogenous degradation process, which also modifies the astrocytic state to create a regeneration-supportive environment in the brain. These findings present the potential of modulating astrocytic clearance of Aß as a powerful therapeutic strategy against AD.

Triterpenoidal Saponins from the Leaves of Aster koraiensis Offer Inhibitory Activities against SARS-CoV-2.

Triterpenoidal saponins have been reported to be able to restrain SARS-CoV-2 infection. To isolate antiviral compounds against SARS-CoV-2 from the leaves of Aster koraiensis, we conducted multiple steps of column chromatography. We isolated six triperpenoidal saponins from A. koraiensis leaves, including three unreported saponins. Their chemical structures were determined using HR-MS and NMR data analyses. Subsequently, we tested the isolates to assess their ability to impede the entry of the SARS-CoV-2 pseudovirus (pSARS-CoV-2) into ACE2+ H1299 cells and found that five of the six isolates displayed antiviral activity with an IC50 value below 10 µM. Notably, one unreported saponin, astersaponin J (1), blocks pSARS-CoV-2 in ACE2+ and ACE2/TMPRSS2+ cells with similar IC50 values (2.92 and 2.96 µM, respectively), without any significant toxic effect. Furthermore, our cell-to-cell fusion and SARS-CoV-2 Spike-ACE2 binding assays revealed that astersaponin J inhibits membrane fusion, thereby blocking both entry pathways of SARS-CoV-2 while leaving the interaction between the SARS-CoV-2 Spike and ACE2 unaffected. Overall, this study expands the list of antiviral saponins by introducing previously undescribed triterpenoidal saponins isolated from the leaves of A. koraiensis, thereby corroborating the potency of triterpenoid saponins in impeding SARS-CoV-2 infection.

Employing the sustained-release properties of poly(lactic-co-glycolic acid) nanoparticles to reveal a novel mechanism of sodium-hydrogen exchanger 1 in neuropathic pain.

Neuropathic pain is caused by injury or disease of the somatosensory system, and its course is usually chronic. Several studies have been dedicated to investigating neuropathic pain-related targets; however, little attention has been paid to the persistent alterations that these targets, some of which may be crucial to the pathophysiology of neuropathic pain. The present study aimed to identify potential targets that may play a crucial role in neuropathic pain and validate their long-term impact. Through bioinformatics analysis of RNA sequencing results, we identified Slc9a1 and validated the reduced expression of sodium-hydrogen exchanger 1 (NHE1), the protein that Slc9a1 encodes, in the spinal nerve ligation (SNL) model. Colocalization analysis revealed that NHE1 is primarily co-localized with vesicular glutamate transporter 2-positive neurons. In vitro experiments confirmed that poly(lactic-co-glycolic acid) nanoparticles loaded with siRNA successfully inhibited NHE1 in SH-SY5Y cells, lowered intracellular pH, and increased intracellular calcium concentrations. In vivo experiments showed that sustained suppression of spinal NHE1 expression by siRNA-loaded nanoparticles resulted in delayed hyperalgesia in naïve and SNL model rats, whereas amiloride-induced transient suppression of NHE1 expression yielded no significant changes in pain sensitivity. We identified Slc9a1, which encodes NHE1, as a key gene in neuropathic pain. Utilizing the sustained release properties of nanoparticles enabled us to elucidate the chronic role of decreased NHE1 expression, establishing its significance in the mechanisms of neuropathic pain.

Visualizing Cancer-Originating Acetate Uptake Through MCT1 in Reactive Astrocytes in the Glioblastoma Tumor Microenvironment.

BACKGROUND: Reactive astrogliosis is a hallmark of various brain pathologies, including neurodegenerative diseases and glioblastomas. However, the specific intermediate metabolites contributing to reactive astrogliosis remain unknown. This study investigated how glioblastomas induce reactive astrogliosis in the neighboring microenvironment and explores 11C-acetate PET as an imaging technique for detecting reactive astrogliosis. METHODS: Through in vitro, mouse models, and human tissue experiments, we examined the association between elevated 11C-acetate uptake and reactive astrogliosis in gliomas. We explored acetate from glioblastoma cells, which triggers reactive astrogliosis in neighboring astrocytes by upregulating MAO-B and MCT1 expression. We evaluated the presence of cancer stem cells in the reactive astrogliosis region of glioblastomas and assessed the correlation between the volume of 11C-acetate uptake beyond MRI and prognosis. RESULTS: Elevated 11C-acetate uptake is associated with reactive astrogliosis and astrocytic MCT1 in the periphery of glioblastomas in human tissues and mouse models. Glioblastoma cells exhibit increased acetate production as a result of glucose metabolism, with subsequent secretion of acetate. Acetate derived from glioblastoma cells induces reactive astrogliosis in neighboring astrocytes by increasing the expression of MAO-B and MCT1. We found cancer stem cells within the reactive astrogliosis at the tumor periphery. Consequently, a larger volume of 11C-acetate uptake beyond contrast-enhanced MRI was associated with worse prognosis. CONCLUSION: Our results highlight the role of acetate derived from glioblastoma cells in inducing reactive astrogliosis and underscore the potential value of 11C-acetate PET as an imaging technique for detecting reactive astrogliosis, offering important implications for the diagnosis and treatment of glioblastomas.

Ultrasonocoverslip: In-vitro platform for high-throughput assay of cell type-specific neuromodulation with ultra-low-intensity ultrasound stimulation.

Brain stimulation with ultra-low-intensity ultrasound has rarely been investigated due to the lack of a reliable device to measure small neuronal signal changes made by the ultra-low intensity range. We propose Ultrasonocoverslip, an ultrasound-transducer-integrated-glass-coverslip that determines the minimum intensity for brain cell activation. Brain cells can be cultured directly on Ultrasonocoverslip to simultaneously deliver uniform ultrasonic pressure to hundreds of cells with real-time monitoring of cellular responses using fluorescence microscopy and single-cell electrophysiology. The sensitivity for detecting small responses to ultra-low-intensity ultrasound can be improved by averaging simultaneously obtained responses. Acoustic absorbers can be placed under Ultrasonocoverslip, and stimuli distortions are substantially reduced to precisely deliver user-intended acoustic stimulations. With the proposed device, we discover the lowest acoustic threshold to induce reliable neuronal excitation releasing glutamate. Furthermore, mechanistic studies on the device show that the ultra-low-intensity ultrasound stimulation induces cell type-specific neuromodulation by activating astrocyte-mediated neuronal excitation without direct neuronal involvement. The performance of ultra-low-intensity stimulation is validated by in vivo experiments demonstrating improved safety and specificity in motor modulation of tail movement compared to that with supra-watt-intensity.

Hypothalamic GABRA5-positive neurons control obesity via astrocytic GABA.

The lateral hypothalamic area (LHA) regulates food intake and energy balance. Although LHA neurons innervate adipose tissues, the identity of neurons that regulate fat is undefined. Here we show that GABRA5-positive neurons in LHA (GABRA5LHA) polysynaptically project to brown and white adipose tissues in the periphery. GABRA5LHA are a distinct subpopulation of GABAergic neurons and show decreased pacemaker firing in diet-induced obesity mouse models in males. Chemogenetic inhibition of GABRA5LHA suppresses fat thermogenesis and increases weight gain, whereas gene silencing of GABRA5 in LHA decreases weight gain. In the diet-induced obesity mouse model, GABRA5LHA are tonically inhibited by nearby reactive astrocytes releasing GABA, which is synthesized by monoamine oxidase B (Maob). Gene silencing of astrocytic Maob in LHA facilitates fat thermogenesis and reduces weight gain significantly without affecting food intake, which is recapitulated by administration of a Maob inhibitor, KDS2010. We propose that firing of GABRA5LHA suppresses fat accumulation and selective inhibition of astrocytic GABA is a molecular target for treating obesity.

Cortical astrocytes modulate dominance behavior in male mice by regulating synaptic excitatory and inhibitory balance.

Social hierarchy is established as an outcome of individual social behaviors, such as dominance behavior during long-term interactions with others. Astrocytes are implicated in optimizing the balance between excitatory and inhibitory (E/I) neuronal activity, which may influence social behavior. However, the contribution of astrocytes in the prefrontal cortex to dominance behavior is unclear. Here we show that dorsomedial prefrontal cortical (dmPFC) astrocytes modulate E/I balance and dominance behavior in adult male mice using in vivo fiber photometry and two-photon microscopy. Optogenetic and chemogenetic activation or inhibition of dmPFC astrocytes show that astrocytes bidirectionally control male mouse dominance behavior, affecting social rank. Dominant and subordinate male mice present distinct prefrontal synaptic E/I balance, regulated by astrocyte activity. Mechanistically, we show that dmPFC astrocytes control cortical E/I balance by simultaneously enhancing presynaptic-excitatory and reducing postsynaptic-inhibitory transmission via astrocyte-derived glutamate and ATP release, respectively. Our findings show how dmPFC astrocyte-neuron communication can be involved in the establishment of social hierarchy in adult male mice.

A neural probe for concurrent real-time measurement of multiple neurochemicals with electrophysiology in multiple brain regions in vivo.

Real-time monitoring of various neurochemicals with high spatial resolution in multiple brain regions in vivo can elucidate neural circuits related to various brain diseases. However, previous systems for monitoring neurochemicals have limitations in observing multiple neurochemicals without crosstalk in real time, and these methods cannot record electrical activity, which is essential for investigating neural circuits. Here, we present a real-time bimodal (RTBM) neural probe that uses monolithically integrated biosensors and multiple shanks to study the connectivity of neural circuits by measuring multiple neurochemicals and electrical neural activity in real time. Using the RTBM probe, we demonstrate concurrent measurements of four neurochemicals-glucose, lactate, choline, and glutamate without cross-talking each other-and electrical activity in real time in vivo. Additionally, we show the functional connectivity between the medial prefrontal cortex and mediodorsal thalamus through the simultaneous measurement of chemical and electrical signals. We expect that our device will contribute to not only elucidating the role of neurochemicals in neural circuits related to brain functions but also developing drugs for various brain diseases related to neurochemicals.