Abstract 18 F-THK5351, initially developed as a positron-emission tomography (PET) tracer for tau pathology, was later shown to display high affinity for monoamine oxidase-B (MAO-B), raising uncertainty about the biological origin of its brain signals in neurodegenerative diseases. To resolve this ambiguity, we implemented a multi-scale validation framework integrating enzyme activity inhibition assays, molecular docking, biolayer interferometry, autoradiography, multiple transgenic and viral animal models, and human PET imaging. THK5351 selectively inhibited MAO-B while sparing MAO-A and exhibited reversible binding kinetics to recombinant MAO-B. Computational modelling localized THK5351 near the MAO-B substrate funnel, revealing moderate binding energy and weaker π–π stacking interactions compared with selective tau tracers. Autoradiographic analysis of human cortical tissue demonstrated that tracer binding was dominated by MAO-B-related signals, with a smaller contribution from tau aggregates, a difference insufficient to produce visually distinguishable patterns in clinical imaging. In APP/PS1 mice, 18 F-THK5351 uptake colocalized with regions of reactive astrogliosis and was abolished by MAO-B inhibition, whereas overexpression of P301L-hTau induced extensive tau deposition without affecting tracer retention. MAO-B knockout reduced both tracer binding and tau phosphorylation, and viral induction of astrogliosis elevated tracer uptake that was reversed by selective MAO-B blockade. In a patient with corticobasal syndrome, tracer signals decreased during selegiline treatment and reappeared after drug withdrawal, mirroring preclinical pharmacological responses. Collectively, these findings demonstrate that 18 F-THK5351 uptake primarily reflects MAO-B-mediated reactive astrogliosis rather than tau pathology, providing mechanistic insight into its signal origin and underscoring the value of cross-scale, multimodal validation in PET tracer development for neurodegenerative disease research.

Abstract Astroglia, an extended class of homeostatic and defensive cells of the central nervous system (CNS), contribute to the pathogenesis of all known neurological and neuropsychiatric disorders. The pathophysiology of astrocytes is complex, mutable, disease and disease-stage specific. In neuroinflammatory lesions and in various chronic conditions, astrocytes undergo an evolutionary conserved defensive remodeling known as reactive astrogliosis, which produces highly heterogeneous reactive astrocytic phenotypes. Broadly, reactive astrogliosis can be classified into proliferative anysomorphic barrier-forming astrogliosis characteristic of traumatic CNS lesions and nonproliferative isomorphic gliosis widely manifested in chronic neuropathologies. In addition, in many pathologies, astrocytes undergo atrophy and asthenia with resulting loss of homeostatic support and neuroprotection precipitating neuronal damage. Reactive and atrophic astrocytes may coexist or emerge in sequence in a disease-stage-dependent manner. Several classes of astrocyte-specific molecules and processes implicated in various diseases of the CNS represent therapeutic targets. Astrocyte-specific therapeutic strategies may improve both disease-preventing and disease-modifying therapeutic outcomes.

Programmed cell death protein 1 (PD-1) and its ligand (PD-L1) are crucial in cancer immune evasion and in modulating neuroinflammation. Although PD-1/PD-L1 signaling is believed to modulate immune and neuronal responses, its role in AD pathophysiology remains unclear, with existing studies reporting inconsistent findings. This systematic review and meta-analysis investigated the effects of PD-1/PD-L1 blockade on AD-related pathology and cognitive behavior in preclinical studies. Additionally, we evaluated the impact of PD-1/PD-L1 inhibitors on cognitive outcomes in clinical studies involving cancer patients. Relevant research was systematically identified using the MEDLINE, Embase, CENTRAL, and Web of Science databases from their inception until July 31, 2025. Overall, 40 studies were included in this meta-analysis, conducted using R software. Preclinical studies revealed that blockade of PD-1 signaling reduces amyloid-beta plaque burden, tau phosphorylation, and astrocyte reactivity in AD mouse models. These pathological improvements were accompanied by enhanced cognitive performance, whereas wild-type mice showed no significant cognitive changes under the same treatment, whereas wild-type mice showed no significant cognitive changes under the same treatment. Furthermore, clinical studies demonstrated the beneficial effect of PD-1 signaling inhibitors on cognitive function in patients with cancer. PD-1/PD-L1 inhibition impacts AD pathology and cognitive function, suggesting its potential as a therapeutic development strategy for AD. Further studies are warranted to clarify the exact mechanisms, opening avenues for future therapies that modulate the PD-1/PD-L1 pathway for AD.

Hemoglobin (Hb) is well known for transporting oxygen in the blood, but its role in the brain remains poorly understood. Here, we identified Hb in the cytosol, mitochondria, and nuclei of hippocampal and substantia nigra astrocytes and dopaminergic neurons. As a pseudoperoxidase, Hb decomposes hydrogen peroxide (H2O2) and mitigates H2O2-induced oxidative damage. However, in Alzheimer’s disease, Parkinson’s disease, and aging, excessive H2O2 diminishes astrocytic Hb, perpetuating a vicious cycle of oxidative stress and neurodegeneration. To counter the harmful effects of aberrant H2O2 production in diseases, we developed KDS12025, a BBB-permeable small molecule that enhances Hb pseudoperoxidase activity 100-fold, even at a low level of Hb. KDS12025 and its analogs achieve this enhancement through its electron-donating amine group, possibly stabilizing the complex between Hb, H2O2, and KDS12025. KDS12025 reduces astrocytic H2O2, alleviates astrogliosis, normalizes Hb, and reverts to a virtuous cycle of redox balance, preventing neurodegeneration without altering the oxygen-transport function of Hb. Gene silencing of Hb abrogates the impact of KDS12025 in both culture and animal models, confirming the necessity of Hb for the effects of KDS12025. KDS12025 extends survival and improves motor function even in severe amyotrophic lateral sclerosis and aging. Furthermore, the enrichment of astrocytic Hb in the nucleolus highlights a novel antioxidative mechanism potentially protecting against nuclear oxidative damage. Our findings suggest that Hb is a new therapeutic target for neurodegenerative diseases, with KDS12025 emerging as a first-in-class approach that enhances Hb pseudoperoxidase activity to reduce H2O2. Increasing Hb pseudoperoxidase activity with KDS12025 mitigates oxidative stress and alleviates neurodegeneration in AD, PD, and ALS patients and increases the degree of aging, with broad applicability for numerous oxidative-stress-driven diseases.

Three years ago, our group reported direct imaging of neuronal activity (DIANA) with high spatiotemporal resolution, but its reproducibility and signal origin remain controversial. Here, we report the results of our reproduction experiments of DIANA fMRI performed to date using forelimb electrical stimulation at various magnetic field strengths in anesthetized mice, along with the characteristics of DIANA signal, called the pseudo-steady state (PSS). Theoretical analysis and Bloch simulations demonstrated that the spatial location and temporal phase of PSS oscillations are primarily determined by frequency-offset, and that their spatiotemporal superposition can generate peak signals in specific regions at specific timing that closely resemble DIANA signals. These findings suggest that if PSS oscillations are the primary source of DIANA signals, it may be premature to interpret them as neuronal responses to sensory stimulation. Further studies are needed to clarify the relationship between DIANA signals and brain activation. ### Competing Interest Statement The authors have declared no competing interest.

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.

BACKGROUND:Astrocytes, one of the most resilient cells in the brain, transform into reactive astrocytes in response to toxic proteins such as amyloid beta (Aβ) in Alzheimer's disease (AD). However, reactive astrocyte-mediated non-cell autonomous neuropathological mechanism is not fully understood yet. We aimed our study to find out whether Aβ-induced proteotoxic stress affects the expression of autophagy genes and the modulation of autophagic flux in astrocytes, and if yes, how Aβ-induced autophagy-associated genes are involved Aβ clearance in astrocytes of animal model of AD. METHODS:Whole RNA sequencing (RNA-seq) was performed to detect gene expression patterns in Aβ-treated human astrocytes in a time-dependent manner. To verify the role of astrocytic autophagy in an AD mouse model, we developed AAVs expressing shRNAs for MAP1LC3B/LC3B (LC3B) and Sequestosome1 (SQSTM1) based on AAV-R-CREon vector, which is a Cre recombinase-dependent gene-silencing system. Also, the effect of astrocyte-specific overexpression of LC3B on the neuropathology in AD (APP/PS1) mice was determined. Neuropathological alterations of AD mice with astrocytic autophagy dysfunction were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through novel object recognition test (NOR) and novel object place recognition test (NOPR). RESULTS:Here, we show that astrocytes, unlike neurons, undergo plastic changes in autophagic processes to remove Aβ. Aβ transiently induces expression of LC3B gene and turns on a prolonged transcription of SQSTM1 gene. The Aβ-induced astrocytic autophagy accelerates urea cycle and putrescine degradation pathway. Pharmacological inhibition of autophagy exacerbates mitochondrial dysfunction and oxidative stress in astrocytes. Astrocyte-specific knockdown of LC3B and SQSTM1 significantly increases Aβ plaque formation and GFAP-positive astrocytes in APP/PS1 mice, along with a significant reduction of neuronal marker and cognitive function. In contrast, astrocyte-specific overexpression of LC3B reduced Aβ aggregates in the brain of APP/PS1 mice. An increase of LC3B and SQSTM1 protein is found in astrocytes of the hippocampus in AD patients. CONCLUSIONS:Taken together, our data indicates that Aβ-induced astrocytic autophagic plasticity is an important cellular event to modulate Aβ clearance and maintain cognitive function in AD mice.

Two years ago, our group reported direct imaging of neuronal activity (DIANA), a functional magnetic resonance imaging (fMRI) technique that directly detects neuronal activity at high spatiotemporal resolution. In this study, we successfully reproduced the DIANA response in medetomidine-anesthetized mice using forelimb electrical stimulation at 11.7 T. More importantly, we showed that multiple neural circuits can be effectively revealed by DIANA fMRI through spatiotemporal activation mapping. The spatiotemporal activation mapping proposed here utilizes the temporal information of the DIANA response, that is, the time when the DIANA response reaches its peak, which is a unique feature that distinguishes it from the activation mapping method used in existing fMRI. Based on DIANA activation areas, we identified several neural circuits involved in forelimb sensory processing in the somatosensory network, which includes multiple brain regions: ventral posterolateral nucleus of the thalamus (VPL), posteromedial thalamic nucleus (POm), forelimb primary somatosensory cortex (S1FL), secondary somatosensory cortex (S2), primary motor cortex (M1), and secondary motor cortex (M2). Additionally, we also identified a pain-related neural circuit involving brain regions of the anterior cingulate cortex (ACC) and mediodorsal nucleus (MD). Interestingly, the spatiotemporal activation mapping also allowed us to identify subregions with different DIANA response times within the same functional region (e.g., VPL, POm, S1FL, and S2). Our study highlights the potential of DIANA fMRI to advance our understanding of sensory information processing throughout the brain and to provide insight into the spatiotemporal dynamics of brain networks at the level of neural circuits. ### Competing Interest Statement The authors have declared no competing interest.

Summary paragraph Hemoglobin (Hb) is well-known for transporting oxygen in red blood cells within blood vessels 1 . Although Hb is also present in the brain 2 , its role remains poorly understood. Here, we show that Hb, found in astrocytes of neurodegenerative animal models and patients, displays significant antioxidant effects through its H 2 O 2 -decomposing peroxidase activity, and a small molecule enhancer boosts this activity, reducing aberrant H 2 O 2 and mitigating H 2 O 2 -induced neurodegeneration. To counteract the harmful effects of aberrant H 2 O 2 -production in Alzheimer’s disease (AD), we developed KDS12025, a blood-brain barrier (BBB)-permeable small molecule that effectively enhances the peroxidase activity of Hb by a hundredfold, especially at a low level of Hb. KDS12025 and its analogs achieve this enhancement through its electron-donating amine group. KDS12025 reduces H 2 O 2 levels in astrocytes, exhibits neuroprotective effects, and reverses memory impairment in AD models. Gene-silencing of Hbβ abrogates KDS12025’s impact in both culture and animal models of AD. Moreover, KDS12025 prevented the death of dopaminergic neurons in a Parkinson’s disease (PD) model without altering the oxygen-transporting function of Hb. KDS12025 extended survival and improved motor function even in the severe amyotrophic lateral sclerosis (ALS) mouse model. Our findings propose Hb as a new therapeutic target for neurodegenerative diseases, with KDS12025 emerging as a first-in-class drug candidate that enhances Hb’s peroxidase activity to reduce H 2 O 2 . Boosting Hb’s peroxidase activity with KDS12025 mitigates oxidative stress and alleviates neurodegeneration in AD, PD, and ALS with broad applicability for numerous oxidative-stress-driven diseases.

Pathological pain is caused by abnormal activity in the neural circuit that transmits nociceptive stimuli. Beyond homeostatic functions, astrocytes actively participate in regulating synaptic transmission as members of tripartite synapses. The perisynaptic astrocytic process (PAP) is the key structure that allows astrocytes to play these roles and not only physically supports synapse formation through cell adhesion molecules (CAMs) but also regulates the efficiency of chemical signaling. Accumulating evidence has revealed that spinal astrocytes are involved in pathological pain by modulating the efficacy of neurotransmitters such as glutamate and GABA through transporters located in the PAP and by directly regulating synaptic transmission through various gliotransmitters. Although various CAMs contribute to pathological pain, insufficient evidence is available as to whether astrocytic CAMs also have this role. Therefore, more in-depth research is needed on how pathological pain is induced and maintained by astrocytes, especially in the PAP surrounding the synapse, and this will subsequently increase our understanding and treatment of pathological pain.

Astrocytes directly participate in learning and memory. However, the structural association between astrocytes and memory-encoding engram neurons after learning remains to be elucidated. We developed astrocyte-enhanced green fluorescent protein reconstitution across synaptic partners (eGRASP) to examine tripartite synapses between astrocytes and engram neurons. Using astrocyte-eGRASP, we found that astrocytes had increased connections to engram neurons after learning. Dendritic spines with astrocytic contacts showed enhanced morphology. Live-cell imaging of astrocyte-eGRASP revealed that astrocytic connections are stabilized by neuronal activity. These results indicate that astrocytes distinguish contact between engram neurons and generate engram-specific contact patterns during learning.

BACKGROUND: NMDA receptor (NMDAR) hypofunction has been implicated in several psychiatric disorders with impairment of cognitive flexibility. However, the molecular mechanism of how NMDAR hypofunction with decreased NMDAR tone causes the impairment of cognitive flexibility has been minimally understood. Furthermore, it has been unclear whether hippocampal astrocytes regulate NMDAR tone and cognitive flexibility. METHODS: We employed cell type-specific genetic manipulations, ex vivo electrophysiological recordings, sniffer patch recordings, cutting-edge biosensor for norepinephrine, and behavioral assays to investigate whether astrocytes can regulate NMDAR tone by releasing D-serine and glutamate. Subsequently, we further investigated the role of NMDAR tone in heterosynaptic long-term depression, metaplasticity, and cognitive flexibility. RESULTS: We found that hippocampal astrocytes regulate NMDAR tone via BEST1-mediated corelease of D-serine and glutamate. Bestl knockout mice exhibited reduced NMDAR tone and impairments of homosynaptic and cc i adrenergic receptor-dependent heterosynaptic long-term depression, which leads to defects in metaplasticity and cognitive flexibility. These impairments in Bestl knockout mice can be rescued by hippocampal astrocyte-specific BEST1 expression or enhanced NMDAR tone through D-serine supplement. D-serine injection in Bestl knockout mice during initial learning rescues subsequent reversal learning. CONCLUSIONS: These findings indicate that NMDAR tone during initial learning is important for subsequent learning, and hippocampal NMDAR tone regulated by astrocytic BEST1 is critical for heterosynaptic long-term depression, metaplasticity, and cognitive flexibility.

Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage indicating blood-brain barrier (BBB) dysfunction. At day 5, edema disappeared with recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct. Oral administration of a reversible monoamine oxidase B inhibitor significantly decreased the astrocyte reactivity and fibrous scar formation. Our result suggests that astrocyte reactivity may be a key target to alleviate the impact of microinfarction.

Cover Illustration: 3D reconstructed image of glial fibrillary acidic protein (GFAP) of scar-forming reactive astrocytes in penetrating brain injury model. The pseudo-colored image represents the gradient volume of reactive astrocytes near the injury site. (See Chun, H., et al, https://doi.org/10.1002/glia.24110.)

Summary Cognitive flexibility is an essential ability to adapt to changing environment and circumstances. NMDAR has long been implicated in cognitive flexibility, but the precise molecular and cellular mechanism is not well understood. Here, we report that astrocytes regulate NMDAR tone through Best1-mediated glutamate and D-serine release, which is critical for cognitive flexibility. Co-release of D-serine and glutamate is required for not only homosynaptic LTD but also heterosynaptic LTD, which is induced at unstimulated synapses upon release of norepinephrine and activation of astrocytic α1-AR during homosynaptic LTP. Remarkably, heterosynaptic LTD at unstimulated synapses during memory acquisition is required for later repotentiation LTP during reversal learning, laying a foundation for flexible memory and cognitive flexibility. Our study sheds light on the pivotal role of astrocytes in orchestrating multiple synapses during memory formation and determining the fate of consolidated memory to be retained as a flexible memory. Highlights Astrocytes regulate NMDAR tone via Best1-mediated glutamate and D-serine release Activation of astrocytic α1-AR induces heterosynaptic LTD via NMDAR tone Heterosynaptic LTD is required for repotentiation LTP and spatial reversal learning Astrocytic regulation of NMDAR tone is critical for metaplasticity and flexible memory

Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage indicating blood-brain barrier (BBB) dysfunction. At day 5, edema disappeared with recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct. Oral administration of a reversible monoamine oxidase B inhibitor significantly decreased the astrocyte reactivity and fibrous scar formation. Our result suggests that astrocyte reactivity may be a key target to alleviate the impact of microinfarction.

The role of astrocytes in brain plasticity has not been extensively studied compared with that of neurons. Here we adopted integrative translational and reverse-translational approaches to explore the role of an astrocyte-specific major water channel in the brain, aquaporin-4 (AQP4), in brain plasticity and learning. We initially identified the most prevalent genetic variant of AQP4 (single nucleotide polymorphism of rs162008 with C or T variation, which has a minor allele frequency of 0.21) from a human database ( n =60 706) and examined its functionality in modulating the expression level of AQP4 in an in vitro luciferase reporter assay. In the following experiments, AQP4 knock-down in mice not only impaired hippocampal volumetric plasticity after exposure to enriched environment but also caused loss of long-term potentiation after theta-burst stimulation. In humans, there was a cross-sectional association of rs162008 with gray matter (GM) volume variation in cortices, including the vicinity of the Perisylvian heteromodal language area (Sample 1, n =650). GM volume variation in these brain regions was positively associated with the semantic verbal fluency. In a prospective follow-up study (Sample 2, n =45), the effects of an intensive 5-week foreign language (English) learning experience on regional GM volume increase were modulated by this AQP4 variant, which was also associated with verbal learning capacity change. We then delineated in mice mechanisms that included AQP4-dependent transient astrocytic volume changes and astrocytic structural elaboration. We believe our study provides the first integrative evidence for a gliogenetic basis that involves AQP4, underlying language-associated brain plasticity.

Reactive astrocytes manifest molecular, structural, and functional alterations under various pathological conditions. We have previously demonstrated that the reactive astrocytes of the stab wound injury model (STAB) display aberrant cellular gamma-aminobutyric acid (GABA) content and tonic GABA release, whereas the active astrocytes under enriched environment (EE) express high levels of proBDNF. However, the role of monoamine oxidase B (MAO-B) in reactive astrogliosis and hypertrophy still remains unknown. Here, we investigate the role of MAO-B, a GABA-producing enzyme, in reactive astrogliosis in STAB. We observed that the genetic removal of MAO-B significantly reduced the hypertrophy, scar formation, and GABA production of reactive astrocytes, whereas the MAO-B overexpression under glial fibrillary acidic protein (GFAP) promoter enhanced the levels of GFAP and GABA. Furthermore, we found that one of the by-products of the MAO-B action, H2 O2 , but not GABA, was sufficient and necessary for the hypertrophy of reactive astrocytes. Notably, we identified two potent pharmacological tools to attenuate scar-forming astrogliosis-the recently developed reversible MAO-B inhibitor, KDS2010, and an H2 O2 scavenger, crisdesalazine (AAD-2004). Our results implicate that inhibiting MAO-B activity has dual beneficial effects in preventing astrogliosis and scar-formation under brain injury, and that the MAO-B/H2 O2 pathway can be a useful therapeutic target with a high clinical potential.

The basal ganglia network has been implicated in the control of adaptive behavior, possibly by integrating motor learning and motivational processes. Both positive and negative reinforcement appear to shape our behavioral adaptation by modulating the function of the basal ganglia. Here, we examined a transgenic mouse line (G2CT) in which synaptic transmissions onto the medium spiny neurons (MSNs) of the basal ganglia are depressed. We found that the level of collaterals from direct pathway MSNs in the external segment of the globus pallidus (GPe) ('bridging collaterals') was decreased in these mice, and this was accompanied by behavioral inhibition under stress. Furthermore, additional manipulations that could further decrease or restore the level of the bridging collaterals resulted in an increase in behavioral inhibition or active behavior in the G2CT mice, respectively. Collectively, our data indicate that the striatum of the basal ganglia network integrates negative emotions and controls appropriate coping responses in which the bridging collateral connections in the GPe play a critical regulatory role.