Journal of Neuroinflammation

Although effective antiretroviral therapy (ART) has substantially reduced the severity of human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND), the condition remains highly prevalent. Understanding HAND has been a challenge due to the lack of small animal models capable of supporting productive HIV infection in the brain. Recent advances in humanized mouse models with engrafted human glial cells now enable systemic HIV infection that extends to the central nervous system, offering a powerful platform to study HAND pathogenesis. In this study, we investigated behavioral alterations and neuropathological changes associated with HIV infection. Using home-cage monitoring, we observed that HIV-infected mice exhibited reduced feeding efficiency, consuming less food despite increased time spent at the feeder, compared to uninfected controls. Additionally, infected animals displayed disrupted circadian rhythms, with a significant correlation between central nervous system viral load and increased locomotor activity during the light cycle. Neuropathological analyses revealed region-specific vulnerability, with the cortex exhibiting pronounced inflammatory and neurodegenerative changes. These findings were supported by transcriptomic profiling, which demonstrated heightened inflammatory and antiviral gene expression in the cortex associated with differentially expressed genes related to neuropathology and behavior deficits. Together, these results highlight distinct region-specific responses to HIV infection in the brain and establish this humanized mouse model as a valuable tool for elucidating the mechanisms underlying HAND and its associated behavioral deficits.

NOD-like receptor family pyrin domain-containing 3 (NLRP3) is a cytosolic regulator of an inflammasome-mediated innate immune response. In the central nervous system (CNS), NLRP3 inflammasome activation has been implicated in multiple neurodegenerative diseases, yet the mechanisms by which it contributes to disease remain unclear. Here, we investigated the CNS effects of chronic NLRP3 activation using a humanized NLRP3 gain-of-function mouse model (hNLRP3D305N). Bulk brain analyses confirmed constitutive inflammasome activation, widespread cytokine induction, and the increased presence of blood-associated proteins suggestive of dysfunction at CNS border sites and the blood-brain barrier (BBB). Furthermore, cerebrospinal fluid (CSF) neurofilament light chain levels were elevated, indicating neuronal damage. Single-cell RNA-sequencing of CD45+ immune cells in the brain demonstrated that microglia adopt distinct reactive states and that peripheral immune cells infiltrate the CNS, with neutrophils emerging as the predominant infiltrating immune cell type. This finding was confirmed by untargeted bulk brain and CSF proteomics that also suggest neutrophil reactivity. Immunohistochemistry further revealed regional neutrophil entry into the brain parenchyma, concurrent with reactive microglia and engulfment of neutrophils, suggesting functional microglia-neutrophil interactions. Collectively, these findings establish a direct pathogenic role for the NLRP3 inflammasome in the CNS independent of other neurodegeneration-related disease pathologies.

The neuronal mechanisms driving progression in neuroinflammatory disorders from early relapse-remitting phases to later neurodegenerative phases remain largely elusive. Functional brain state shifts towards hyperactivity, persisting beyond relapses, represent an early maladaptive response. Here, in remission stage of an experimental autoimmune encephalitis (EAE) mouse model of RRMS, we identified a reduced excitability upon optogenetic stimulation in the brain stem, the area of active disease, while in the cortex a persistent cortical neuronal hyperactivity and synaptic remodeling emerged, accompanied with an increase of markers of early apoptosis. In contrast, hippocampal circuits, which undergo a functional state shift without hyperactivity, do not show increased apoptosis. Visual cortical networks showed a deterioration of the accuracy of encoding visual information and a decrease in the behavioural visual discrimination ability in mice. In RRMS patients in remission, we identified a reduced visual colour discrimination, indicating both the presence and the clinical relevance of early brain state maladaptation that may contribute to progression independent from relapse activity (PIRA). SummaryIn a RRMS model and in patients, impaired visual processing was reported, indicating brain state maladaptations, associated with persistent cortical hyperactivity, brain stem hypoactivity, synaptic remodeling, and apoptosis. These maladaptations might contribute to relapse-independent disease progression through sustained network dysfunction.

Growing evidence supports a strong association between periodontitis and Alzheimers disease (AD), yet the mechanisms linking these conditions remain poorly defined. In neurodegenerative disorders, including AD, microglia are often characterized by increased lipid droplet (LD) accumulation, heightened activation, and impaired function. In this study, we examined whether Porphyromonas gingivalis (Pg), a keystone periodontal pathogen, promotes LD accumulation in microglia and disrupts their function. We found that Pg infection induces robust LD accumulation in BV2 microglial cells and in microglia from Pg-infected App KI mice. This Pg-driven LD buildup was closely associated with elevated reactive oxygen species (ROS) production, impaired phagocytic ability, and altered activation. Notably, pharmacological inhibition of LD with a triglyceride synthesis inhibitor effectively reversed Pg-induced LD accumulation, mitigated ROS production, and restored phagocytic function, thus underscoring the critical role of lipid metabolism in regulating microglial function. These findings support a model in which, in the context of periodontitis, systemic dissemination of periodontal pathogens promotes LD accumulation in microglia, and this metabolic alteration exacerbates microglia dysfunction via a self-reinforcing cycle of excessive oxidative stress and impaired phagocytosis, potentially accelerating AD progression.

Traumatic brain injury (TBI) induces rapid neuroinflammatory responses not only in the brain but also in anatomically and immunologically connected central nervous system (CNS) compartments, including the retina. In our study, we investigated retinal microglial activation, retinal ganglion cell (RGC) calcium dynamics, and caspase-3 activation in adult mice subjected to severe traumatic brain injury using the Marmarou impact-acceleration model at 24 and 48 h post-injury. Carrying out Ca{superscript 2}-imaging, immunohistochemistry, and ex vivo time-lapse microscopy, we found robust microglial activation in both the superficial and deep retinal layers following TBI, accompanied by increased microglial motility. RGCs exhibited a transient surge in degeneration-induced spontaneous activity at 24 h, followed by a marked reduction below control levels at 48 h, consistent with early degenerative changes. Activated caspase-3 levels were significantly elevated in both microglia and other retinal cell types at both time points, indicating ongoing apoptotic effects. Together, these findings demonstrate that TBI rapidly triggers inflammatory and apoptotic mechanisms in the retina, which are detectable within the first 48 hours. Our results highlight the retina as a sensitive indicator of early CNS pathology after traumatic injury and underscore the potential of retinal analysis for monitoring TBI-induced neurodegeneration for future clinical implementation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/734783v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@5bc694org.highwire.dtl.DTLVardef@14a4ce4org.highwire.dtl.DTLVardef@fe2d32org.highwire.dtl.DTLVardef@149419d_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundMicroglial heterogeneity is a fundamental feature of brain homeostasis and pathology. The purpose of this study was to investigate the complexity of microglial plasticity by characterizing specialized oligodendrocyte-like microglial subsets. MethodsThe study was performed utilizing single-cell transcriptomics analyses and immunofluorescence staining to identify and profile microglial subpopulations. Additionally, spatial transferring and morphological analyses were conducted to determine the anatomical distribution and structural features of these specific cells. ResultsWe identified a distinct microglial subset termed dual-phenotype microglia (DPM), which co-expresses microglial and oligodendrocyte markers. DPM consisted of two subtypes with distinct functions: myelin-associated DPM (mDPM) and neuron-associated DPM (nDPM). Spatial and morphological evaluations revealed that mDPMs were sparsely distributed across the whole brain and exhibited a highly ramified architecture, whereas nDPMs were enriched in the hippocampal dentate gyrus. Mechanistically, we found that mDPM function was driven by the Sox10 regulon to modulate myelin maintenance and axonal ensheathment, while nDPM was orchestrated by Glis2, facilitating essential neuron-glia crosstalk and synaptic regulation. Furthermore, we demonstrated that nDPM and mDPM were predicted to undergo significant alterations in multiple sclerosis and Alzheimers disease. Notably, mDPMs were selectively enriched in active multiple sclerosis lesions, revealing that DPM were closely related to neuropsychiatric disorders. ConclusionsBy comprehensively characterizing the morphology, molecular signatures, and spatial logic of these oligodendrocyte-like microglial subsets, our study elucidated the complexity of microglial plasticity. These findings provided new insights into their diverse roles in central nervous system health and disease. Graphical abstractIdentification, Molecular Profiling, and Functional Modeling of Dual-Phenotype Microglia (DPM). (1) Discovery: Identification of the dual-phenotype microglia (DPM) population through single-cell transcriptomics. (2) Molecular Signatures: The transcriptomic identity of DPM subtypes is governed by specific regulatory networks. (3) Distribution & Pathology: Spatial mapping reveals divergent anatomical logic and disease relations for DPM subtypes. (4) Mechanism/Theory: A proposed functional model of mDPMs as "metabolic relay" and support units. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/724239v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@b7db1dorg.highwire.dtl.DTLVardef@9265e7org.highwire.dtl.DTLVardef@1605d82org.highwire.dtl.DTLVardef@19b048f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microglia are the brains resident immune cells that exhibit complex signaling behavior, including phagocytic activity in response to threats and prolonged neuronal activity. Adenosine triphosphate (ATP) is a chemoattractant for microglia. In the nucleus accumbens (NAc), ATP is co-packaged and released with DA, and microglia express dopamine (DA) receptors and ATP receptors. The present work examines microglia chemotactic motility for these transmitters using iontophoresis and multiphoton microscopy approaches in NAc brain slices from GFP-monocyte labeled transgenic mice. ATP chemoattraction was more regularly observed than DA chemoattraction, and DA chemoattraction occurred in only a small subset of microglia. The DA chemoattraction of this subset was blocked by DA D1 antagonism. Microglia are reactive oxygen species (ROS) scavengers. Application of glucose oxidase produces mild but consistent increases in ROS and induced inflammatory-related changes in microglial morphology and motility. Glucose oxidase application decreased DA release but had variable effects on ATP release. The toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) transitioned microglia from ramified to amoeboid morphology over a period of 4 hours, and increased DA and ATP release across this same period. These studies highlight the complex relationship between local immune activation and DA terminal functionality.

Microglia constitute a major innate defense mechanism against prion infection; however, the molecular pathways regulating microglial responses during disease progression remain incompletely understood. Galectin-3 (Gal3), a {beta}-galactoside-binding lectin associated with reactive microglia in multiple neurodegenerative disorders, has been implicated in phagocytosis, inflammatory signaling, and lysosomal homeostasis. Here, we investigated the role of Gal3 in prion disease pathogenesis using prion-infected mice. Gal3 expression was undetectable in healthy brain but became upregulated beginning at late preclinical stages, increasing with disease progression. Gal3 localized predominantly to a subpopulation of reactive IBA1-positive microglia, particularly within the thalamus, and inversely correlated with expression of the homeostatic microglial markers P2Y12 and TMEM119, consistent with acquisition of a reactive phenotype. Microglia engaged in neuronal envelopment displayed elevated Gal3 expression during terminal disease. Constitutive deletion of Gal3 significantly accelerated clinical disease progression without altering total PrPSc accumulation, reactive gliosis, neuronal envelopment, or overall microglial and astrocytic activation. However, Gal3 deficiency markedly reduced microglial uptake of PrPSc, resulting in a lower intracellular-to-extracellular PrPSc ratio, supporting a role for Gal3 in phagocytic sequestration of prions. In contrast, Gal3 deficiency did not impair lysosomal activity, lysosomal membrane integrity, or expression of genes involved in lysosomal repair pathways. Likewise, selective inhibition of autophagy in myeloid cells exerted only minor effects on disease progression. Collectively, these findings identify Gal3 as a sensitive marker of reactive microglia that contributes to microglial uptake of PrPSc and exerts a protective role during prion disease progression.

Microglia represent the immune component of the central nervous system (CNS) that displays dynamic responses to injury and disease. Across the developing and mature CNS, microglia emerge as immunocompetent cells that continuously survey their surroundings to maintain tissue homeostasis and respond to threats. There remains a gap in 3D in vitro models that contain microglia and can provide both developmental and mature functional hallmarks. Using a 3D neural multicellular model, cortical microtissues, derived from primary rat cortical cells, we conducted live imaging to monitor microglia dynamics from early, middle, and late stage microtissue maturation. We optimized a within-micromold imaging approach that allows for live microglia imaging without removing microtissues from their culturing environment. We confirm that microglia exhibit baseline surveillance characterized by relatively stationary somas and highly dynamic cell processes that continuously extend and retract. Following proinflammatory challenges, microglia engulf lipopolysaccharide particles, accompanied by dynamic shifts in motility patterns; and rapidly respond to laser-induced tissue damage through process extension, whole-cell displacement, and local recruitment. Lastly, we show that microtissue age in culture strongly influences both baseline and directed motility profiles. Collectively, these studies demonstrate that within a 3D microenvironment, microglia exhibit pronounced changes in morphology, surveillance area, motility, and injury response across microtissue maturation. Microtissues can serve as a valuable in vitro platform for both microglia developmental studies and investigations of brain inflammation related to CNS injuries, infections, and diseases.

HIV-associated neurocognitive disorders (HAND) have become a major clinical concern, particularly among the aging HIV-1-seropositive population, which is generally characterized by persistent viral reservoirs and a lower level of chronic inflammation. NLRP3 inflammasome activation exhibits its unique role in the progression of many chronic inflammatory diseases. Furthermore, pyroptosis, an inflammatory form of programmed cell death, has been implicated in numerous neurological diseases. However, the mechanisms linking EcoHIV infection, microglial pyroptosis, and NLRP3 inflammasome activation remain incompletely understood. In this study, EcoHIV was retro-orbitally injected into C57BL/6J wild-type mice and analyzed at 14-, 30-, 60-, and 90-days post-infection to establish a NeuroHIV model. Additionally, in vitro, BV2 microglial cell line was infected with EcoHIV and treated with MCC950, an inhibitor of the NLRP3 inflammasome, for three days. Pyroptosis marker GSDMD, NLRP3 inflammasome components, Caspase-1 (a marker of inflammasome activation), HLA-DR (an immune activation marker), Programmed-death 1 (PD-1, an immune checkpoint molecule), and Ki67 (a cellular proliferation marker) were assessed by immunofluorescence staining. Results showed that EcoHIV-infected mice showed a peak in NLRP3 expression at 14 days post-infection, compared with controls, followed by a modest decline at 30 days, while GSDMD expression increased progressively across 14 and 30 days. These findings demonstrate dynamic changes in microglial pyroptosis and NLRP3 inflammasome activation over the course of EcoHIV infection. In vitro, EcoHIV-infected BV2 cells exhibited significantly increased EcoHIV-eGFP fluorescence compared with controls, confirming the utility of BV2 cells as an in vitro model of microglial EcoHIV infection. Expression levels of GSDMD and NLRP3 were elevated following infection, indicating enhanced pyroptosis and neuroinflammation. Treatment with MCC950 significantly reduced the expression of GSDMD, NLRP3, HLA-DR, PD-1, and Ki67, suggesting that inhibition of NLRP3 inflammasome activity suppresses both pyroptosis and microglial activation and proliferation. Together, elucidating the interplay between microglial pyroptosis and NLRP3 inflammasome activation may provide new insights into the pathogenesis and potential therapeutic strategies for NeuroHIV in the aging HIV-1-seropositive population.

Progressive neurodegeneration in the central nervous system (CNS) in multiple sclerosis (MS) is driven by chronic inflammatory demyelination. Neutrophils are increasingly recognized as versatile innate immune cells with potentially underappreciated roles in CNS inflammation, but their contribution to MS pathology remains poorly understood. Interestingly, we observed foamy neutrophils in active CNS lesions of MS patients. Therefore, we investigated the ability of human neutrophils to internalize myelin debris and assessed how this impacts their functional phenotype. Neutrophils exhibited efficient myelin uptake, peaking between 3 and 6 hours, predominantly through complement opsonization and internalization via complement receptor 3. Prolonged exposure to high concentrations of myelin induced a pro-inflammatory phenotype, marked by increased production of reactive oxygen species, neutrophil extracellular traps, and inflammatory mediators such as CXCL8 and CCL3. Gene expression analysis revealed a dose-dependent inflammatory signature after myelin uptake, characterized by gradual upregulation of CXCL8 and decreased ARG1 expression, suggesting a shift toward a pro-inflammatory neutrophil phenotype. These findings provide novel insights into the role of neutrophils in myelin clearance and inflammation in the CNS, highlighting complement receptor 3-mediated uptake and downstream pro-inflammatory activation as key mechanisms.

Reactive astrocytes contribute to neuroinflammation and synaptic dysfunction, but it remains unclear whether transient inflammatory stimulation causes a persistent reactive state after the initial inflammatory stimulus is removed. Here, we investigated whether transient exposure to a defined inflammatory cytokine/complement cocktail induces a persistent reactive astrocyte state and examined the signaling mechanism underlying its maintenance. Human astrocytes were exposed to the inflammatory stimulus and subsequently subjected to stimulus washout, followed by time-course analyses to compare the reversibility of inflammatory gene expression after stimulus removal. Following washout, the expression of several inflammatory response genes, including CXCL10 and NF-{kappa}B-associated genes such as NFKBIA, TNFAIP3, and RELB, returned toward baseline levels. In contrast, C3 expression remained elevated, indicating persistence of a post-inflammatory C3-high astrocyte state after withdrawal of the inflammatory stimulus. Pharmacological inhibition of JAK signaling reduced persistent C3 expression to near-baseline levels, supporting the involvement of JAK-dependent signaling in maintenance of this persistent state. Together, these findings suggest that transient inflammatory stimulation induces a post-inflammatory persistent C3-high astrocyte state that is maintained even after broader inflammatory gene responses have subsided. This persistent C3-high component is pharmacologically attenuated by JAK inhibition, identifying JAK-dependent pathways as modulators of persistent astrocyte inflammatory reactivity.

HIV-1 infection of the brain occurs early in acute infection and results in neuroinflammation and - when untreated - in cognitive impairment, yet the mechanisms by which microglia become infected remain poorly defined. Evidence from simian immunodeficiency virus (SIV) studies supports a model in which infected CD4+ T cells disseminate HIV-1 to tissue macrophages, but this has not yet been confirmed for human microglia. Here, we used human monocyte-derived microglia (MDMi) and autologous HIV-1-infected primary CD4+ T cells to investigate viral transmission and immune cell interactions. Transcriptional profiling of MDMi confirmed microglia signature genes such as CX3CR1, P2RY12 and C1QB, and surface staining showed expression of CD4 and the HIV-1 coreceptor CCR5. Compared to cell-free infection, direct cell-to-cell contact between MDMi and HIV-1-infected CD4+ T cells markedly enhanced productive infection of MDMi. HIV-1 infection downmodulated the "dont-eat-me" signal CD47 and increased phosphatidylserine on the surface of primary CD4+ T cells. Consequently, HIV-1 infection of primary CD4+ T cells increased microglia-CD4+ T cell interactions and resulted in enhanced phagocytosis by MDMi. Together, this supports a mechanism where HIV-1 facilitates cell-to-cell spread from primary CD4+ T cells to microglia, which has important implications for therapeutic targeting of HIV-1 brain reservoir seeding.

Zika virus (ZIKV) encephalitis induces cytokine-mediated cognitive deficits which persist long-term. Here, we determined if NOD2-mediated conversion of monocytes from Ly6CHi inflammatory to Ly6CLo anti-inflammatory phenotypes during ZIKV encephalitis preserves neural correlates of learning and memory within the hippocampus. Short-term administration of the NOD2 agonist, muramyl dipeptide (MDP), during peak ZIKV infection prevents synapse elimination and loss of adult hippocampal neurogenesis, without impacting CNS virologic control. Transcriptomic analyses of forebrain immune cells in MDP-treated mice revealed functional modulation of infiltrated monocytes and T cells, reducing their expression of pro-inflammatory cytokines, with limited effects on microglia, compared to controls. Notably, NOD2 activation in peripheral immune cells alone balances innate immune signals, preserving synapses, and increasing macrophage phagocytic capacities that do not target synapses. Our findings identify infiltrating Ly6CHi monocytes as key drivers of long-term cognitive dysfunction following ZIKV encephalitis and as potential therapeutic targets for limiting synapse loss. HighlightsO_LINOD2 activation via MDP phenotypically shifts monocyte subsets from inflammatory to anti-inflammatory during the acute phase of ZIKV encephalitis. C_LIO_LIShort-term MDP administration increases phagocytic machinery and reduces inflammatory cytokine/chemokine production within macrophage populations. C_LIO_LISynapse elimination is attenuated within the hippocampus of ZIKV-infected animals treated with MDP. C_LIO_LIMDP derived effects are mediated through peripherally derived immune cells. C_LI

Brain organoids are three-dimensional cultures derived from human pluripotent or embryonic stem cells that recapitulate key genetic, biochemical, and molecular features of the human brain. They provide a powerful platform for studying human brain development and modeling genetic neurological disorders. However, their application to age-dependent neurodegenerative diseases remains limited, largely due to the absence of standardized methods for incorporating functional microglia, critical regulators of neuroinflammation and disease progression. Here, we describe a strategy for generating neuroimmune assembloids, brain organoids containing functional glial cells capable of mounting inflammatory responses. By introducing hematopoietic progenitor cells into developing brain organoids, we enable their in situ maturation into microglia-like cells that persist in culture for up to one month. These cells exhibit hallmark microglial behaviors, including morphological remodeling, migration, phagocytosis and transcriptional changes in response to inflammatory stimuli. Together, these immunocompetent-like brain organoids provide a promising and versatile platform for investigating neuroimmune interactions and neuroinflammatory mechanisms underlying age-related neurodegenerative diseases.

Systemic inflammation drives neurodegeneration, yet its differential effects across neural tissues and genetic backgrounds remain poorly understood. We performed RNA-sequencing on brain, optic nerve head (ONH), and retina from four genetically diverse mouse strains (B6, CAST, NZO, WSB) following lipopolysaccharide (LPS)-induced systemic inflammation. The ONH mounted the largest response to LPS (9510 DEGs), followed by retina (5152) and brain (4586). A conserved core of 1444 DEGs across all tissues was enriched for innate immune and acute-phase pathways. Tissue-specific responses were apparent; the retina downregulated phototransduction and visual perception genes; ONH exhibited bidirectional remodeling with upregulated proteasome and ribosome biogenesis and suppressed lipid metabolism and lysosomal function; yet the brain displayed no significant pathway level enrichment. Genetic background strongly modulated the LPS response across the three tissues; the retina exhibited the greatest strain-dependent divergence. Interestingly, differing genetic context affected the ONH response to LPS the least despite its markedly larger response to LPS overall. In totality, both genetic and physical context dictate the neuroinflammatory response to LPS.

Phagocytic and immune-like cells have been observed in the satellite envelope of neuronal somata in peripheral sensory ganglia of many species for several decades. These cells likely play an important role in normal function of sensory neurons and they may also play an important role in neuronal dysfunction and neurodegeneration seen with neuropathy. Recent findings have described a satellite macrophage population transcriptomically similar to microglia in peripheral ganglia of some mammalian species. The function of these cells, and the mechanisms by which they may influence neurons in neuropathy are unclear. We sought to understand the phenotype and localization of these cells in the human dorsal root ganglion (hDRG) using large-scale single nucleus and spatial transcriptomic datasets from individuals with and without a history of peripheral diabetic neuropathy. We observed a large population of macrophages that express classical microglia makers such as TMEM119 and P2RY12 in the hDRG, as previously described. Our findings confirm that these microglia-like cells (MLCs) localize to the satellite envelope around neuronal somata, yet are transcriptomically distinct from all glial cell types characterized in the hDRG. These MLCs exhibit changes in abundance and localization with diabetic painful neuropathy (DPN) in both the hDRG and sural nerves suggesting that they are not exclusively localized to the DRG. We conclude that microglia-like cells are likely the resident tissue macrophage (RTM) of the hDRG, and perhaps the peripheral nervous system (PNS) given their localization to the sural nerve and other ganglia, where they are predicted to regulate homeostatic neuronal functions and response to injury. HighlightsO_LIMLCs are likely the RTM of hDRGs C_LIO_LIMLCs localize to the satellite envelope and recede with Nageotte nodule formation C_LIO_LIMLC activation state and signaling shift with diabetic neuropathy C_LIO_LIMLCs are also present in other ganglia and sural nerve C_LI

Heat stroke is characterized by profound central nervous system dysfunction and vascular abnormalities. Previous studies have demonstrated the marked vulnerability of the CNS to thermal stress, resulting in neuronal injury and glial activation. However, the metabolic mechanisms linking acute injury to chronic neurological long-term effects remain understood. The neuropathological changes are closely associated with neuroinflammatory and metabolic disturbances, including dysregulation of the kynurenine pathway, whose metabolites modulate neurotoxicity, neuroprotection, and immune responses. Here, we present the first comprehensive characterization of kynurenine pathway metabolomic profile across both plasma and brain tissue in a mouse model of heat stroke. Using a validated and sensitive LC-MS/MS method, we simultaneously measured and quantified 13 analytes (kynurenine, kynurenic acid, quinolinic acid, nicotinic acid, picolinic acid, xanthurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, indole-3-acetic acid, indole-3-lactic acid, 5-hydroxyindoleacetic acid and neopterin). The findings reveal a biphasic metabolic response, characterized by an acute serotonergic disruption and reduced neuroprotective capacity, followed by chronic activation of the kynurenine pathway, depletion of central serotonin metabolites, and metabolic signatures consistent with gut microbiota dysbiosis. The acute phase is marked by a transient imbalance favoring neurotoxic kynurenine pathway metabolites, whereas the chronic phase reflects sustained pathway activation. Notably, the plasma-brain dissociation of 5-hydroxyindoleacetic acid emerged as the most prominent cross-compartment finding, suggesting a potential biomarker of central serotonergic depletion and a mechanistic link between peripheral and central metabolic changes, with implications for therapeutic targeting during the subacute recovery phase.

Neurological and neuropsychiatric symptoms, collectively termed neuroPASC, are among the most prevalent Post-Acute Sequelae of COVID-19 (PASC). Neuroinflammation - particularly microglia reactivity - has been implicated in neuroPASC. Current insights are largely derived from post-mortem tissues of acutely infected patients and may not reflect PASC-related neuropathology. We previously established a PASC model in which SARS-CoV-2-infected mice developed persistent behavioral alterations and prolonged neuroinflammation for up to 120 days post-infection (dpi) in the absence of viral neuroinvasion. Here, we extended these results to a longitudinal single-cell RNA sequencing analysis of brain immune cells collected at 0, 6, 30, and 100 dpi. We identified a coordinated contribution of infiltrating and resident myeloid cells to the initiation and persistence of neuroinflammation. In specific, microglia displayed sustained expansion of subclusters characterized by inflammatory, stress response, and metabolic signatures. Border-associated macrophages upregulated monocyte attractants during acute infection. Concurrently, monocytes and neutrophils showed marked brain recruitment and mounted transient inflammatory responses at 6 dpi, potentially triggering long-term microglial reactivity. Together, these findings provide a high-resolution atlas of brain myeloid immune dynamics during neuroPASC and highlight a central role for microglia in sustaining chronic neuroinflammation.

BackgroundGut dysbiosis has been increasingly implicated in post-stroke cognitive impairment (PSCI), yet the causal contribution and therapeutic potential of gut microbiota-derived metabolites remain unclear. This study aimed to identify key microbiota-derived metabolites involved in PSCI and to elucidate their underlying mechanisms. ResultsWe found that both PSCI patients and middle cerebral artery occlusion (MCAO) mice exhibited distinct gut microbial alterations, characterized by a marked reduction in tryptophan-metabolizing bacteria and indole-3-propionic acid (IPA), a gut microbiota-derived tryptophan metabolite. Exogenous IPA administration alleviated PSCI-like phenotypes in MCAO mice. Mechanistically, IPA preserved tyrosine hydroxylase-positive (Th) fibers and catecholamine levels in the dorsal hippocampus. Further analyses showed that IPA binds to the adaptor protein Ywhab, promotes ERK activation, and enhances neuronal survival, thereby counteracting neuronal apoptosis-associated inflammation and subsequent Th fiber degeneration. ConclusionThese findings identify IPA as a gut microbiota-derived neuromodulator that mitigates PSCI by preserving dorsal hippocampal catecholaminergic transmission. IPA may therefore serve as a promising predictive biomarker and therapeutic candidate for PSCI.