Journal of Neuroinflammation

BackgroundTraumatic brain injury (TBI) is increasingly recognized as a chronic condition with lasting systemic consequences. Beyond persistent neuroinflammation, long-term TBI disrupts peripheral immune homeostasis, increasing susceptibility to infection and organ dysfunction, particularly in older patients. The voltage-gated proton channel Hv1, expressed in microglia and peripheral immune cells, regulates oxidative injury via modulation of NADPH oxidase activity. Yet few animal studies extend long enough to recapitulate the lifelong trajectory of human TBI, leaving the long-term effects of Hv1 deficiency on systemic immune homeostasis unresolved. MethodsYoung adult (3-month-old) male wild-type (WT) and Hv1 knockout (Hv1KO) mice were subjected to a moderate controlled cortical impact (CCI), and survival was monitored for up to 18 months post-injury, with endpoint analyses performed at 21 months of age. After neurological behavioral assessments, spleen, lung, liver, gut, ipsilateral cortex, and blood samples were collected for flow cytometry, qPCR, NanoString nCounter Panels, and in vivo plasma transfer studies. ResultsHv1 deficiency resulted in significantly increased mortality following TBI, starting at 14 months post-injury, compared with WT/TBI mice. No significant difference in survival was observed between the two sham groups. At 18 months post-injury, Hv1KO mice exhibited significant weight loss and splenomegaly. qPCR revealed an approximately 30-fold increase of pan-bacterial 16S rRNA levels in the spleens of Hv1KO/TBI mice, but not in the lungs or liver. Furthermore, chronic TBI in the Hv1KO mice led to a compromised intestinal tight junction and mucus barrier integrity, accompanied by aberrant activation of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway in the spleen. Transcriptomic profiling of the spleen, liver, and lung revealed distinct post-injury immune signatures in Hv1KO mice. In contrast, surviving Hv1KO/TBI mice showed modest behavioral resilience and a partially neuroprotective cortical transcriptomic profile. Lastly, systemic transfer of plasma from WT/TBI or Hv1KO donors into naive young adult mice altered immune responses in the spleen, lung, and brain. ConclusionsHv1 plays a critical role in maintaining peripheral immune integrity and antibacterial defense throughout the chronic course of TBI. Despite conferring modest neuroprotection through attenuation of microglial-mediated oxidative stress, Hv1 deficiency exacerbated systemic phagocyte dysfunction and significantly reduced long-term survival.

Brain development, especially developmental myelination, continues through young adulthood. Concordantly, children may be particularly vulnerable to neural-immune challenges. To investigate the consequences of major childhood immune challenges, juvenile mice were exposed to respiratory influenza (H1N1) infection. White matter-specific microglial reactivity accompanied by oligodendrocyte loss was evident until two months following infection. Mice exhibited hyperlocomotion and impaired attention, but not anxiety-like behavior, at one month following infection. Linking the oligodendroglial and behavioral deficits, genetic disruption of oligodendrocyte development at the same juvenile timepoint recapitulated this behavioral phenotype. Microglial reactivity and oligodendrocyte numbers normalized by young adulthood. However, myelin development was disrupted, with persistently decreased myelinated axon density and reduced myelin sheath thickness. Hyperlocomotion resolved, but anxiety-related behaviors emerged at two months after infection. At 6 months, anxiety resolved but cognitive deficits persisted. Elevated CSF chemokines and microglial chemokine expression prompted testing the role of the multi-chemokine receptor CCR3. CCR3 inhibition rescued these cellular and behavioral aberrations after juvenile H1N1 infection. Together, these findings underscore the potential for disruption of myelin development and lasting cognitive and neuropsychiatric sequelae following major immune challenges during the juvenile period and highlight chemokine signaling as an important therapeutic target.

Immune checkpoint inhibitor (ICI) combinations that block cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) signaling have revolutionized cancer care but also exert a range of immune-related adverse events (irAE) in various tissues, including the brain. Our understanding of the mechanisms of irAE in the brain is still evolving, and we recently demonstrated that ICI (blockade of CTLA-4 and PD-1) perturbs hippocampal-dependent memory function by derailing neuro-immune homeostasis and compromising synaptic integrity. However, the spatial patterns and the cell-type-specific molecular mechanisms underlying ICI-related brain dysfunction remain not well-defined. To address this gap, we performed spatial transcriptomic profiling of the hippocampal region using multiplexed error-robust fluorescence in situ hybridization (MERFISH) to map gene expression at single-cell resolution. By integrating spatial single-cell data with bulk RNA-seq, we define the distribution of microglia, astrocytes, synaptic, and neuroinflammatory markers, and determine how ICI reshapes hippocampal cellular composition in a syngeneic murine melanoma model. MERFISH revealed upregulation of microglial, astrocytic, oligodendrocytic, and T cell markers post-ICI treatment, revealing unique pathways driving neuroinflammation, synaptic function, and cellular signaling. Furthermore, immunofluorescence analysis of postmortem brains from patients treated with ICI corroborates our findings of ICI-related immune activation of microglia. Finally, using a conditional deletion model, we show that T cells are indispensable for ICI-driven microglial activation. Altogether, our study provides a high-resolution spatial framework for understanding irAEs in brain function and a T cell-microglia crosstalk axis as a driving mechanism of dysregulated neuro-immune homeostasis during ICI.

BackgroundHypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal mortality and neurodevelopmental impairments. Following brain hypoxia-ischemia (HI), microglia face substantial metabolic stress; and upon phagocytosis, they become overloaded with lipids derived from engulfed dead neurons and myelin debris. It is unclear how microglia respond to and process the lipid cargo, and whether lipid accumulation may affect microglia function following neonatal HI. MethodsThe postnatal day 10 mice were subjected to HI using the Vannucci model. Lipid droplets (LD) were assessed by histology and immunofluorescent staining. Single-nucleus RNA sequencing (snRNA-seq) was performed using brain tissue from HI-injured and sham-operated mice at 72 hours after HI. LD-accumulating microglia (LDAM) were identified by a specific LD marker gene perilipin 2 (Plin2). Differential gene expression was analyzed between Plin2-positive and Plin2-negative microglia after HI. Human HIE brain sections were also examined for LD accumulation. The dynamic changes of PLIN2-expressing microglia and infiltrating monocyte-derived macrophages (MDM) at 24 hours, 72 hours and 7 days after HI were compared using flow cytometry. In addition, mouse BV2 microglia were subjected to oxygen-glucose deprivation (OGD) to study phagocytosis and cytokine expression. ResultsLipid droplets accumulated primarily in microglia after HI in neonatal mice and in human HIE brain. LD were not found in astrocytes or neurons. Plin2-expressing LDAM emerged as new microglia clusters after HI. Compared with microglia without LD, LDAM showed a distinct transcriptional profile with upregulation of genes linked to microglial activation, enhanced cholesterol and lipid processing, and a shift towards phagocytic and pro-inflammatory state. Blocking LD biogenesis reduced elevated phagocytosis and IL-1{beta} expression in BV2 cells following OGD. ConclusionOur study revealed that microglia accumulate lipid droplets as part of their metabolic responses to HI in the neonatal brain. Microglial lipid droplet formation is associated with a pro-inflammatory phenotype at early stage after HI, and increased phagocytosis in vitro. The lipid metabolic changes may regulate microglial function and influence HI outcomes.

Brain tuberculosis remains associated with high mortality, and many survivors exhibit cognitive impairments. Progress in understanding the disease is hindered by the lack of human models. In this study, human neural organoids were infected, revealing that a subpopulation of neural progenitor cells (NPCs) is directly infected by apoptotic cell receptors expressed by NPCs, mediating bacterial uptake. Phagocytosed bacteria were localized in late endosomes, lysosomes, and the cytoplasm. Cytoplasmic bacteria frequently formed cords, indicating limited control of bacterial expansion. Immunostaining demonstrated that infected NPCs produce a type I interferon (IFN) response, corroborated by increased expression of type I IFN and IFN-regulated genes detected by RNA sequencing. Pathways related to innate immune response, cell death, and proliferation were also activated following Mycobacterium tuberculosis (Mtb) uptake by NPCs. The addition of color-coded microglia and monocytes to 3D neural organoids and NPCs revealed cross-infection of NPCs and other phagocytes by Mtb, suggesting a mechanism by which NPCs may access the bacteria. Infection of NPCs resulted in increased cell death, inhibition of neural differentiation, and reduced proliferation, effects that were partially mitigated by anti-IFN treatment. Differentiated neurons were not infected. These findings indicate that brain organoids and NPC-based in vitro platforms provide a novel approach for studying brain tuberculosis. Decreased NPC function may contribute to brain tuberculosis-induced cognitive disease.

Microglia, the resident immune cells of the central nervous system, undergo dynamic transcriptional remodeling across embryonic and postnatal development. However, the precise transcriptional programmes governing these transitions, and the role of oxidative stress pathways such as NRF2/Hmox1 in shaping microglial maturation, remain incompletely understood. Here, we characterized the transcriptional landscape of mouse microglial development using pseudobulk RNA-sequencing data, spanning five developmental stages, from embryonic day 17 to postnatal day 60. We identified four distinct transcriptional programmes (homeostatic, phagocytic, NRF2/Hmox1 oxidative stress-responsive, and Apoc1-associated) whose relative activities shift coordinately across development. Early developmental microglia were dominated by phagocytic and NRF2/Hmox1-associated gene expression, while mature microglia progressively acquired a homeostatic transcriptional identity marked by Tmem119 and P2ry12. Pseudotime trajectory analysis confirmed a continuous developmental axis along which the phagocytic programme declined, homeostatic programme increased, and NRF2/Hmox1 activity peaked at intermediate stages. Differential expression analysis distinguished Tmem119+ homeostatic microglia from Tmem119- populations, and early developmental from mature microglial states. Additionally, chemokine receptor expression, including Cxcr4 at early timepoints, suggested a role for chemokine signaling in microglial migration and tissue integration during brain development. Collectively, these findings support a model in which microglial maturation proceeds along a transitional regulatory role during brain development.

Alzheimers disease (AD) is one of the most common neurodegenerative disorders, yet the environmental drivers that accelerate its progression remain poorly defined. Traffic-related air pollution is emerging as a modifiable AD risk factor, but how inhaled particles perturb microglial clearance of amyloid beta (A{beta}) is unknown. Microglia are the principal A{beta}-clearing phagocytes of the brain. Here, we showed that exposure of primary mouse microglia and human induced pluripotent stem cell-derived microglia (iMGLs) to 3-100 {micro}g/mL diesel exhaust particles (DEP) disrupted microglial homeostasis, induced morphological abnormalities, increased reactive oxygen species, impaired lysosomal degradation, and led to a concentration-dependent loss of phagocytic capacity. Importantly, DEP markedly reduces A{beta} uptake in both species. Transcriptomic profiling revealed a DEP-induced, non-canonical state characterized by metabolic reprogramming, broad suppression of inflammatory pathways, antigen-presentation, chemokine, and species-specific remodeling during subsequent A{beta} challenge, including defective chemotaxis, cell cycle, and cytoskeletal signatures. These data show that DEP profoundly alters microglial transcriptional and metabolic states, leading to impaired A{beta} clearance, which could, thereby, further contribute to AD progression.

Vascular cognitive impairment and dementia (VCID) is increasingly recognized as a major contributor to cognitive decline; however, the mechanisms through which vascular dysfunction drives innate immune dysregulation remain poorly understood. In this study, we explore the impact of VCID on the cerebral innate immune landscape, focusing on innate lymphoid cells (ILCs) and neutrophils, two key players in neuroinflammation and brain immune homeostasis. Using a murine model of VCID induced by bilateral common carotid artery stenosis (BCAS) with modifications in C57BL/6 mice, we investigated innate immune cell distribution, polarization, and functional profiles using flow cytometry and immunofluorescence staining. Our findings reveal a compartment-specific shift in ILC populations, with a reduction of ILC2s in the meninges and concurrent expansion in the choroid plexus, accompanied by altered cytokine production. Furthermore, VCID drove a marked shift in neutrophil polarization toward a pro-inflammatory N1-like phenotype in both the meninges and choroid plexus. Critically, immunofluorescence staining of hippocampal brain sections confirmed that activated N1-like neutrophils, characterized by elevated IL-1{beta} and MPO and reduced IL-10, infiltrate the hippocampal parenchyma in VCID, suggesting a spatially progressive innate immune response spanning from CNS border compartments to brain tissue. These results identify a novel innate immune signature in VCID, compartment-specific ILC redistribution, pro-inflammatory neutrophil polarization at CNS borders, and parenchymal neutrophil infiltration in the hippocampus, which may collectively amplify neuroinflammation and accelerate cognitive decline, identifying potential therapeutic targets for vascular-related dementia.

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.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease, with progressive neurodegeneration that is refractory to current therapies. Here, we identify microglia aging as a major factor responsible for age-related loss of beneficial demyelinating and remyelinating brain functions. Brain transcriptomics and in situ spatial gene transcription analysis revealed significant oligodendrocyte loss in both young and aged mice during cuprizone-induced experimental demyelination, but impaired microglial activation in aged mice. Age-related defects in microglial activation were associated with reduced clearance of dead myelin and accumulation of lipid droplets, and impaired remyelination, implying exhaustion of microglial function in the aged mouse brain. Transcriptomic analysis of human brain samples from MS donors with matched disease course and severity and with chronic active and inactive lesions, validated that microglial activation is strongly reduced with increasing age. To investigate microglial responses after repeated demyelinating insults and their direct impact on myelin integrity, we established an experimental model of repeated demyelinating episodes in young and aged mice to recapitulate MS features. Notably, aged mice developed a progressive neuroinflammatory response following sequential demyelinating episodes, in contrast to the alternating cycles of demyelination and remyelination reminiscent of relapsing-remitting MS that were observed in young mice. Microglia depletion and repopulation using a CSF1R antagonist recovered demyelination-remyelination capacity in aged mice. The results indicate that microglia aging is a major determinant in the pathogenesis of progressive MS, and that microglia replacement represents a promising therapeutic approach.

Reactive astrogliosis is a prominent feature of prion diseases, yet the molecular mechanisms regulating astrocyte activation and their contribution to disease progression remain poorly understood. Signal transducer and activator of transcription 3 (Stat3) is a master regulator of reactive astrocytes in numerous neurological disorders, but its role in prion disease has not been established. Here, we investigated the contribution of astrocytic Stat3 signaling to prion pathogenesis using an inducible astrocyte-specific Stat3 knockout mouse model. Stat3 expression was elevated across multiple neuroinflammatory conditions but was most strongly induced during prion disease. Among four mouse-adapted prion strains (ME7, RML, 22L, and SSLOW), the magnitude of Stat3 activation closely paralleled the severity of neuroinflammation. Astrocyte-specific Stat3 deletion was evaluated in mice infected with either the highly inflammatory SSLOW strain or the less inflammatory 22L strain. Stat3 deletion had no detectable effect on disease progression in SSLOW-infected mice but modestly delayed disease onset and behavioral decline in male mice infected with the 22L strain, particularly when knockout was induced before prion inoculation. Despite its limited effect on survival, astrocyte-specific Stat3 deletion consistently attenuated astrocyte reactivity, as evidenced by reduced vimentin expression, delayed cortical GFAP induction, and lower GFAP expression in recombined astrocytes at the single-cell level, demonstrating a cell-autonomous role for Stat3 in promoting reactive astrogliosis. In contrast, PrPSc accumulation and overall microglial activation remained unchanged, indicating that astrocytic Stat3 signaling is dispensable for prion replication and does not substantially influence the global microglial response. Tamoxifen-induced recombination occurred in only 40-70% of astrocytes, resulting in partial and region-dependent Stat3 deletion that likely underestimated the impact of astrocytic Stat3 loss. Together, these findings identify Stat3 as an important regulator of astrocyte reactivity during prion disease but demonstrate that its contribution to disease progression is limited and highly context-dependent, varying with the inflammatory milieu, timing of pathway inhibition, and biological sex. Our results highlight the redundancy of inflammatory signaling networks driving chronic prion neurodegeneration and suggest that targeting astrocytic Stat3 alone is unlikely to substantially alter disease progression.

Sickness is an organismal response to inflammation, yet its immune, metabolic, neural, and behavioral components are often studied separately and predominantly in male C57BL/6 mice. In this study, we characterized these responses to systemic lipopolysaccharide (LPS) in female BALB/c mice. Mice received intraperitoneal LPS at moderate concentrations and outcomes were assessed during the acute and resolving phases of endotoxemia. LPS caused rapid disappearance of resident peritoneal macrophages, followed by neutrophil accumulation and increased circulating TNF- and IL-6. In the liver, LPS induced inflammatory, acute-phase, and anti-inflammatory transcripts while suppressing genes involved in lipid, cholesterol, and xenobiotic metabolism. Hepatic glutathione was reduced, whereas total superoxide dismutase activity was unchanged. These peripheral responses were followed by transient hypothermia, reduced food intake, and body weight loss. Regional brain mapping showed increased c-Fos labeling in the area postrema, nucleus of the solitary tract, external lateral parabrachial nucleus, paraventricular nucleus of the hypothalamus, and arcuate nucleus. In parallel, LPS selectively promoted IBA1-positive area in the median eminence and arcuate nucleus, whereas several other regions showed no changes, indicating that neuronal and microglial responses are regionally distinct. Behaviorally, LPS reduced locomotion and exploration, increased freezing, and increased forced-swim immobility. Changes in spatial exploration were most pronounced during the acute phase, whereas locomotor suppression and passive stress-coping persisted longer and varied in magnitude with the timing of inflammatory challenge. Together, these findings show that systemic LPS produces a coordinated sickness state in female BALB/c mice that links peripheral inflammation and hepatic metabolic and redox changes with region-specific neuronal and microglial responses, altered thermoregulation and feeding, and behavioral suppression.

BackgroundA significant number of people with HIV-1 still experience neurocognitive impairments (NCI), despite effective antiretroviral treatment. HIV-NCI is diverse and multifactorial, with mechanisms that cause its development and progression still not fully understood. We examined early HIV-related changes in brain stability and studied neuroimmune interactions at the single-cell level to better understand how NCI develops. MethodsTo model changes in brain homeostasis, we developed an advanced human iPSC-derived 3D cerebral assembloid model that includes microglia, by co-developing neural progenitor cells with tdTomato-tagged and CD34+ cell-derived microglial precursors. Assembloids were infected with a macrophage R5-tropic HIV-1 strain NL-AD8. Viral spread was measured using a proviral DNA assay, qPCR for HIV RNA, and 3D immunostaining for Tat protein. Single-cell transcriptomics with tdTomato lineage tracing revealed HIV-1 induced disturbances and cell-type-specific responses. The niche net algorithm was used to identify ligand-receptor interactions between microglia and the brain microenvironment during homeostasis and HIV infection. ResultsHighly ramified tdTomato+ IBA-1+ microglia were evenly distributed throughout the assembloids within 15 days of culture. Single-cell transcriptomics identified microglia, excitatory/inhibitory neurons, astrocytes, and oligodendrocyte precursors within the assembloids. Neurons in microglia-containing assembloids upregulated genes related to neurotransmission, synaptogenesis, and neuronal development compared to neurons in organoids without microglia. Niche net analysis showed microglia-derived neurotropic ligands supported neuronal and astrocytic differentiation. The R5-tropic HIV-1 specifically targeted microglia, inducing a reactive phenotype that transmitted interferon and pro-inflammatory signals to nearby cells and increased MHC-I antigen-presentation genes. Notably, neuroprotective ligands from non-glial cells and bystander microglia in the assembloids attempted to counteract HIV-related inflammation and promote neural repair. ConclusionsOur microglia-containing assembloid model replicates in vivo neurodevelopmental interactions, allowing high-resolution analysis of homeostatic and HIV-induced responses across different brain cell types. Homeostatic microglia support neuronal health, while HIV infection triggers a reactive state that spreads inflammatory signals within the brain environment. The presence of multiple glial and non-glial populations uncovered previously unknown crosstalk, including bystander microglial phenotypes and neuroprotective signaling mechanisms that counteract inflammation. These findings emphasize early HIV responses that balance injury and adaptation, offering insights for developing therapies that target microglial activation, boost neuroprotection, and address HIV reservoirs in the brain.

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.

West Nile virus (WNV) is a mosquito-borne orthoflavivirus that causes severe encephalitis, for which no approved antiviral therapies or human vaccines are currently available. Disease-associated microglia (DAM) represent a microglial state associated with neuroprotective functions in neurodegenerative diseases. We previously showed that DAM-like cells are localized in the vicinity of WNV-infected cells in the mouse brain and surmised that these cells may respond to WNV-infected cells. However, the functional significance of this spatial association has since remained unclear. Given the previous reports linking interleukin-34 (IL-34) to the activation of DAM-like cells, we investigated whether IL-34 promotes DAM-like responses and whether these responses are associated with protection against WNV infection. Transcriptomic analysis of IL-34-treated HMC3 human microglial cells revealed upregulation of markers characteristic of DAM, including TREM2, APOE, FABP5, and FTH1. IL-34-treated HMC3 cells suppressed WNV replication in co-culture with WNV-infected SH-SY5Y human neuroblastoma cells apparently in a cell-cell contact-dependent manner, independent of secreted factors. In WNV-infected mice, IL-34 administration improved survival, reduced viral titers, and decreased neuronal apoptosis. IL-34 also increased the abundance of CD11c- and SPP1-positive DAM-like cells, predominantly in the vicinity of WNV-infected cells, in the mouse brain. These findings indicate that IL-34 promotes the activation of DAM-like cells and enhances protective responses in WNV encephalitis. IMPORTANCEWest Nile virus (WNV) is a major cause of viral encephalitis worldwide, with no approved antiviral treatment or human vaccine currently available. Microglia, the resident immune cells of the brain, play key roles in responding to viral infection. However, the mechanism through which specific microglial activation states contribute to protection in viral encephalitis remains poorly understood. In this study, we show that interleukin-34 promotes microglial responses resembling disease-associated microglia (DAM) and is associated with enhanced protection against WNV infection in cell culture and mouse models. The study findings suggest that activation of DAM-like cells may contribute to protective host responses against neurotropic viral infection and provide new insights into the role of specific microglial states in viral encephalitis.

Chronic cocaine use is associated with neuroinflammation and cognitive dysfunction, but the underlying mechanisms remain unclear. We previously identified oral enrichment of Streptococcus parasanguinis (SP) and other species in individuals with cocaine use disorder (CUD), and here demonstrate that cocaine selectively enhanced SP growth in vitro. To investigate causality, antibiotic-pretreated wild-type C57BL/6 mice received chronic oral inoculation of SP, S. salivarius, Neisseria flavescens, or vehicle. SP-treated mice exhibited spatial memory impairment, increased brain IL-1{beta}, and non-region-specific microglial activation, without detectable bacterial translocation into the brain. While amyloid-associated signaling changes were observed across all bacterial treatment groups, only SP induced cognitive deficits and neuroinflammation. Untargeted metabolomics identified distinct SP-associated oral-to-brain metabolite signatures, including cysteine S-sulfate (CSS) and altered histamine-associated metabolites. CSS and histamine induced neuroinflammatory and amyloid-associated responses in vitro. Together, these findings identify a cocaine-associated oral pathobiont that promotes neuroinflammation and neurodegeneration, suggesting a novel oral microbiome-brain axis in CUD.

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

Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by demyelination, neurodegeneration, and progressive neurological disability. Galanin, a neuropeptide with immunomodulatory properties, signals through G protein-coupled receptors, among which galanin receptor 2 (GALR2) has been implicated with neuroprotective and anti-inflammatory functions. A rare homozygous single nucleotide variant in GALR2 (rs61745847; p.W249L) has been identified in a patient diagnosed with relapsing-remitting MS, however, the biological relevance of this variant in neuroinflammation remains unknown. Here, we investigated the impact of the orthologous GALR2 W248L mutation using a knock-in mouse model and experimental autoimmune encephalomyelitis (EAE). GALR2 W248L knock-in (KI) mice exhibited a more severe clinical course of EAE, accompanied by enhanced inflammatory infiltration, exacerbated demyelination, and increased microglial activation in the spinal cord compared with wild-type (WT) mice. Despite comparable lymphoid and myeloid cell frequencies in the central nervous system, alterations in microglial density and morphology suggested an important contribution of the innate immune system to disease exacerbation in the KI mice. Ex vivo analyses revealed that bone marrow-derived macrophages from KI animals exhibited a pronounced shift toward a pro-inflammatory phenotype, characterized by enhanced M1 polarization, impaired M2-associated responses, and increased NLRP3 inflammasome activation. In parallel, live-cell imaging of primary hippocampal neurons demonstrated reduced galanin binding in mutant cells, consistent with impaired GALR2 functional availability at the plasma membrane. Together, these findings identify GALR2 as a modulator of the neuroinflammatory response and indicate that disruption of galanin-GALR2 signaling promotes sustained innate immune activation, highlighting the relevance of this pathway for MS pathogenesis and its potential as a therapeutic target in neuroinflammatory disorders.

Although the brain was traditionally considered immune-privileged, recent studies show immune factors play key roles in brain function. Dysfunction of these factors is linked to neurodevelopmental disorders, but mechanisms remain unclear. Using a maternal immune activation (MIA) mouse model, we investigated immune-related genes in neurodevelopmental disorder pathogenesis. MIA mice showed increased locomotor activity and disrupted prepulse inhibition. RNA-seq and qPCR analyses revealed persistent increases in major histocompatibility complex class II (MHCII) expression and persistent decreases in GABAergic synapse-related gene expression, particularly glutamate decarboxylase (Gad) expression, in dopaminergic regions. These expressions were negatively correlated, and immunohistochemistry showed MHCII at postsynaptic GABAergic synapses on dopaminergic neurons. Patch-clamp recordings confirmed reduced mIPSC frequency in MIA mice. MHCII knockout mice showed opposite phenotypes, while MHCII overexpression in dopaminergic neurons decreased Gad expression. These results suggest MIA-induced MHCII upregulation enhances pruning of GABAergic synapses on dopaminergic neurons, leading to behavioral deficits.

PurposePerioperative exposure to the volatile anesthetic isoflurane (ISO) has been associated with cognitive and olfactory deficits and may increase the risk of Alzheimers disease (AD). Apolipoprotein E4 (APOE4), the strongest genetic risk factor for AD, contributes to disease pathogenesis through disrupted lipid homeostasis. However, whether and how isoflurane interacts with APOE genotype to influence neurological vulnerability remains unclear. MethodsYoung adult, presymptomatic humanized APOE4 and APOE3 knock-in mice underwent laparotomy under 2 h of isoflurane anesthesia. Microglia and astrocytes were isolated from the olfactory bulb (OB) and hippocampus (HI) by magnetic-activated cell sorting. Lipid composition, transcriptional responses, and functional outcomes were assessed using lipidomic, bulk RNA-seq, and longitudinal behavioral testing. In vivo and ex vivo electrophysiological recordings evaluated neuronal excitability and synaptic transmission in both regions. ResultsBy day 7 post-anesthesia, cell type-specific lipidomic profiling of both OB and HI revealed more pronounced lipid perturbations in microglia and astrocytes from APOE4/ISO mice than from APOE3 mice, characterized by elevated free fatty acids, increased lipid peroxidation, triglyceride depletion, and reduced hippocampal hexosylceramides and cardiolipins. Electrophysiological recordings showed greater olfactory circuit dysfunction in APOE4/ISO mice, accompanied by persistent odor memory deficits, transient olfactory sensitivity loss, early motor coordination impairments, and delayed cognitive deficits. RNA sequencing of the OB identified downregulated lipid metabolism and atherosclerosis-related pathways. ConclusionThese findings establish a mechanistic link between APOE4-dependent glial lipid dysregulation, olfactory circuit dysfunction, and delayed cognitive impairment following isoflurane anesthesia and surgery, highlighting lipid homeostasis as a potential therapeutic target.