Journal of Neuroinflammation

Background and ObjectivesHerpes simplex virus type 1 (HSV-1) is a neurotropic pathogen capable of invading the central nervous system (CNS) and increasingly associated with chronic neuroinflammation, cognitive impairment, and neurodegenerative disease. While microglia orchestrate the initial immune response to HSV-1, the molecular mechanisms that regulate their sustained neuroinflammatory activity in vivo remain poorly understood. MethodsTo define the transcriptional and epigenetic mechanisms that shape microglial responses during acute HSV-1 infection in vivo, we have, for the first time, integrated single-nucleus RNA sequencing, chromatin accessibility profiling, and spatial transcriptomics in a physiologically relevant intranasal HSV-1 infection model. ResultsSingle-cell multiome analysis of CD11b nuclei identified transcriptionally and epigenetically distinct microglial and macrophage populations. HSV-1 infection redistributed monocyte-lineage states, with a marked overrepresentation of interferon (IFN)-responsive microglia and macrophage-associated populations. These states exhibited differential amplification of STAT1/2-, IRF1-, and CEBPB-centered regulons, distinguishing IFN-responsive microglia from macrophage-enriched populations rather than reflecting uniform activation. Homeostatic microglial gene signatures (e.g., ApoE, Cst3) were reduced in response to HSV-1 infection. Spatial transcriptomics localized HSV-1 antigen to discrete brainstem regions, which were enriched for predicted STAT-, IRF-, and CEBPB-regulated targets identified through single-nuclei analysis. DiscussionUsing a multiomic framework, we demonstrate that HSV-1 infection drives transcriptional and epigenetic remodeling of microglial populations, characterized by a dominance of IFN-responsive states and a loss of homeostatic signatures. These findings provide mechanistic insight into how localized viral infection can reprogram microglial regulatory landscapes to maintain persistent HSV-1-associated neuroinflammation, contributing to long-term neurological vulnerability and neurodegenerative disease risk.

CommentaryElucidating how normal aging increases vulnerability to neurodegeneration remains a major gap in our understanding of disease risk and progression. The dorsal striatum serves as the primary input nucleus of the basal ganglia and is a key region implicated in multiple neurodegenerative diseases (NDDs) (1). In Colon et al. 2025 (2), we examined the impact of normal aging on neuroinflammatory signaling and perineuronal net (PNN) homeostasis within the dorsal striatum. We observed age-associated shifts in the inflammatory landscape and evidence of increased microglial activation, yet PNN homeostasis was largely preserved (2). PNNs are highly organized extracellular matrix (ECM) specializations that preferentially enwrap the soma and proximal dendrites of fast-spiking GABAergic parvalbumin (PV) interneurons, where they contribute to the regulation of synaptic plasticity and provide protection against oxidative stress (3,4). Building on these findings, we developed a working hypothesis to explain the apparent preservation of PNN homeostasis despite an aging-associated pro-inflammatory environment. The shift toward a pro-inflammatory milieu, together with increased gliosis and phagocytic activity, would be expected to impact the maintenance and integrity of perineuronal nets. The observed increase in phagocytosis-related markers may reflect microglia-directed activity as well as contributions from additional central nervous system (CNS) cell populations. Microglia are specialized embryonic-derived myeloid cells that serve as the resident immune cells of the brain and contribute to PNN homeostasis under physiological conditions (5). In Colon et al. 2025, we observed evidence of microgliosis (e.g., morphological changes, Iba1, Trem2) along with elevated expression of markers associated with phagocytosis (e.g., Cd68) and extracellular matrix-modifying proteases (e.g., Mmp9, Adam17) capable of cleaving key PNN components (2). Importantly, Cd68 expression is not exclusive to microglia and has been detected in brain infiltrating macrophages, reactive astrocytes, and neutrophils during inflammation (6-8). Thus, increased Cd68 levels may not solely reflect microglial phagocytic activation but may also reflect astrocyte reactivity and phagocytic phenotypes. Furthermore, astrocytes are the most abundant glial cell in the brain, and they play a major role in maintaining CNS homeostasis by regulating extracellular neurotransmitter concentrations, providing metabolic support, contributing to the synthesis and remodeling of PNN components, and modulating neuronal communication through their involvement in the tetrapartite synapse (9-12). Astrocytes can also phagocytosis microglial debris, myelin, and synapses (7). To better define the cellular source of phagocytic activity and its relationship to PNN remodeling in aging, we performed immunostaining for microglia (Iba1+), astrocytes (GFAP+), phagolysosomal activity (CD68+), and PNNs using Wisteria floribunda agglutinin (WFA+), enabling us to assess the spatial relationship between phagocytosis and PNN components.

IntroductionThe major histocompatibility complex class II (MHC-II) pathway is central to adaptive immunity and immune tolerance, and age-related erosion of these mechanisms is increasingly recognized as a driver of chronic neuroinflammation. The HLA-DRB1*15:01 allele--the strongest genetic risk factor for multiple sclerosis in Caucasians--has been implicated in shaping pathogenic CD4 T-cell responses and broader neuroimmune vulnerability, yet how this allele modulates age- and sex-dependent neuroimmune processes within the central nervous system (CNS) remains poorly defined. MethodsWe investigated the impact of HLA-DRB1*15:01 expression using a humanized mouse model (HLA mice) and wild-type (WT) controls. Male and female mice were analyzed at 6, 9, and 15 months of age, with endocrine stratification in females. Behavioral testing, flow cytometry, immunofluorescence, and multiplex cytokine analyses were used to assess cognitive performance, glial activation and oxidative stress, astrocyte-microglia IL-3/IL-3R signaling, endothelial activation, selective immune cell accumulation at CNS borders, tissue organization, and hippocampal cytokine profiles. ResultsHLA mice developed age- and sex-dependent cognitive impairment, most pronounced in aged females. HLA-DRB1*15:01 expression promoted progressive microglial activation, characterized by increased CD14 and CD68 expression, elevated mitochondrial oxidative stress, altered astrocyte phenotypes, and enhanced IL-3/IL-3R signaling. Hippocampal axonal and myelin organization was disrupted in aged HLA mice, and this disruption was spatially associated with increased microglial presence. At CNS interfaces, HLA mice exhibited selective immune remodeling, including increased accumulation of CD4 T cells and NK1.1CD3 natural killer T (NKT) cells, particularly in females, accompanied by endothelial activation, as evidenced by elevated ICAM-1 and E-selectin expression. Hippocampal cytokine profiling revealed selective, sex-biased alterations, including increased IL-12p70 and reduced IL-10 and IL-2, without broad induction of classical inflammatory cytokines. ConclusionTogether, these findings demonstrate that HLA-DRB1*15:01 drives a coordinated, age- and sex-dependent neuroinflammatory program linking behavioral dysfunction, glial activation and oxidative stress, selective immune cell recruitment, endothelial activation, tissue remodeling, and targeted cytokine imbalance. This integrated phenotype provides mechanistic insight into how this major MS risk allele confers vulnerability to chronic neuroinflammation during aging, with heightened impact in females.

Viral pathogens cause neurologic sequelae during acute and post-acute phases of infection. CD8+ T cells are hypothesized to contribute to these effects, but the mechanisms through which they act are poorly understood. We posited that viral infections and/or antiviral immune responses induce DNA damage, which may underlie neuronal dysfunction. Using a model of neurotropic flavivirus infection, we found that genes associated with interstrand crosslinking (ICL) DNA damage were upregulated post-infection, temporally congruent with T cell infiltration. Using an in vitro co-culture system, our results demonstrate that CD8+ T cells induced ICL-like damage in primary neurons, independent of antigen-specific interactions or direct contact. Human transcriptomic data also showed overexpression of genes associated with ICL damage in the brains of people with Parkinsons disease, Alzheimers disease, and multiple sclerosis, which are neurologic diseases characterized by neuroinflammation. Together, these data indicate that CD8+ T cells cause genotoxic DNA damage in neurons, which may underlie the neurologic dysfunction seen in neurodegenerative conditions. SummaryResults indicate that CD8+ T cells induce interstrand crosslinking-like DNA damage in neurons independent of antigen-specificity in a mouse model of viral infection, in vitro primary cell culture system, and human neurologic diseases. These findings provide insight on the mechanistic connection between neuroinflammation and neurologic dysfunction.

Highly aggressively proliferating immortalized (HAPI) cells were initially described as a spontaneously immortalized rat cell line isolated from a mixed neonatal rat glial population. It was demonstrated that HAPI cells are phagocytic, stain for macrophage-/microglia-specific markers like CD11b and GLUT5, and exhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-) release. These characteristics led to their widespread use as a rat microglial cell line. Here, we report that HAPI cells are mouse cells, not rat cells, but further establish that they have a microglia-like identity and properties useful for in vitro modeling. Cell line authentication by short tandem repeat (STR) profiling, a method that detects identifying DNA signatures, indicates that HAPI cells are a 100% match for SIM-A9 cells, a mouse microglial cell line reported to be spontaneously immortalized from primary cell culture. We find that both HAPI cells and SIM-A9 cells express the microglia-selective gene Tmem119, as well as the microglia-/macrophage-selective marker Cx3cr1, supporting a microglial origin. Like primary rodent microglia or macrophages, HAPI cells respond to combined stimulation with LPS and the Type II interferon, interferon-gamma (IFN-{gamma}), with a pro-inflammatory morphology, NO production, NO-dependent suppression of mitochondrial oxygen consumption, and increased extracellular acidification (an indicator of glycolysis). The Type I interferon, interferon-alpha (IFN-), also reduces mitochondrial oxygen consumption when administered alone or in combination with LPS. Overall, results indicate that HAPI cells are SIM-A9-related mouse cells of microglial origin and support their continued use to study microglial behavior in vitro, including immunometabolism.

BackgroundTraumatic brain injury (TBI) initiates a rapidly evolving neuroinflammatory response; however, the temporal relationship between early innate immune activation, T cell polarization, and neurobehavioural recovery remains poorly understood. Here, we hypothesize that interleukin-1{beta} (IL-1{beta}) is a critical upstream mediator that polarizes T cells towards pro-inflammatory and cytotoxic effector functions following TBI. MethodsUsing a controlled cortical impact model in adult male C57BL/6J mice, we mapped post-injury immune dynamics and investigated whether targeting key innate inflammatory compartments influenced subsequent T cell programming and neurological outcomes. We conducted longitudinal immune profiling by multiparameter spectral flow cytometry and quantitative polymerase chain reaction up to 10 days post-injury. Antibody-based immune depletion strategies were used to investigate neutrophil and monocyte contributions to the post-traumatic T cell response, while pharmacological inhibition of NLRP3 inflammasome by MCC950 treatment was used to investigate the contribution of IL-1{beta}. ResultsTBI elicited a structured early innate immune response, marked by rapid chemokine induction, followed by temporally distinct infiltration of neutrophils, monocytes, and dendritic cells. Neutrophils and monocytes were the predominant early IL-1{beta}-producing infiltrating populations. This was followed by a delayed adaptive phase characterized by sustained recruitment of T cell subsets (CD4+, CD8+, {gamma}{delta}+), alongside dynamic effector cytokine production (IL-17, IFN-{gamma}). Neutrophil depletion altered the early myeloid composition but did not result in durable improvements in T cell effector responses or neurobehavioral outcomes. Depletion of CCR2-dependent inflammatory monocytes reduced acute monocyte accumulation and attenuated early downstream T cell responses; however, these effects were not sustained and only resulted in modest neurobehavioural benefits. In contrast, inhibition of the NLRP3 inflammasome suppressed microglial IL-1{beta} production, without significantly altering leukocyte recruitment or subacute T cell effector phenotypes. These phenotypic changes were associated with improvements in motor and cognitive function recovery. ConclusionWe show that early monocyte IL-1{beta} signalling actively regulates downstream T cell infiltration and effector function after TBI. In addition, inhibition of NLRP3 inflammasome after TBI attenuates microglial IL-1{beta}-associated immune activation and results in behavioural improvement despite ongoing leukocyte recruitment, indicating that targeting the nature and cellular source of IL-1{beta} signalling can dissociate immune cell burden from neurological outcomes. Collectively, our findings identify myeloid IL-1{beta}-linked pathways as a viable bridge between innate and adaptive immunity post-TBI, and underscore cellular compensation as a critical design consideration for next-generation immunotherapies.

Nerve growth factor (NGF) exerts neuroprotective effects in the retina, and accumulating evidence indicates that microglia represent a key cellular target of NGF/TrkA signaling. However, evidence showing that the NGF/TrkA signaling in microglia is required for downstream neuroprotective actions remains unresolved. Here, we directly addressed this question by pharmacologically depleting microglia and assessing the impact on NGF pathway activity and retinal integrity. Adult C57BL/6J mice were treated with the CSF1R inhibitor PLX5622 for three weeks, resulting in a robust ([~]77%) depletion of retinal microglia. Microglial ablation induced marked structural and cellular alterations, including significant loss of retinal ganglion cells (RGCs) and thinning of retinal layers, in the absence of any other lesion or insult. Residual microglia exhibited layer-specific phenotypic changes, with a phagocytic profile in the ganglion cell layer and a more ramified morphology in the outer plexiform layer. Strikingly, microglial depletion led to a profound decrease of NGF signaling, with a strong reduction in total and phosphorylated TrkA, and decreased p75NTR levels, in retinal extracts. The amount of TrkA expression is strongly correlated with microglial levels, supporting a primary role of microglia in sustaining NGF signaling in the retina. Together, these findings demonstrate that microglia are required for NGF/TrkA signaling and identify these cells as essential mediators of NGF-dependent neuroprotection in the retina.

While dopamine is a monoamine neurotransmitter best known for its roles in reward, motivation, and motor function in the central nervous system, its actions extend beyond neurons and can influence non-neuronal cells via epigenetic mechanisms. An increasing body of literature corroborates that dopamine signaling is important in immune cells, which express dopamine receptors (DRD1-DRD5) as well as the molecular machinery for dopamine synthesis and metabolism. Dopamine can regulate inflammatory activity, cell trafficking, and disease pathology, yet the epigenetic mechanisms underlying these effects remain poorly understood. Here, we show that in primary human monocyte-derived macrophages, dopamine increases DNA methylation at the IL-1{beta} proximal promoter in a DNMT-dependent manner, while concurrently upregulating IL-1{beta} gene expression. Dopamine also increases the expression of key epigenetic regulators, including TET2, HDAC2, and HDAC6, suggesting coordinated changes in both DNA methylation and histone modifications that shape inflammatory transcription. Importantly, baseline dopamine receptor expression and donor demographics, including sex and age, influence the magnitude of these epigenetic responses, highlighting inter-individual variability in macrophage sensitivity to dopaminergic signaling. These findings establish dopamine as a modulator of macrophage inflammation via epigenetic remodeling and provide a mechanistic framework for understanding how peripheral immune cells respond to dopaminergic cues. By linking dopamine signaling, epigenetic regulation, and innate immunity, this work identifies potential targets for therapeutic intervention and supports the use of accessible human immune cells to investigate dopaminergic dysregulation in neuroimmunological disorders.

Background and PurposeSubarachnoid hemorrhage (SAH) triggers a complex immune response that critically influences early brain injury (EBI) and long-term outcomes. However, the precise spatiotemporal dynamics and heterogeneity of immune cell infiltration and microglial reprogramming remain poorly understood. We aimed to construct a high-resolution immune atlas to delineate cell states, lineage trajectories, and spatial niches following SAH. MethodsWe integrated single-cell RNA sequencing (scRNA-seq) of CD45+ immune cells with spatial transcriptomics (ST) in a murine endovascular perforation SAH model. Immune landscapes were profiled at 24 hours (acute phase) and 72 hours (subacute phase) post-injury, compared with sham controls. Advanced bioinformatics integrated transcriptional signatures with spatial localization to map macrophage, neutrophil, and microglial dynamics. ResultsOur atlas reveals a coordinated immune transition from acute inflammation to reparative processing. We identified five macrophage, four neutrophil, and eight microglial subsets with distinct spatiotemporal patterns. Notably, we discovered a SAH-specific inflammatory microglial population (MG_03; Spp1+/Lpl+) that clusters at the rupture site during the acute phase. This subset is transcriptionally distinct from disease-associated microglia (DAM) in other neurodegenerative conditions. Trajectory analysis suggests MG_03 acts as a signaling hub for immune recruitment before transitioning toward proliferative and reparative states (MG_06-08) that disperse into the parenchyma by 72 hours. ConclusionsThis study provides the first comprehensive spatiotemporal immune atlas of SAH, highlighting the distinct role of the Spp1+ MG_03 subpopulation in early injury sensing. These findings offer a roadmap for identifying precise therapeutic windows and targeting specific immune subsets to mitigate EBI. Graphical AbstractExperimental workflow for single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) in a mouse SAH model induced by endovascular perforation. Brain tissue from the ipsilateral (injured) hemisphere was collected from sham and at 24 h and 72 h post-SAH. For scRNA-seq, CD45 immune cells were isolated prior to library preparation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/703421v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@4a1bc9org.highwire.dtl.DTLVardef@16664c4org.highwire.dtl.DTLVardef@1619725org.highwire.dtl.DTLVardef@a0d34_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mild traumatic brain injuries (mTBI) can substantially impact quality of life, and repetitive mTBIs (rmTBI) can amplify injury effects compared to a single injury. However, effective clinical treatments remain elusive, largely due to an incomplete understanding of the underlying injury mechanisms. Neuroinflammation has emerged as a key contributor to worse functional outcomes after mTBI/rmTBI. While microglia are traditionally viewed as primary mediators of post-injury inflammation, accumulating evidence suggests neurons play an immunomodulatory role in initiating the rmTBI inflammatory cascade through activation of intracellular proinflammatory pathways like p38 MAPK and secretion of cytokines that, in turn, stimulate microglial activation. Here, we tested whether inducible neuronal p38 knockout protects against functional, immune, and cerebrovascular consequences of a weight-drop closed head injury model of rmTBI. A battery of functional assays was conducted 4 weeks post-injury, and tissues were collected at both 4 hours and 4 weeks following final CHI. In males, neuronal p38 knockout protected against injury-induced depressive-like behavior, hyperactivity, synaptic loss, microglial reactivity, cytokine upregulation, and reduction in cerebral blood flow. In females, neuronal p38 knockout protected against risk-taking behavior and partially protected against cytokine upregulation but had limited effect on microglial reactivity and cerebral blood flow. Together, these findings identify neuronal p38 as a sex-dependent driver of rmTBI-associated neurological consequences, and they support neuronal p38-immune signaling as a mechanistically relevant therapeutic target for future studies.

This study describes the distribution of non-reactive brain-resident microglia densely populated along the borders of the lateral ventricles and choroid plexus in premature rabbit pups during early forebrain development. Following intraventricular hemorrhage (IVH), microglia become activated, proliferate, and migrate deeper into parenchymal regions. During this process, activated microglia exhibit a global expansion with a disproportionally elevated proinflammatory M1 nomenclature phenotype from 25% to 50% of the total; that shift was reduced by sulforaphane (SFN; Nrf2-antioxidant response element [ARE] activator of anti-inflammatory pathways) plus deferoxamine (DFN; iron chelator) treatment. Transcriptome analysis identified over expression of pro-inflammatory calcium-binding proteins S100A8 and S100A12 (intracellular damage signals), as well as chemokines CXCL8 and CXCL10 by neurons and microglia. The combination treatment of SFN-DFN mitigated M1 infiltration, suppressed the magnitude of inflammation and reduced ferroptosis after IVH in the developing postnatal brain. Moreover, SFN-DFN treatment reversed most dysregulated genes in inflammation and iron homeostasis networks, revealing potential molecular targets for additional pharmacologic interventions after IVH. We propose that reducing the toxic microcellular environment will attenuate both the injurious inflammatory responses and improve recovery of the trajectory toward normal brain development. Additionally, suppression of proinflammatory molecules and iron toxicity should promote better survival as well as salutary effects of "living stem cell therapy" as we have previously shown.

Atherosclerosis is linked to an increased risk of cognitive decline, with chronic inflammation being a common feature of both pathologies. IL-12 activates STAT4 to regulate myeloid cell functions, and blockade of this pathway alleviates cognitive impairment in Alzheimers models. However, the mechanisms connecting vascular pathology to neuroinflammation remain unclear. Here, we examine whether STAT4 functions as a common mediator of neuroinflammation in atherosclerosis. We demonstrate that LysMCre-specific STAT4 deficiency ameliorates deficits in long-term memory in low-density lipoprotein-deficient (Ldlr-/-) mice fed a high-fat diet (HFD-C). STAT4 deficiency moderately reduces Ser199-phosphorylated Tau burden. Atherosclerosis alters brain immune composition, characterized by increased numbers of CD45+ leukocytes, activated microglia, and activated T and B cells, whereas STAT4 deficiency attenuates these effects. Nanostring gene-expression pathway analysis further highlights the importance of STAT4 in regulating multiple neuroinflammatory pathways and the Rhodopsin-like receptor signaling, which is associated with synaptic plasticity. LysMCre-specific STAT4 deficiency supports microglial efferocytosis in atherosclerotic Ldlr-/- mice and increases the number of efferocytotic macrophages. Accordingly, STAT4 deficiency also reduced neuronal death. Overall, our data reveal an important role for myeloid-driven STAT4 expression in the pathogenesis of cognitive decline associated with atherosclerosis, mediated through impaired efferocytosis and enhanced leukocyte activation, leading to increased brain neuroinflammation.

Toll like receptor 9 (TLR9) is an endosomal-lysosomal innate immune receptor which recognizes microbial and host-derived double-stranded DNA (dsDNA) to induce cellular immunity. TLR9 pathway has been implicated in Alzheimers disease (AD) pathology via its ability to modulate peripheral immune cells but its exact role in the central nervous system (CNS) remains unclear. Here we show that extranuclear dsDNA accumulating in human AD brain and in a mouse model of AD, together with microglia around amyloid-{beta} (A{beta}) plaques stimulates TLR9 in vitro. Loss of TLR9 inhibits the transition of homeostatic microglia to reactive microglia, a cell type associated with a chronic pro-inflammatory state, promoting their reprogramming into an anti-inflammatory state, thereby restoring lysosomal integrity required for A{beta} peptides degradation. Predisposition to lysosome permeabilization could be mimicked with repeated TLR9 stimulation using CpG-A ODNs in a human microglial cell line and was rescued with TLR9 inhibition. Furthermore, lack of TLR9 protects mice developing AD from cognitive defects and neuronal loss. A{beta} pathology is also reduced specifically in male mice. Altogether, our results have identified a critical role of TLR9 in AD, where it modulates microglial activation and lysosome function to interfere with the progression of AD. These findings suggest that targeting TLR9 signaling and lysosomal function may be a relevant strategic approach to reduce AD pathology.

BackgroundAlzheimers disease (AD) is the most prevalent neurodegenerative disease, currently devoid of a cure. ADs clinical manifestations stem from a multitude of dysfunctional cellular processes, regulated by mitochondria-endoplasmic contact sites (MERCS), which undergo physical alterations and malfunction in AD brain. Despite ongoing research, the understanding of MERCS in AD remains in its nascent stages. We postulate that these subcellular interfaces are responsible for AD progression. Neuroinflammation contributes significantly to neurodegeneration and is primarily driven by microglia, the innate immune cells in the brain. In AD, activated microglia secrete pro-inflammatory cytokines that compromise neuronal vitality. The production of these cytokines is promoted by NLRP3 inflammasome. Although inflammasome activation has been observed at MERCS, the underlying MERCS-mediated mechanisms governing regulation of inflammasome activation remain to be elucidated. MethodsPrimary microglia were isolated from 3-4 months old wild-type (WT) and AppNL-G-F mice (AD). MERCS ultrastructure was analyzed by transmission electron microscopy. Mitochondrial Ca2+ level and metabolic function were assessed using Rhod-2 AM fluorescence and Seahorse extracellular flux analysis respectively. Inflammasome activation was induced by lipopolysaccharide and nigericin and evaluated by IL-1{beta} ELISA, caspase-1 activity assay, and ASC immunocytochemistry. MERCS were genetically modulated via siRNA-mediated knockdown of MERCS-associated proteins, and ER-to-mitochondria Ca{superscript 2} transfer was pharmacologically inhibited using Xestospongin C and MCU-i11. Microglial A{beta} phagocytosis was quantified using fluorescence-conjugated A{beta}1-42. ResultsAD microglia exhibited increased MERCS number and contact length, accompanied by a reduction in mitochondria-ER proximity. These structural changes were associated with elevated mitochondrial Ca2+ levels and enhanced respiratory activity, indicating metabolic reprogramming and functional change. Structural and functional decrease of microglial MERCS attenuated NLRP3 inflammasome activation and restored inflammasome-associated impairments in A{beta} phagocytosis. Pharmacological inhibition of Ca2+ channels at MERCS identified ER-to-mitochondria Ca2+transfer as a key regulatory mechanism for inflammasome activation. ConclusionsOur findings identify microglial MERCS remodeling as an early event in AD and establish ER-mitochondria coupling as an upstream regulator of energy metabolism, inflammation, and A{beta} clearance. Targeting MERCS may therefore represent a promising strategy to modulate neuroinflammation while preserving essential microglial functions in AD.

Traumatic brain injury (TBI) elicits robust neuroinflammation and oxidative stress, coupled with an acute inhibition of macro-autophagy (autophagy) in neurons and microglia. Rubicon (Rubcn), a Beclin1 interacting protein that suppresses autophagy and mediates LC3-associated phagocytosis and endocytosis (LAP/LANDO), influences inflammatory signaling in metabolic, neurodegenerative, and inflammaging diseases; yet its role in acquired brain injury has not been defined. Using a controlled cortical impact model, we investigated the role of Rubicon in acute neuroinflammatory responses following injury by comparing wild-type and Rubcn-mutant mice. Bulk-RNA sequencing of injured cortex revealed attenuated induction of inflammatory pathways and reduced activation of pro-inflammatory microglial/macrophage phenotype in injured Rubcn-mutant mice. Rubcn-mutant mice demonstrated less pronounced inhibition of autophagy during the acute phase of injury. Although the inflammatory dicerences were transient, Rubicon mutant mice exhibited improved motor coordination and gait stability during recovery. Proteomic analyses revealed the presence of a truncated Rubicon protein in the mutant mice and identified the negative regulator of reactive oxygen species (NRROS) as a novel interactor of Rubicon. Consistent with this interaction, Rubcn-mutant mice displayed markedly reduced oxidative damage, indicated by decreased lipid peroxidation after injury. Together, these findings indicate that Rubicon promotes acute neuroinflammatory and oxidative stress responses following TBI by modulating autophagy and ROS production. Rubicon mediated pathways may serve as therapeutic targets that ocer a neuroprotective strategy to improve outcomes after TBI.

Excessive iron accumulation is a pathological feature of several neurodegenerative diseases (NDDs) and a growing body of evidence suggests that ferroptosis, an iron-dependent form of regulated cell death (RCD) driven by lipid peroxidation, is implicated in their pathogenesis. Microglia, the brains resident immune cells, buffer iron overload but become susceptible to ferroptotic death, exacerbating neuroinflammation and neuronal loss. To uncover the molecular events leading to microglial ferroptosis, we established a human microglial ferroptosis model using the HMC3 cell line. This model recapitulates core features of ferroptosis, including increased reactive oxygen species (ROS) and peroxidation of lipids at the membrane, both rescued by Ferrostatin-1 (Fer-1). We used this model to perform integrated multi-omics profiling and identified significant dysregulation in lipid species, notably an accumulation of sterols, including oxysterols such as the 7-oxo-cholesterol, alongside the oxidation of polyunsaturated fatty acid (PUFA) characteristic of ferroptosis. Transcriptomic and proteomic analyses corroborated these findings, revealing the upregulation of genes and proteins involved in the mevalonate pathway and cholesterol metabolism. Importantly, the increased expression of some of these key metabolic genes was also reversed by Fer-1 treatment, indicating their role in a pre-ferroptotic signature. Our model provides a novel platform for investigating early molecular events in microglia ferroptosis. Integrating these findings into future investigations could uncover new protective mechanisms against microglia ferroptosis at the crossroad between ROS level mitigation and sterol metabolism.

Although congenital Zika virus (ZIKV) syndrome is well-characterized, the neurodevelopmental consequences of postnatal infection are less understood. Here we used a rhesus macaque model to investigate the developmental consequences of ZIKV infection during infancy on the brain and behavior, building on our prior research. Male and female infant rhesus macaques infected with ZIKV at 1 month of age were compared to sex-, age-, and rearing-matched uninfected controls and infants treated with the TLR3 agonist PolyIC as a control for activation of the innate immune system. Longitudinal behavioral assessments revealed alterations in emotional regulation following ZIKV exposure, including poor state control scores obtained from the Infant Neurobehavioral Assessment Scale early after ZIKV infection and longer-term displays of increased hostility during an acute stressor. While attachment bonds to caregivers were preserved, ZIKV-infected infants showed sex-specific alterations in behavioral regulation during caregiver separation compared to controls. At 3 months of age, MRI scans revealed larger total cerebrospinal fluid (CSF) volume and reduced volumes in visual processing regions in ZIKV-infected infants compared to controls. Postnatal ZIKV exposure also resulted in sex-specific brain structural alterations with males exhibiting amygdala hypertrophy, whereas ZIKV-infected females had volumetric reductions in temporal-limbic and temporal-auditory cortices. These findings demonstrate that postnatal ZIKV infection disrupts the development of sensory, social and emotion-regulatory systems and CSF function, highlighting the critical need for long-term monitoring of exposed children. One-Sentence SummaryPostnatal Zika virus infection disrupts emotional regulation and alters brain development in infant rhesus macaques, revealing a critical window of neurodevelopmental vulnerability that extends beyond the fetal period.

BackgroundRecent studies revealed key immunological mechanisms within the central nervous system (CNS) that contribute to Alzheimers disease pathology. Additionally, analyses of human AD datasets have also associated viral encephalitis exposure (i.e., viral-induced neuroinflammation) with the development of AD and dementia, highlighting the need to better understand how viral encephalitis and neuroimmune mechanisms within the brain may impact AD pathologies such as A{beta} plaque deposition. Intracranial infection of susceptible mice with the neurotropic JHM strain of murine coronavirus (JHMV) results in acute encephalomyelitis characterized by viral infection of glia and a robust inflammatory response comprised of monocytes/macrophages and T cells that aid in controlling viral replication. MethodsTo determine how coronavirus-induced encephalitis may impact established A{beta} plaque deposition, we intracranially inoculated JHMV into aged 5xFAD model of amyloidosis. We utilize immunohistochemical and biochemical analysis to assess the impact on existing A{beta} pathology. We also utilize spatial transcriptomic imaging to explore how viral encephalitis affects cellular responses to plaque pathology with single-cell resolution. ResultsIn aged 5xFAD mice, JHMV-induced encephalitis at 12 days p.i. resulted in minimal changes to overall A{beta} protein within the brain. However, viral encephalitis induces CD4+ and CD8+ T cell infiltration and more Lgals3/MAC2-expressing macrophages surrounding dense-core A{beta} plaques, which appear more compacted in JHMV-infected 5xFAD brains compared to uninfected 5xFAD controls. We compared gene expression within JHMV-infected 5xFAD mice and uninfected controls to identify distinct cellular responses to A{beta} plaques that differed. Utilizing differential gene expression and pathway analysis, we found that viral encephalitis increased the proportion of myeloid cells in the 5xFAD brain, which also showed down-regulated disease-associated (DAM) pathways involving A{beta} clearance, response to lipids, and macrophage activation within the post-encephalitis 5xFAD brains. ConclusionsTogether, these findings suggest an attenuated myeloid cell response to A{beta} plaque burden in 5xFAD mice following acute viral encephalitis. Future experiments aim to further dissect inflammatory mechanisms between infiltrating myeloid cells, T cells, and the progression of A{beta} and tau pathology. Data derived from these experiments will further elucidate the viral-induced neuroimmune mechanisms that affect AD pathology and offer an opportunity to determine how these neuropathologic changes, such as subsequent neuronal damage, occur.

Pathogens such as Porphyromonas gingivalis (P. gingivalis) may lead to Alzheimers disease (AD), but how they get into the brain and cause AD remains unclear, especially their level in blood is usually low. AD involves early breakdown of the blood-brain barrier (BBB). BBB disruption may allow blood-derived neurotoxic components to penetrate the BBB and enter the brain. We investigated whether BBB disruption permits P. gingivalis to enter the brain and accelerate disease-related changes. Using animal models, human BBB organoids and bEnd.3 cells, we found that a leaky BBB, especially through increased caveolin-1-dependent transcytosis, allowed P. gingivalis to cross into the brain. The bacteria triggered the hyperphosphorylation of Tau protein, increased A{beta} levels, and sustained neuroinflammation via activated glial cells. These findings indicate that BBB dysfunction facilitates P. gingivalis invasion, creating a vicious cycle of infection and neurodegeneration that may drive AD progression.

Astrocytes play key roles in maintaining brain homeostasis, metabolism, and neurovascular integrity, yet their diversity and age-related modulation remain insufficiently understood, particularly across primate lineages. While rodent studies have generated extensive knowledge, notable species differences highlight the need for comparative analyses in non-human primates. The gray mouse lemur (Microcebus murinus), a small primate widely used in aging research, offers a valuable but underexplored model for studying astroglial aging. In this study, we characterized astrocyte distribution, morphology, and reactivity in 17 mouse lemurs aged 1.0-11.5 years using GFAP and vimentin immunohistochemistry. We identified marked regional and morphological heterogeneity, with dense astrocytic labeling in white matter, hippocampus, and sparse but diverse cortical populations. Distinct astrocyte subtypes--including fibrous, protoplasmic, projection, pial and subpial interlaminar, radial glia-like cells, tanycytes--were documented. Varicosity-bearing processes were common across multiple astroglial subtypes and may indicate altered physiological states. Quantitative analyses revealed pronounced age-related increases in astrocytic reactivity, particularly in white matter and interlaminar astrocytes. Cortical and hippocampal changes were comparatively modest. These findings indicate region-specific astrocytic vulnerability during aging and support the translational value of the mouse lemur for investigating glial aging in primates. Main PointsO_LIThe mouse lemur is the smallest primate on earth with a key role to understand primate brain characteristics. C_LIO_LIWe characterized seven different astrocyte subtypes: from fibrous to primate-specific astrocyte as interlaminar astrocytes in different brain regions of this primate. C_LIO_LIVaricosities were reported in different astrocyte subtypes found close to brain borders. C_LIO_LIMain age-related changes concerned fibrous astrocytes in the white matter and interlaminar astrocytes at the cortical border. C_LI