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.

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.

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.

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.

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.

The impact of microglia antigen presentation on CNS infiltrating CD8 T cells responses during neurotropic virus infection has been difficult to define. Using Theilers murine encephalomyelitis virus (TMEV) infection of neurons as a model system, our laboratory has previously determined that H-2Db restricted, but not H-2Kb restricted CD8 T cells are required for viral clearance, demonstrating the role of discrete MHC class I alleles. To determine the extent microglia antigen presentation impacts brain-infiltrating CD8 T cells, our laboratory generated novel single MHC class I conditional knockout mice in which H-2Kb or H-2Db can be specifically deactivated in TMEM119+ microglia with tamoxifen administration. During TMEV infection, conditional knockout of H-2Kb in microglia reduced antigen-specific CD8 T cell proliferation in the brain. Meanwhile, mice with deletion of Db in microglia had reduced levels of perforin in antigen-specific CD8 T cells. Furthermore, microglia specific deletion of H-2Db reduced CD8 T cell numbers in the brain and preserved blood-brain barrier (BBB) integrity. Microglial Db restricted antigen presentation was also essential for the reactivation of CD8 tissue resident memory (TRM) cells and BBB integrity during memory recall responses. These findings further our understanding of how brain infiltrating antiviral CD8 T cell responses are impacted by microglia, as well as define how this cellular interaction contributes to BBB disruption during neuroinflammation. These findings also have high significance to our understanding of how microglia impact CD8 TRM cell populations that reside in the brain long after virus infection is cleared. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/722741v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@9302a9org.highwire.dtl.DTLVardef@193b5f4org.highwire.dtl.DTLVardef@8eb036org.highwire.dtl.DTLVardef@1d2cd6a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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

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.

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.

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.

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.

The effect of sortilin inhibition on acute inner retinal neurodegeneration induced by optic nerve crush was investigated. Pharmacological sortilin inhibition using intravitreal delivery of a polyclonal antibody or a small-molecule inhibitor was evaluated in C57BL/6JRj male mice subjected to unilateral crush. Inner retinal thickness was evaluated by optical coherence tomography, and retinal ganglion cell density was determined in retinal flat mounts. Furthermore, the effect of constitutive sortilin deficiency was examined using Sort1-/- mice. Changes in protein and mRNA levels of sortilin, p75NTR, and associated injury markers were analyzed. Neither pharmacological inhibition or constitutive loss of sortilin protected against inner retinal thinning or retinal ganglion cell loss following optic nerve crush. A transient 1.4-fold increase in p75NTR mRNA was observed early after injury, accompanied by a two-fold increase in protein levels. While sortilin expression remained largely unchanged, sortilin deficiency was associated with an altered baseline retinal state, including increased GFAP, p75NTR, and proBDNF levels. Following optic nerve crush, the induction of p75NTR was significantly attenuated in sortilin-deficient retinas compared with wild type, without affecting the extent of RGC degeneration. In summary, sortilin inhibition does not preserve inner retinal structure following optic nerve crush, but modulates glial activation, inflammatory signaling, and proneurotrophin dynamics. These findings indicate that sortilin-dependent pathways are not key drivers of optic nerve crush-induced neurodegeneration but may be more relevant in disease contexts characterized by chronic stress and neuroinflammation.

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.

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.

While Apolipoprotein E4 (APOE4) is the greatest known genetic risk factor for late-onset Alzheimers disease, its mechanistic role in the brain-resident macrophage, microglia, remains elusive. Microglia are important in the clearance of pathology in disease, heavily relying on lysosome functionality; therefore, we sought to understand the impact of APOE4 on microglial function. APOE44 microglia have been shown to have lipid accumulation, yet the mechanisms leading to this accumulation are unknown. Using induced pluripotent stem cell-derived microglia, we found that the APOE4 haplotype resulted in transcriptional state shifts in microglia, suppressing activated-response microglia (ARMs) and promoting a G2 senescent-like state. We found that APOE44 microglia accumulate cholesterol esters and provide less lipid support to fibroblast-induced neurons, decreasing their synaptic connections. APOE44 microglia secrete significantly less lipoproteins, leading to the accumulation of lipoproteins within the cells including the lysosomes. APOE44 microglia exhibit impaired lysosomal acidification and degradation capacity. Further, our results elucidated that APOE44 microglia are proinflammatory and shift away from fatty acid oxidation towards glycolysis, due to dysfunctional mitochondria. Taken together, our findings indicate that a loss-of-function in lipoprotein secretion drives intracellular lipid accumulation, including within lysosomes, ultimately disrupting the lysosome-endoplasmic reticulum-mitochondrial axis. This drives a proinflammatory and metabolically compromised microglial phenotype with impaired neuro-supportive functions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/724612v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@18d6a2org.highwire.dtl.DTLVardef@b3644dorg.highwire.dtl.DTLVardef@17e3716org.highwire.dtl.DTLVardef@1529caf_HPS_FORMAT_FIGEXP M_FIG C_FIG