Journal of Neuroinflammation

Microglia are essential immune cells of the brain parenchyma which are responsible for maintaining homeostasis and responding to brain injury. When activated, they induce immune signaling and become phagocytic. In the context of neurodegenerative diseases, they exhibit both pro- and anti-inflammatory properties. Human endogenous retroviruses (HERVs) are the remnants of ancient retroviral germline infections that integrated into the human genome, and their expression has been directly linked to microglial-mediated neurodegenerative diseases. Despite this, HERV expression is not consistently included in analyses characterizing microglia. Investigating the function of microglia across different diseases is critical for elucidating mechanisms of disease progression and for the development of effective therapeutic interventions. Approaches to studying microglia include isolation from post-mortem brain tissues, differentiation from peripheral blood monocytes or differentiation of induced pluripotent stem cells (iPSC). Ex vivo brain derived microglia (BdMG) are considered most physiologically relevant, but constraints including the unpredictable timing of availability, labor-intensive protocols, and low yields reduce their practicality. Existing alternatives are iPSC-derived microglia (iMG) and monocyte-derived microglia (MDMi) models that circumvent sample accessibility and scalability issues. Here, we explored the transcriptome and proteome of these three microglia sources and identified a common microglia surface marker expression profile, establishing that MDMi and iMG exhibit similar phenotypes relative to BdMG. We also identified a common HERV expression profile across BdMG, iMG and MDMi, indicating deeper similarities across different sources. Altogether, our findings indicate that given the phenotypic similarities of the 2 models, MDMi represent a practical and reliable model for investigating microglial function with iMG models serving for more molecular based approaches, such as CRISPRa/i.

Microglial activation is a central component of neuroinflammatory responses in many brain pathologies. Increasing evidence indicates that microglial phenotype is tightly linked to cellular metabolism, with pro-inflammatory activation associated with enhanced glycolytic flux. Lactate, traditionally considered a metabolic substrate, has recently emerged as a signaling molecule capable of modulating immune responses. However, its direct impact on microglial inflammatory activation remains incompletely understood. In the present study, we investigated the effects of lactate on microglial phenotype under inflammatory conditions using primary rat microglial cultures stimulated with lipopolysaccharide (LPS). Microglial activation was assessed through the expression of phenotypic markers, cytokine production, and secreted chemokine profiles. LPS stimulation induced a strong pro-inflammatory response characterized by increased CD86 expression, elevated TNF-alpha secretion, and enhanced release of several pro-inflammatory chemokines. Post-treatment with sodium L-lactate significantly attenuated these inflammatory responses, reducing pro-inflammatory marker expression and cytokine secretion, while restoring the anti-inflammatory marker CD206. To explore the relevance of these findings in a pathological context, the effects of lactate were further examined in a neonatal rat model of hypoxia-ischemia. Sodium L-lactate administration after injury reduced microglial activation and promoted a shift toward an anti-inflammatory phenotype in cortical regions, whereas hippocampal microglia showed a more limited response. Together, these results demonstrate that lactate directly modulates microglial inflammatory activation and cytokine production in vitro and suggest that lactate-mediated metabolic signaling may contribute in vivo to the regulation of neuroinflammatory responses.

Chronic pain is frequently accompanied by anxiety and other affective disturbances, but the molecular and cellular changes linking peripheral injury to persistent alterations in brain function remain incompletely defined. The hippocampal extracellular matrix (ECM) regulates synaptic plasticity, cellular signaling, and neuroimmune interactions and may therefore contribute to pain-related central plasticity. We used a tibial fracture/cast immobilization model to examine temporal changes in hippocampal ECM, glial morphology, and affective behavior following peripheral injury. Behavioral testing was performed at 3, 7, and 20 weeks after injury, while high-dimensional morphometric analysis of microglia and astrocytes was performed at 1, 3, 7, and 20 weeks. Hippocampal ECM associated molecular signals were mapped at the chronic stage using decellularization coupled with MALDI-TOF mass spectrometry imaging. Injured mice developed mechanical hypersensitivity and reduced exploration in two anxiety related paradigms during the early and chronic stages, with these behavioral phenotypes no longer detected at 20 weeks. At 7 weeks, injury was associated with changes in hippocampal ECM-associated peptide signals, including reduced signals corresponding to four ECM-associated proteins. Microglia and astrocytes exhibited distinct, time-dependent changes in density and multidimensional morphology, with some alterations persisting after behavioral measures returned toward control levels. In vitro, BV2 microglia exhibited rigidity-dependent changes in morphology, phagocytic behavior, and calcium signaling. Together, these findings define a temporally evolving hippocampal ECM and glial phenotype following peripheral injury and identify matrix biomechanics as a candidate regulator of microglial function.

BackgroundTherapeutics that modulate peripheral and central immune responses are emerging as promising approaches for neurodegenerative disease. However, the biological context in which these interventions are deployed may critically influence therapeutic outcomes. An attenuated Brucella melitensis 16M strain and a similar strain engineered to express the metabolite indole have previously demonstrated therapeutic efficacy in preclinical models of cancer and autoimmunity through immune system modulation. Here, we tested whether repeated administration of these bacterial strains could modify pathological outcomes in a mouse model of tauopathy. MethodsAttenuated Brucella strains were administered to transgenic mice overexpressing mutant tau protein on a human APOE4 knock-in background via intraperitoneal injection every 5 weeks starting at 6 months of age. Mice were analyzed at 9.5 months of age for tau pathology, neurodegeneration, and changes in innate and adaptive immune responses. ResultsBrucella strains distributed predominantly in the periphery following repeated intraperitoneal treatment in transgenic mice, with minimal detection in the brain and no detection in the dural meninges. Repeated administration of either bacterial strain was well tolerated, with no evidence of adverse safety events. Surprisingly, repeated treatment with the engineered Brucella strain exacerbated neurodegenerative pathology, resulting in reduced hippocampal volume and increased phosphorylated tau pathology compared with control- and non-engineered Brucella strain -treated animals. Immunohistochemical analyses revealed increased numbers of hippocampal CD4+ T cells following treatment with either Brucella strain relative to PBS controls. However, only treatment with the engineered Brucella increased microglial reactivity within the hippocampus. ConclusionThese findings demonstrate that peripheral administration of an engineered bacterial therapeutic can differentially influence neuroimmune responses and disease progression. These results underscore the importance of disease context when developing bacteria-based immunomodulatory therapies for neurodegenerative disorders.

INTRODUCTION: Alzheimer's disease (AD) disproportionately affects women, but the underlying mechanisms are not fully understood. We compare sex-specific molecular changes across AD with and without dementia at single-cell resolution. METHODS: We performed sex-stratified single-nucleus transcriptomic analysis of human AD to dissect sex-specific differences in dementia progression, using the SEA-AD cohort for discovery and the ROSMAP and NBB datasets, together with 5xFAD mouse models, for validation. Donors were classified as no-dementia controls, no-dementia AD, or dementia AD, followed by donor-level pathway analysis, de novo microglial clustering, communication modeling, and cytokine-response enrichment RESULTS: Females with AD dementia showed enhanced inflammatory pathway activity across glial, neuronal and vascular-associated cell types, with the strongest enrichment in microglia. This female-biased program centered on IL6-JAK-STAT3 signaling and increased STAT3 expression in microglia and astrocytes. These results were validated in the ROSMAP and NBB datasets and conserved in the 5xFAD mouse model. Microglial clustering resolved 18 states and revealed sex-divergent disease remodeling, including female-specific expansion of redox-reactive and CD163+ macrophage-primed states. Cell:cell communication analysis identified female-biased TGF{beta}/GRN signaling, male-biased CD99 signaling, and dementia-associated SPP1-integrin communication with the vasculature. Microglial cytokine-response analysis identified IL-2 response as a dementia-associated signature in both sexes, alongside a female-biased IL-17/interferon response and a male-biased IL-7 response. DISCUSSION: These findings identify a disease-emergent, female-biased STAT3-centered neuroinflammatory program in human and mouse AD, linking sex-specific microglial remodeling to cytokine responsiveness and glial-vascular communication. The results suggest that sex-specific changes in neuroinflammation pathways in microglia and other cell types are associated with progression to dementia.

The skull bone marrow axis has recently emerged as an important source of immune cells during neuroinflammation, including in the experimental autoimmune encephalomyelitis (EAE) , the mouse model of multiple sclerosis. The cribriform plate bone marrow (cpBM), is a portion of the ethmoid bone of the skull of mice, and is positioned next to nasal regions. At the cribriform plate olfactory nerves pass through the skull and recent evidence supports that it represents an important immune niche with links to CSF outflow and connections to cpBM. Previous data supports cellular composition of the ethmoid bone marrow in mice is altered in neuroinflammation suggesting it senses inflammatory cues, but the exact nature of these changes is still unclear. Here using scRNAseq and flow cytometry of the ethmoid bone marrow region we test how the region responds to EAE. In addition we also compare the cpBM region with systemic bone marrow in the femur to understand region specific differences. We report that neuroinflammation creates a regulatory response at these sites and implicates the cpBM in controlling these effects. A better understanding of the cribriform niche may offer new treatments for neuroinflammatory disease

Maternal systemic infection during pregnancy is associated with placental dysfunction even when pathogens do not directly invade the placenta, but the cellular pathways linking antecedent infection to persistent placental alterations remain poorly defined. Here we used spatial transcriptomics to resolve trophoblast-, fetal endothelial, and Hofbauer cell-enriched niches in placentas collected at term after clinical recovery from antepartum pyelonephritis. We profiled 168 spatially defined areas from 14 placentas and integrated transcriptional findings with blinded histopathology and quantitative extracellular matrix analysis. Pyelonephritis was associated with strikingly compartment-specific transcriptional responses. Hofbauer cell-enriched niches showed the most extensive alterations, with coordinated enrichment of extracellular matrix remodeling and vascular developmental programs, including increased ELN expression (log2FC = 0.84, FDR = 0.033) and increased expression of genes governing collagen deposition, elastic-fiber assembly, matrix maturation and turnover. Quantitative analysis of 1,790 fetal vessels independently identified altered perivascular matrix composition, with Hofbauer cell-enriched ELN expression correlating with greater perivascular elastin content ({rho} = 0.67, P = 0.008). Fetal endothelial-enriched regions exhibited complementary alterations in vascular remodeling and endothelial identity, including increased PLAC8 (log2FC = 1.36, FDR = 0.047), whereas trophoblast-enriched regions showed little detectable transcriptional response. Histopathology further demonstrated increased lesions associated with maternal vascular malperfusion and perivillous fibrin deposition. Together, these findings identify the villous immune-stromal-vascular microenvironment as a persistent site of placental remodeling following maternal infection and suggest that clinical recovery from maternal illness may not coincide with normalization of placental biology.

Background: Glioblastoma (GBM) adapts to therapy through coordinated malignant-cell and microenvironmental responses, but the mechanisms linking treatment-associated inflammation to tumor-cell phenotypic plasticity remain poorly defined. Here, we investigated whether preoperative temozolomide exposure is associated with inflammatory-repair remodeling and a ROCK-linked therapeutic vulnerability in recurrent GBM using a window-of-opportunity clinical trial cohort. Methods: We integrated patient-resolved single-cell transcriptomics, spatial RNA profiling and multiplex protein imaging with experimental perturbations and orthotopic glioblastoma models. In the presurgical window-of-opportunity subgroup of NCT05236036, four patients with recurrent glioblastoma received additional preoperative temozolomide (TMZ; 150 mg/m2/day for 5 days) before resection and were compared with three recurrent comparators who did not receive additional preoperative TMZ. Results: Across seven tumors, malignant-cell inflammatory activity covaried with integrin-binding and wound-healing programs (median partial {rho} = 0.45 and 0.42, respectively). Tumors exposed to preoperative TMZ showed enrichment of inflammatory, chemokine, adhesion and wound-healing programs (GSEA q < 0.05), with the direction of enrichment preserved in all leave-one-patient-out analyses. In two tumors exposed to preoperative TMZ, CXCL12 localized to vascular territories, whereas chemokine and wound-repair programs increased near injury-reference regions. Across seven specimens analyzed by multiplex protein imaging, MYL9 abundance correlated with local CXCL12 ({rho} = 0.61), phosphorylated MYPT1 ({rho} = 0.48) and nuclear phosphorylated STAT3 ({rho} = 0.45), with positive associations in every specimen. Experimentally, TMZ increased CXCL8 and reactive-state markers and enhanced subsequent scratch closure, while CXCL8 and CXCL12 increased MLC2 phosphorylation. Fasudil attenuated TNF-NF-{kappa}B, inflammatory-response and IFN-{gamma}-response programs in TMZ-treated cells and reduced scratch closure and p-MLC2 in complementary assays. Across three orthotopic models, fasudil plus TMZ prolonged survival versus TMZ alone (model-stratified HR, 0.21; 95% CI, 0.07-0.61; P = 0.001). Conclusions: These findings link treatment-associated inflammatory-repair programs with cytoskeletal remodeling and support further translational evaluation of fasudil plus TMZ in glioblastoma.

Traumatic brain injury (TBI) is one of the world's leading causes of death and disability and a major risk factor for dementias. The primary dementia associated with TBI is chronic traumatic encephalopathy (CTE), a neurodegenerative disease classified as a tauopathy, in which toxic tau molecules lead to disease pathologies and degeneration. The processes that lead to tauopathy and subsequent dementia after TBI remain unclear. Here, we built upon the finding that seizures after TBI may be a mechanism leading to tauopathy, by dissecting the functions of the metabotropic glutamate receptor 5 - cellular prion protein (mGluR5-PrPC) pathway. We delivered TBI to larval in a blast paradigm, and quantified aggregation of Tau via a genetically-encoded Tau-GFP fusion reporter. Zebrafish larvae lacking prp2 (homolog of mammalian cellular Prion Protein, PrPC) displayed a 168% increase in post-traumatic seizures activity after TBI. An mGluR5 agonist (CHPG) reduced post-traumatic seizures, whereas an mGluR5 antagonist (MPEP) increased post-traumatic seizures. Moreover, agonizing mGluR5 reduced tau aggregation and antagonizing mGluR5 increased tau burden. Larvae seizing from convulsants, rather than TBI, were treated with CHPG/MPEP and provided a similar pattern of outcomes, suggesting seizures may be a factor needed for mGluR5 activity to influence tau aggregation. The PrPC-mGluR5 pathway is proposed as one candidate pathomechanism linking TBI to subsequent seizures and tauopathy, and thus it warrants investigation as a target for prophylactic interventions.

Neuroinflammation is a hallmark of several chronic neurodegenerative disorders, including Parkinson's disease (PD) and other synucleinopathies, and is closely associated with microglial activation. The inflammasome adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) undergoes oligomerization upon inflammasome activation, resulting in the formation of ASC specks. These structures are recognized by microglia as damage-associated molecular patterns (DAMPs), thereby promoting inflammatory signaling. Recent studies have demonstrated that extracellular ASC specks associate with amyloid-{beta} (A{beta}), and that uptake of ASC-A{beta} composites by microglia amplifies pro-inflammatory responses. Although ASC specks have also been shown to potentiate -synuclein (Syn) preformed fibril-induced pathology, their interaction with monomeric Syn and their effects on inflammasome activation remain poorly understood. Here, we co-stimulated murine SIM-A9 microglia with ASC-Syn assemblies and evaluated their effects on NLRP3 inflammasome activation. Electron microscopy revealed a close association between ASC fibrils and Syn monomers. Exposure of microglia to ASC-Syn enhanced NLRP3 inflammasome activation, as evidenced by increased ASC speck formation, caspase-1 activation, and secretion of IL-1{beta} and IL-18, when compared to ASC or Syn alone. In addition, immunohistochemical analyses of post mortem brain tissue from patients with dementia with Lewy bodies demonstrated ASC expression in regions exhibiting Syn pathology and showed ASC co-localization with Syn within Lewy bodies. Collectively, these findings identify ASC-Syn assemblies as potent amplifiers of NLRP3 inflammasome signaling and suggest a mechanism through which they may contribute to sustained neuroinflammation in synucleinopathies.

Microglia, the tissue resident immune cells of the central nervous system, are critical regulators of postnatal neural circuit refinement. Innate immune receptors on microglia such as toll-like receptor 4 (TLR4) are best characterized in the context of inflammation, however, endogenous TLR4 ligands are generated during typical developmental processes. Here, we investigated whether TLR4 signaling in CX3CR1-expressing myeloid cells contributes to microglial morphology, synaptic engulfment, and behavioral development in mice under basal conditions. TLR4 conditional knockout altered maternal separation-induced ultrasonic vocalizations without impacting social preference or anxiety-like behaviors. Conditional TLR4 knockout in CX3CR1-expressing myeloid cells markedly increased microglial ramification and cell volume within the paraventricular nucleus (PVN) of the hypothalamus. TLR4 conditional knockout also reduced microglial engulfment of vGlut2-positive presynaptic material, while overall excitatory synapse numbers remained unchanged. Together, these findings demonstrate that TLR4 signaling in CX3CR1-expressing myeloid cells contributes to microglial morphology, presynaptic engulfment, and neonatal communicative behavior under basal conditions.

Early parental experiences can be translated into biological signals that are transmitted to offspring. These signals influence developmental trajectories to both increase offspring vulnerability following adverse experiences or promote resilience when environments are supported and enriched. Among the potential routes of parental-offspring communication, milk-derived extracellular vesicles (EVs) transferred through nursing are particularly interesting because they carry bioactive molecular cargos capable of influencing recipient cells. Previously, we found that milk-derived EV cargo is remodeled by both adverse and positive experiences and that environmental enrichment (EE) can rescue adversity-associated effects, including EV-miRNA cargo changes. Environmental programming of EVs during lactation may therefore have functional consequences on offspring development and adaptability. Here, we tested whether milk-derived EVs from our rat EE model are sufficient to rescue offspring outcomes following maternal immune activation (MIA). Milk-derived EVs isolated from donor EE dams were administered directly to MIA-exposed pups, mitigating MIA-associated reductions in social behavior. EE-EV administration also promoted social behavior in non-MIA exposed male offspring. To investigate microglia as a cellular target, EE-EVs were administered to LPS-activated BV2 microglial cells. EE-EV exposure attenuated LPS-associated transcriptional alterations across inflammatory pathways, including cytokine, chemokine, and NF-B signaling, while modulating genes associated with cellular homeostasis and metabolic responses. Together, these results 1) support milk-derived EVs as a biologically active interface through which environmental experience may be communicated to offspring, and 2) identify microglial transcriptional responses as a potential cellular pathway underlying these effects.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a crucial neuroinflammatory mediator in ischemic stroke and alcohol-induced memory impairment. We have recently discovered that amyloid{beta} causes microglia to release eCIRP, prompting us to investigate its role in Alzheimer's disease (AD). We found that eCIRP levels were significantly elevated in the cerebrospinal fluid (CSF) and plasma of AD patients compared with age-matched non-AD subjects, as well as in hTau.P301S mice (a model of AD tauopathy) compared with wildtype control mice. Plasma eCIRP strongly correlated with astrocyte activation marker glial fibrillary acidic protein (GFAP) in AD patients. eCIRP induced neurotoxic astrocyte-specific genes and astrocyte release of proinflammatory and neurotoxic factors in C8-D1a cells, primary murine astrocytes and intracerebroventricular eCIRP-injected C57BL/6 mice brains. In particular, eCIRP increased Complement 3, an astrocytic marker involved in neuroinflammation-associated neurodegeneration. Primary astrocytes from TREM-1 knockout mice were resistant to eCIRP induction of neurotoxic astrocytes. eCIRP increased TREM-1 expression and activation in astrocytes. Blocking CIRP/TREM-1 interaction using peptide M3, effectively attenuated eCIRP's induction of neurotoxic astrocytes. Thus, eCIRP strongly correlates with astrocyte reactivity in AD patients, and eCIRP induces neurotoxic astrocytes via TREM-1, which is attenuated by M3, suggesting a novel therapeutic opportunity to target neurotoxic astrocytes in AD.

Abstract Background: Chronic subdural hematoma (cSDH) remains a significant and increasingly prevalent cause of morbidity in older adults. Disease progression is sustained by de novo formed highly vascularized pseudomembranes and a persistent cycle of fluid accumulation and hematoma expansion. Although inflammatory and angiogenic pathways have been implicated in cSDH pathogenesis, the endothelial mechanisms underlying persistent vascular leak remain poorly understood. We investigated the cSDH vascular microenvironment in human patients and examined the role of vascular endothelial cadherin (VECAD) remodeling in cSDH endothelial dysfunction. Methods: Human cSDH membranes and fluid were evaluated by multiplex profiling, histology, immunofluorescence, immunoprecipitation, western blotting and exploratory bulk RNA sequencing. A human brain endothelial cell (HBEC) line was exposed to patient-derived cSDH fluid to assess VECAD remodeling, endothelial barrier permeability, and proliferation. Results: Multiplex profiling of patient-derived cSDH fluid detected mediators associated with endothelial cell (EC) activation, extracellular matrix remodeling, inflammation, and angiogenesis. In patient pseudomembranes, discontinuous VECAD coincided with erythrocyte extravasation. Two hyperdense (high-risk) specimens showed more extensive disruption than the hypodense (low-risk) specimen while vessel counts appeared similar. Bulk RNA sequencing identified an angiogenic environment and enrichment in cadherin regulation. A soluble VECAD (sVECAD) isoform was detected in cSDH fluid. Exposure of HBECs to patient-derived cSDH fluid increased EC proliferation, cytoplasmic VECAD (cVECAD) accumulation, and EC barrier permeability. Concurrent dexamethasone or minocycline treatment attenuated VECAD cleavage. Conclusions: VECAD disruption is a prominent feature of cSDH and is associated with EC barrier dysfunction. These findings support further evaluation of soluble VECAD as a candidate biomarker and of junction-preserving pathways as potential therapeutic targets.

Cholinergic neurons in the dorsal motor nucleus of the vagus (DMN) in the brainstem are a key source of efferent vagus nerve fibers that regulate vital functions, including heart rate and inflammation. Whether the integrity of DMN cholinergic neurons is affected during Alzheimer's disease (AD) remains unknown. Here, in female and male mice with experimental AD (5xFAD), which exhibit age-dependent memory impairment, basal forebrain cholinergic neurodegeneration, and microglial alterations, we observe a reduction in cholinergic neuron density in the DMN at 6 and 10 months of age. Furthermore, while an important physiological function of DMN cholinergic signaling, such as suppression of heart rate, is preserved in control mice upon electrical DMN stimulation, the extent of suppression diminishes with age in both female and male 5xFAD mice. In addition, while electrical DMN stimulation lowers pro-inflammatory cytokine levels in control mice subjected to endotoxemia, this anti-inflammatory effect is diminished with age in 5xFAD mice, with females showing earlier dysfunction at 6 months. These results reveal previously unrecognized age-dependent cholinergic deficits in the DMN and disrupted brain - to - periphery vagus nerve circuits in experimental AD. These findings advance our understanding of AD mechanisms and are of interest for the development of conceptually novel therapies.

Axonal degeneration is a principal driver of irreversible neurological disability in multiple sclerosis (MS), yet current treatments fail to target this neurodegenerative phase directly. Sterile alpha and TIR domain-containing protein 1 (SARM1) has emerged as an executioner of programmed axon destruction and is overexpressed in grey and white matter MS tissue, making its inhibition a promising therapeutic strategy. However, the effects of SARM1 inhibition on localized neurodegeneration versus systemic and central inflammation remain poorly understood in complex autoimmune environments like MS. Here, we investigated SARM1 pathology using global SARM1-/- knockout (SARM1-/-) mice, AAV-mediated CRISPR knockdown and overexpression of SARM1 in retinal ganglion cells (RGCs), and the small-molecule SARM1 inhibitor 5-iodoisoquinoline (5IIQ), across optic nerve crush (ONC) and experimental autoimmune encephalomyelitis (EAE) models. While global SARM1-/- did not alter the overall clinical course of EAE, it revealed a complex phenotype characterized by an altered peripheral inflammatory cytokine profile and persistent CNS immune infiltration, alongside preserved axonal and myelin integrity. RGC-restricted SARM1 knockdown partially preserved RGCs and axons during EAE, whereas SARM1 overexpression worsened both retinal function and axonal injury. Pharmacological inhibition with 5IIQ preserved axonal integrity and restored visual function in both the ONC and EAE models, as confirmed by electrophysiology, and reduced serum neurofilament light chain (NfL) in EAE, without affecting demyelination. Together, these findings demonstrate that SARM1 inhibition uncouples axonal self-destruction from demyelination and gross neuroinflammation while providing robust structural and functional neuroprotection. This study establishes SARM1 as a viable target for neuroprotective co-therapies designed to complement existing immunomodulatory and remyelinating regimens in MS and related neurodegenerative disorders.

Midbrain organoids (MOs) can be powerful tools to study brain diseases, yet one shortcoming is the lack of microglia due to their mesodermal developmental origin. Previous studies have incorporated iPSC-derived microglia (iMG), but their immature phenotype restricts the applicability to model age-related neuroinflammatory processes relevant to diseases such as Parkinson's Disease (PD). To address this, we have incorporated MOs with human postmortem PD microglia, iPSC-derived microglial progenitors (iMP), and iMG. Postmortem microglia had a higher infiltration capacity resulting in more microglia per organoid, while iMP had a higher migration capacity reaching the core of the organoid. Compared to iPSC-derived cells, more infiltrated postmortem PD microglia were HLA Class II positive and CD68 positive. This novel approach of integrating postmortem microglia into organoids opens new avenues for modelling age-related neuroinflammatory diseases.Ongoing methodological advancements like our study will offer the field a valuable tool for studying human microglia biology in both health and disease.

Neuropathic pain involves spinal microglial activation and metabolic changes.Forsythoside B (FB) has anti-inflammatory effects, but its role in neuropathic pain and microglial glycolysis remains unclear. We investigated the effects and mechanisms of FB in mice with chronic constriction injury (CCI) and in LPS-stimulated BV2 microglia.FB reduced mechanical allodynia and thermal hyperalgesia in CCI mice. FB also reduced the spinal levels of HIF-1, HK2, PKM2, LDHA, and CD86. It decreased the proportion of IBA1+/CD86+ cells and the levels of IL-1{beta}, IL-6, and TNF-. In LPS20 stimulated BV2 microglia, FB reduced glycolysis-related proteins, proinflammatory markers, and cytokine release. FB also reduced glycolytic activity and partly restored basal mitochondrial respiration. FB did not change Hif1a mRNA levels. However, it Manuscript Click here to access/download;Manuscript;Manuscript.docx increased HIF-1 ubiquitination and promoted its proteasome-dependent degradation. HIF-1 overexpression partly reversed the effects of FB on glycolytic enzymes, CD86 expression, and proinflammatory cytokine production. Immunoprecipitation followed by mass spectrometry and UbiBrowser analysis identified USP7 as a candidate HIF27 1-associated deubiquitinase. Molecular docking and cellular thermal shift assays supported a possible interaction between FB and USP7. FB reduced the association between USP7 and HIF-1. USP7 knockdown produced effects similar to those of FB on HIF-1 ubiquitination and downstream glycolytic and inflammatory responses.Overall, FB alleviated neuropathic pain and reduced proinflammatory microglial responses. These effects may involve the promotion of HIF-1 degradation through modulation of the USP7-HIF-1 regulatory axis.

Multimodal data indicates that microglia contribute to the pathophysiology of anorexia nervosa (AN). Here, we investigated microglial modulation of hypothalamic circuits, key regulators of energy balance, hypothesising an implication in the prolonged starvation and low body weight of AN. First, we generated microglia as well as hypothalamic and cortical neurons from patient-derived induced pluripotent stem cells (iPSCs) to discover upregulation of synapse-related genes in hypothalamic neurons, reduced microglial uptake of hypothalamic synaptic structures, and a microglial unresponsivness to the satiety hormone glucagon-like peptide-1. In an AN animal model (anx/anx mouse), spatial transcriptomics indicated hypothalamic microglial activation and disrupted microglia-synapse signaling. Despite an increased microglia density in both the arcuate nucleus (ARC) and the dorsomedial hypothalamus region (DMH), microglia displayed a decreased per cell uptake of synaptic material in Arc. Together, these data suggest that microglial responses shape hypothalamic circuits with possible implications for the maintained negative energy balance of AN.

Microglial activation in response to aggregated -synuclein (-syn) accumulation is a pathological hallmark of Parkinson disease (PD). A distinct activated microglial state, disease-associated microglia (DAM), has been identified across multiple neurodegenerative disorders, including PD. However, the transcriptional and epigenetic programs governing DAM differentiation remain unclear. Here, we show that neuronal -syn overexpression drives progressive microglial state transitions toward DAM, accompanied by increased phagocytic activity and enhanced cytokine and chemokine production. Integrative transcriptomic and chromatin accessibility analyses reveal a hierarchical NF-{kappa}B--AP-1--IRF regulatory program underlying these state transitions. Microglial NF-{kappa}B p65 (RelA) or IRF4 deficiency impairs microglial progression toward DAM, with RelA and IRF4 governing early microglial activation and subsequent DAM differentiation, respectively. Enrichment of NF-{kappa}B, AP-1, and IRF motifs in DAM in human PD supports conservation of this regulatory program across species. Together, our findings establish RelA and IRF4 as sequential, stage-specific regulators that coordinate -syn-induced DAM differentiation in PD.