Microglia are increasingly recognized as key regulators of neural circuit development and putative contributors to the pathophysiology of neuropsychiatric disorders such as schizophrenia (SCZ). However, the functional impact of SCZ-associated genes in microglia remains largely unexplored. Here, we performed an arrayed CRISPR targeting screen of 30 SCZ-associated genes predicted to be differentially expressed in human microglia-like cells. Target genes were prioritized based on post-mortem transcriptomic relevance and predicted ontology-based roles in phagocytosis pathways. We quantified phagocytic activity and morphological changes following gene targeting using high-content confocal imaging. Key targets, including CYFIP1, MSR1, TREM2, SYK, ITGB2, ITGAM, and IRF8, modulated phagocytosis and altered morphological properties consistent with activation states, validating their functional roles in microglia. To elucidate transcriptional impact, we further applied a multiplexed RNA sequencing platform across gene targets. These analyses revealed gene-specific transcriptional signatures, implicating divergent pathways related to phagocytic, activation, cytoskeletal, and lysosomal function. Together, these findings demonstrate the utility of CRISPR-based functional genomics in characterizing microglia function and identifying new target genes and mechanisms that may underlie their contributions to SCZ pathophysiology.

Microglia engulf dying neurons through efferocytosis, a critical function in both development and disease. How microglia process the engulfed neuronal material-especially lipids-remains poorly understood, despite its central role in neurodegeneration. Thus, we developed HuZIBRA, a scalable in vivo xenotransplantation model in which human iPSC-derived microglia-like cells (iMGLs) are introduced into the developing zebrafish brain (zf-hiMG), a system characterized by high levels of neuronal cell death and amenable to precise genetic and pharmacological manipulation. We show that human microglia-like cells recognize and engulf apoptotic zebrafish neurons, indicating conserved efferocytic mechanisms. In these cells, engulfed neuronal material accumulates into a distinct, lipid-rich intracellular compartment, the gastrosome, which we also observed in iMGLs placed in a human brain-like environment. The size of the human gastrosome dynamically reflects neuronal cell death levels and is regulated by key genes, including TREM2 and SLC37A2. Pharmacological inhibition of the cholesterol transporter NPC1 induces gastrosome expansion and lipid accumulation, recapitulating pathological features of Niemann-Pick disease type C. Thus, HuZIBRA provides a powerful in vivo platform to uncover cell-autonomous adaptive responses of human microglia to apoptotic and metabolic stress, with the gastrosome emerging as a key integrator of neuronal debris processing and disease-relevant lipid metabolism.

Somatic cancer variants enriched in Alzheimer's disease microglia-like cells drive inflammatory and proliferative states.

Dissecting microglial contributions to neurodegenerative disease pathophysiology using human pluripotent stem cells.

Microglia are the primary immune cells of the central nervous system and maintain tissue homeostasis through phagocytosis and regulation of inflammatory signalling. Although these functions are well established, the molecular mechanisms that control microglial activation during neurodegeneration remain poorly understood. We focused on the Purkinje Cell Degeneration (PCD) mouse, which carries a loss-of-function mutation in Ccp1 that disrupts tubulin post-translational modifications essential for cytoskeletal stability. Because cytoskeletal dynamics are fundamental for microglial motility, phagocytosis, and proliferation, the Ccp1 mutation offers a model to directly examine how intrinsic cytoskeletal defects alter microglial behaviour and how these alterations manifest within regions undergoing distinct patterns of neurodegeneration. To this end, we combined in vitro and in vivo approaches. Microglia were isolated from neonatal cortex and adult cerebellum and olfactory bulb, and microglia-like cells were generated from bone marrow-derived haematopoietic stem cells. In vivo microglial depletion was achieved with the CSF1R inhibitor PLX5622. Immunohistochemistry quantified microglial density, morphology, and marker expression; transcriptomic profiling assessed identity and functional pathways; and functional assays evaluated phagocytosis, motility, and proliferation. Motor behaviour tests were performed to determine whether microglial dysfunction contributes to circuit-level impairments. Statistical analyses used parametric or non-parametric tests according to distribution. Ccp1-deficient microglia exhibited intrinsic deficits in phagocytosis, motility, and proliferation, independent of overt neuronal loss. These impairments were amplified in degenerating regions, where microglia adopted a predominantly anti-inflammatory rather than pro-inflammatory activation profile. This atypical state suggests a maladaptive response that may compromise tissue homeostasis and intensify disease progression. Consistent with this, animals showed altered motor behaviour, indicating functional consequences of microglial dysfunction. Together, these findings identify Ccp1 as a key regulator of microglial homeostasis and demonstrate how cytoskeletal disruption can reshape microglial responses in neurodegenerative environments, providing mechanistic insight and potential therapeutic targets.

Lnc-USP28-6/ZBTB16 axis orchestrates NLRP3 inflammasome activation and α-synuclein SUMOylation to drive Parkinson's disease pathogenesis.

Microglia maintain brain homeostasis via iC3b-mediated synaptic pruning. The Arp2/3 complex has been implicated in iC3b-mediated macrophage phagocytosis, but it is unclear whether it is similarly required in microglia in the CNS. We examined the question of CR3-dependent clearance of iC3b in microglia using a combination of in vitro and in situ physical confinement studies. Arp2/3 inhibition decreased iC3b phagocytosis and cell motility in vitro. Furthermore, microglia-like cells remove immobilized iC3b from the substrate in an Arp2/3-dependent fashion, in a process reminiscent of trogocytic synaptic pruning. We also used a novel approach to immobilize an iC3b gradient onto a substrate and demonstrate Arp2/3-dependent haptotactic migration toward increasing iC3b concentrations. While Arp2/3-deficient microglia robustly respond to ATP via chemotaxis within mouse hippocampal slices, they demonstrate a persistent inability to stably interact with iC3b-coated beads. The present study establishes new approaches to systematically interrogate molecular pathways relevant to synaptic pruning, advances the understanding of iC3b phagocytosis as a haptotactic response, and confirms that the Arp2/3-dependent haptotactic response is important for microglia function in the CNS microenvironment.

SummaryAlzheimer’s disease (AD) arises from pathological interactions among diverse brain cell types, but cell-specific proteomic changes remain underexplored. Here, we present deep proteomic profiling of sorted or proximity-labeled brain cells from AD mouse models (5xFAD and AppNL-G-F) at multiple ages, quantifying 13,411 proteins in microglia (three subtypes), astrocytes, oligodendrocyte precursor cells, and neurons. We identified 3,028 differentially abundant proteins across these cell types, the majority of which were not detected in bulk proteomic datasets, and constructed cell type-specific networks to define functional modules and hub proteins. Comparison with transcriptomic data revealed that ~30% of proteomic changes are RNA-independent. Further analyses uncovered cross-cell type signaling proteins conserved in human AD brains, such as pleiotrophin (Ptn), which is transcriptionally enriched in astrocytes but accumulates in microglia. Importantly, recombinant PTN directly activates induced microglia-like (iMG) human cells. Thus, these findings provide a comprehensive cell type-resolved proteomic atlas of AD models, highlighting novel intra- and intercellular signaling events.

Cerebral ischemia-reperfusion (I/R) injury is the main cause of early complications and adverse outcomes after treatment such as myocardial infarction and acute ischemic stroke. In this study, we aimed to explore the functions of insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) and tripartite motif-containing 45 (TRIM45) in neuron injury after cerebral I/R injury. HMC3 cells were exposed to oxygen-glucose deprivation and reoxygenation (OGD/R) to mimic cerebral I/R injury in vitro. Western blot and qRT-PCR were conducted for gene expression. NLR family pyrin domain containing 3 (NLRP3) inflammasome activity was analyzed by western blot. ELISA kits were utilized to determine the concentrations of inflammatory cytokines. Flow cytometry was used to analyze iNOS+ cells, CD206+ cells and neuron apoptosis. Methylated RNA Immunoprecipitation (meRIP) assay and RIP assay were adopted to analyze the relation between TRIM45 and IGF2BP1. CCK-8 assay and TUNEL assay were adopted for the viability and death of neurons. Mice model of middle cerebral artery occlusion (MCAO) was used to explore the function of IGF2BP2 in cerebral I/R injury. IGF2BP1 level was upregulated in HMC3 cells. IGF2BP1 overexpression promoted NLRP3 inflammasome activation and pro-inflammatory phenotype in OGD/R-stimulated HMC3 cells. Mechanically, IGF2BP1 modulated TRIM45 expression through m6A methylation modification. IGF2BP1 knockdown inhibited NLRP3 inflammasome activation and pro-inflammatory phenotype in OGD/R-stimulated HMC3 cells by m6A methylation modification of TRIM45. Inhibition of IGF2BP1 improved the viability and suppressed the death and apoptosis of neurons in the co-culture system of microglia-like and neurons by regulating TRIM45 expression. Inhibition of IGF2BP1 improved the neurotoxicity of proinflammatory HMC3 cells in co-cultured neurons via reducing the m6A methylation of TRIM45. However, the number of biological replicate samples was relatively small (n = 3) and the results in this study were preliminary study.

Microglia are macrophage-like brain resident immune cells known to express numerous Alzheimer's disease risk genes. Here we generated a human induced pluripotent stem cell (iPSC) derived microglia cell culture model for use in neuroimmune modeling and therapeutic testing. We generated iPSC lines using episomal reprogramming for subsequent stepwise differentiation of iPSC-derived microglia (iMG) without commercial kits. We characterized the responses of this model to immunogenic stimuli and recombinant TREM2 antibodies. The iMG expressed several key microglia signature genes and are morphologically and transcriptionally dynamic. iMG rapidly phagocytosed myelin debris and strongly changed expression of lipid homeostasis genes. iMG expressed TREM2 and increased TREM2 levels in response to IL-4. Recombinant TREM2 antibody treatment impaired iMG myelin phagocytosis and upregulated chemokines. We validated our iMG model system for the evaluation of biological responses of human microglia-like cells to stimuli and pharmacological agents for their transcriptional and functional impacts.

Exposure to lead is associated with microglial dysfunction and the development of neuroinflammation. This contributes to accelerated neurodegeneration. Even low doses of this element modulate inflammatory responses and might contribute to central nervous system dysfunction. Extracts from the mushroom Hericium erinaceus (HE) possess well-documented neurotropic properties; however, its potential neuroprotective mechanisms under conditions of environmental neurotoxicity remain poorly defined. In this study, we investigated the effects of HE on inflammatory signaling in a microglia-oriented in vitro model using THP-1-derived macrophages exposed to low levels of lead (3.5 µg/dL). In our study, Pb exposure did not increase tumor necrosis factor (TNF) alpha levels but reduced monocyte chemoattractant protein-1 (MCP-1) secretion and altered cyclooxygenase (COX) expression, indicating immune response modulation rather than inflammatory activation. Under combined Pb and HE exposure, a marked shift in cyclooxygenase expression toward COX-2 at both the gene and protein levels was observed, accompanied by increased PGE2 production; these effects were dose-dependent. The inflammatory signaling was modulated rather than amplified. Also, TNF alpha levels were elevated after combined treatment, whereas gene expression responses were dose-dependent. MCP-1 secretion was fine-tuned toward control values, consistent with macrophage morphological changes, while IL-6 levels were increased. Overall, these findings indicate that Hericium erinaceus exerts immunomodulatory effects in microglia-like cells under low-level lead exposure, supporting its neuroprotective potential through modulation of neuroinflammatory signaling.

Microglia, the brain's resident macrophages, can be reconstituted by surrogate cells - a process termed 'microglia replacement'. To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this system's power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutières syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo - phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.

Microglia, the brain’s resident macrophages, can be reconstituted by surrogate cells – a process termed ‘microglia replacement’. To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this system’s power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi–Goutières syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo – phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.

Dysfunctional microglia contribute to the pathology of numerous neurological diseases. Depletion of harmful microglia and repopulation with healthy progenitors represents a new therapeutic option for neurodegenerative diseases with an urgent need for effective treatments. However, repopulation with patient-derived progenitors could result in similar dysfunction over time. We therefore propose obtaining microglia-like cells (MLCs) derived from healthy donors for allogeneic transplantation. We hypothesize that the immunosuppressive phenotype of MLCs, combined with the brain´s high immune tolerance, would enable effective engraftment. Additionally, the allogeneic origin of MLCs may increase immune tolerance, with additional therapeutic outcomes, particularly in multiple sclerosis (MS). MLCs were generated from MHC-mismatched mouse strains in vitro and exposed to IL-10/IL-4/TGF-β or LPS/IFN-γ to induce specific immunophenotypes. Phagocytosis and T cell proliferation assays assessed MLC responses to pathogenic insults that could arise in autoimmune contexts. Microglial depletion was achieved using Cx3cr1CreER/−R26DTA/− mice or PLX3397 treatment. A protocol administering MLCs directly into the brain via the intracisterna magna was optimized to facilitate repopulation of the microglial niche. Repopulation with MHC-mismatched IL-10/IL-4/TGF-β-polarized MLCs was tested in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, with immune profiling of cellular populations conducted using flow cytometry. BALB/c- and C3H-derived MLCs developed more pronounced anti-inflammatory profiles than did C57BL/6 MLCs, and promoted tolerogenic phenotypes when encountering MHC-mismatched C57BL/6 T cells. In vivo experiments demonstrated a partial repopulation of an emptied microglia-niche by pre-differentiated MLCs administered intracisternally (i.c) into the CNS. The adoptive transfer of MHC-mismatched MLCs led to enhanced immune tolerance mechanisms and the amelioration of disease progression in the EAE mouse model. Our findings support the therapeutic potential of anti-inflammatory MHC-mismatched MLCs in promoting immune tolerance within autoimmune neuropathologies. Specifically, disease progression was attenuated and tolerogenic mechanisms were activated in the MS mouse model. While a polarization protocol towards an anti-inflammatory phenotype confers MLCs with beneficial features in a pro-inflammatory disease context, the MHC-mismatch interaction within the host´s CNS promotes additional tolerogenic processes.

Autologous hematopoietic stem/progenitor cell (HSC)-gene therapy (GT) represents a promising therapeutic option for progranulin (PGRN)-related neurodegenerative diseases due to mutations in the PGRN encoding gene (GRN), such as frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis 11 (CLN11). These conditions are characterized by a deficiency in PGRN, have no cure, and represent an unmet medical need. We report on the efficacy and feasibility of an HSC GT approach that used a lentiviral vector encoding the human GRN complementary DNA to transduce HSCs that then were transplanted into a Grn-/- mouse model, which mirrors both FTD and CLN11 phenotypes. Two promoters, one with medium-low strength (HLA-DRA gene-based promoter regulated by inflammation) and the other with medium-high strength [ubiquitous phosphoglycerate kinase (PGK) promoter], were compared for HSC transduction. Moreover, intravenous and intracerebroventricular HSC administration were compared. Under all tested conditions, a partial reconstitution of PGRN production by microglia-like cells (MLCs) derived from genetically corrected Grn-/- HSCs was observed, which uniformly led to a correction of lipid accumulation, reduced gliosis, and improved social recognition in Grn-/- mice. Therapeutic effects were similarly achieved with either of the promoters and administration routes and particularly also when the PGRN-expressing cells and their MLC progeny had engrafted exclusively in the central nervous system (CNS) after intracerebroventricular transplantation. These findings suggest that a durable yet modest restoration of PGRN expression in the CNS is sufficient to correct pathology.

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder with prominent neurological manifestations, most notably epilepsy, and is frequently accompanied by a wide range of neuropsychiatric comorbidities. Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway plays a central role in TSC pathology, disrupting both general brain development and specific molecular processes such as metabolism. While much attention has focused on neurons and astrocytes in these TSC-related alterations, the contribution of microglia remains relatively underexplored. In this study, we first analysed the transcriptomic profiles from resected TSC brain tissue and identified evidence of calcium (Ca2+) dysregulation in TSC microglia. In order to investigate the functional consequences, we then examined induced pluripotent stem cell (iPSC) derived microglia-like (iMGL) cells from TSC patients. Our findings reveal that these iMGL cells displayed markedly altered Ca2⁺ signalling, characterized by impaired store-operated calcium entry (SOCE) and an increase in mitochondrial Ca2⁺ uptake. These changes are accompanied by elevated mitochondrial respiratory activity, suggesting a shift in metabolic state. In addition, TSC iMGL cells displayed increased phagocytic activity and an altered inflammatory responsiveness, consistent with a dysregulated microglial activation state. Supporting these functional alterations in iMGL cells, transcriptomic analysis of TSC brain tissue revealed upregulation of several genes associated with lipid metabolism, phagocytosis, and innate immune activation, with partial overlap with stage 2 disease-associated microglia (DAM)-like programs. Together these findings suggest that microglial dysfunction may represent a relevant component of TSC pathophysiology.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00401-026-02986-8.

Atypical antipsychotics alter microglial functions via astrocyte-derived extracellular vesicles.

Nuclear ASC speck formation in microglia is associated with inflammasome priming and is exacerbated in LRRK2-G2019S Parkinson's disease.

SummaryAltered microglial lipid metabolism is heavily implicated in Alzheimer’s disease (AD) and aging. Recently, protocols were developed to generate human induced pluripotent stem cell-derived microglia-like cells (iMGL) to study microglial function in vitro, including embryoid body-based methods and induced transcription factor (iTF)-dependent approaches. Here, we performed comparative lipidomics on iMGL from these methods and report major differences in multiple lipid classes, including triglycerides (TGs), a storage form of fatty acids implicated in microglial reactivity. TGs are strongly increased in iTF microglia due to the absence of a media supplement (B-27). Supplementing iTF microglia with B-27, or its component L-carnitine, reduces TGs and promotes a homeostatic state. B-27 also renders iTF microglia metabolically responsive to immune stimuli. Overall, our data show that iMGL differentiation methods have a major impact on microglial lipidomes and warrant attention when studying AD and neuroinflammatory processes involving lipids.

BackgroundMicroglia are emerging as critical regulators of neurovascular function in health and Alzheimer’s disease (AD), yet their interactions with the human neurovascular unit (NVU), particularly brain endothelial cells, remain incompletely understood. Current in vitro NVU platforms typically exclude microglia and lack perfusable vascular networks with physiologically relevant architecture. Here, we established complementary two-dimensional (2D) and three-dimensional (3D) NVU models to investigate microglia-endothelial and microglia-neurovascular interactions.MethodsHuman induced pluripotent stem cell derived-neurons (iNs), astrocytes (iAs), and microglia-like cells (iMGLs) were incorporated into a soft-lithography based engineered microvessel system to establish a multicellular neuroimmune-vascular model. To specifically evaluate iMGL-endothelial cell (EC) interactions, iMGL were co-cultured with primary human brain microvascular endothelial cells (HBMECs) and junctional protein localization was evaluated using immunofluorescence. The barrier integrity of engineered microvessels containing iMGL was evaluated using dextran permeability. Our 2D and 3D systems were stimulated with tumor necrosis factor-α (TNFα) to evaluate whether iMGL would promote or attenuate EC inflammation and barrier breakdown.ResultsIncorporation of iNs, iAs, and iMGLs into a perfusable vascular model enabled a more complete representation of NVU cellular diversity and promoted neuronal health. In monolayer co-culture with iMGL, HBMECs enhanced the junctional localization of tight and adherens junction proteins through both contact-dependent and paracrine mechanisms. Following an inflammatory challenge, iMGLs reduced endothelial inflammatory activation, suggesting a protective role in response to AD-relevant inflammatory conditions. Finally, when embedded in 3D collagen matrices surrounding perfusable endothelialized lumen networks, iMGLs reduced dextran permeability and preserved endothelial barrier integrity following TNFα challenge.Conclusions:Together, these findings establish a 3D perfusable neuroimmune-vascular model that enables the dissection of microglial contributions to neurovascular function in health and disease.