Biology subjects

Chen, R.

Publications and source records attributed to Chen, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

STING agonist-mediated endothelial cell activation drives NK cells and neutrophils-dependent pulmonary inflammation

Stimulator of interferon genes (STING) agonists and derivative molecules have been extensively developed for tumor immunotherapy. However, systemic exposure toxicity risks have constrained clinical trial progression and even threatened patient lives. Currently, systematic toxicity assessments for STING agonists remain lacking, with the mode of action for major organ injury yet to be elucidated. Here, we focused on STING agonist-induced lung injury, revealing that systemic administration of STING agonists caused pulmonary hemorrhage, inflammatory alterations, and respiratory dysfunction. Through single-cell RNA sequencing and immune deletion studies, we found that lung endothelial cells could be stimulated by STING agonists and then secreted chemokines and IL-15 to recruit and activate NK cells. NK cells could induce endothelial cell apoptosis via IFN-{gamma}. Tbx21+ NK subpopulations, which activated by endothelial cells, could produce chemokines to recruit neutrophils. Neutrophils secreted IL-1{beta} through positive feedback pathways and form neutrophil extracellular traps during lung injury. This study elucidates the critical role of the endothelial cell-NK cell-neutrophil axis in mediating STING agonist-associated pneumonia, offering insights for developing intervention strategies for STING agonist toxicity.

pharmacology and toxicology↗

bioRxiv2026

MERFISH 2.0, an ultra-sensitive single-cell spatial transcriptomics imaging chemistry across diverse tissue types

Spatial transcriptomics has emerged as a transformative approach for elucidating tissue architecture, cellular heterogeneity, and disease mechanisms by preserving the spatial context of gene expression in cells. Despite these advances, many spatial transcriptomic methods underperform in archival or clinically relevant specimens, particularly formalin-fixed, paraffin-embedded (FFPE) tissues, where RNA degradation and crosslinking hinder transcript detection. To address these challenges, we developed Multiplexed Error Robust Fluorescence In Situ Hybridization 2.0 (MERFISH 2.0), an optimized spatial transcriptomic imaging chemistry to enhance profiling of fragmented and highly crosslinked RNA. Across diverse human and mouse tissues preserved as fresh-frozen, fixed-frozen, and FFPE specimens, MERFISH 2.0 substantially increased transcript detection sensitivity by up to [~]8-fold relative to MERFISH 1.0 while preserving quantitative concordance (Pearson r [≥] 0.8 across tissues). In archived fresh-frozen human brain samples, MERFISH 2.0s enhanced sensitivity improved transcript recovery, enhanced cell type resolution and spatial analyses. In low quality archival FFPE human breast cancer specimen, MERFISH 2.0 revealed additional cell populations, novel cell clusters, refined tumor-immune architecture, and increased detection of gene-gene and cell-cell interactions relative to MERFISH 1.0, underscoring the impact of improved sensitivity on downstream spatial analysis. By substantially expanding robust transcript detection to degraded and archival samples, MERFISH 2.0 enables scalable, cohort-level spatial transcriptomic analysis across clinically relevant tissue collections.

genomics↗

bioRxiv2026

Characterization of the Gut Microbiota and Serum Metabolomics in Patients with Type 2 Diabetes Mellitus and Newly Diagnosed Acute Coronary Syndrome

Biomarkers for the early identification of acute coronary syndrome (ACS) risk remain inadequately investigated, particularly in patients with type 2 diabetes mellitus (T2DM), for whom timely clinical intervention may substantially enhance prognostic outcomes. The gut microbiota and serum metabolites may serve as pivotal mediators in the occurrence and progression of ACS among patients with T2DM, and whether these factors can be used for the precise discrimination of patients with T2DM complicated by ACS remains to be explored. Overall, 76 participants were enrolled (38 patients diagnosed with T2DM complicated by ACS and another 38 with T2DM without ACS). 16S rRNA sequencing combined with untargeted LC-MS metabolomics revealed a dysregulated gut-serum axis in patients with T2DM complicated by ACS: enrichment of proinflammatory microorganisms (Enterococcus spp.), reduction of butyrate producers (Butyricimonas spp.) and concomitant dysregulation of circulating lipid metabolites-upregulation of PC(16:0/9:0 (CHO)) and arachidonic acid alongside downregulation of cholesterol sulfate. By integrating multiomics data and applying various feature selection methods, we subsequently identified six key biomarkers. The final constructed combined model robustly distinguished patients with T2DM complicated by ACS from those with T2DM alone (AUC = 0.983), outperforming the other single omics models. Our study revealed that the gut microbiota and related serum metabolites serve as key mediators in the onset and progression of ACS among patients with T2DM, and demonstrated their potential value as noninvasive biomarkers for the early diagnosis of T2DM complicated by ACS. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSWe combined 16S rRNA sequencing with untargeted LC-MS metabolomics to dissect the gut-serum axis in patients with T2DM complicated by newly diagnosed ACS. We identified distinct gut microbial and serum metabolic signatures that distinguish ACS progression within the T2DM population. A multiomics classifier integrating clinical, microbial, and metabolic variables achieved robust diagnostic performance (AUC = 0.983) and outperformed single omics models. What Are the Clinical Implications?Integrated multiomics biomarkers facilitate the early identification of ACS progression in patients with T2DM, offering novel avenues for precision prevention, dynamic clinical surveillance, and individualized therapeutic strategies. Dysregulations of the gut microbiota and serum metabolome may play an important role in the development and progression of ACS in patients with T2DM.

microbiology↗

bioRxiv2026

Substantial Deceleration of Adaptation of HIV-1 Within 1,500 Generations in an Experimental Evolution: A Genomic Perspective

Numerous experimental evolution studies have suggested that adaptation rate of microbial populations evolving in stable environments decline over time. Despite the generality of this phenomenon across different domains of life, the timing and magnitude of decline in adaptation can vary greatly based on the idiosyncrasies of the biological system. To investigate the characteristics of adaptation deceleration in a fast-evolving virus, we propagated HIV-1 in two human T-cell lines (MT-2 and MT-4) for approximately 4.8 years and tracked its genome evolution through next-generation sequencing. The curated sequencing data covering the whole-genome can be accessed and explored via LTEEviz, an interactive web application. Time-resolved sequencing data uncovered that despite constant fixation rate of 0.085 (MT-2) and 0.042 (MT-4) mutations per generation, the fixation kinetics of adaptive mutations changed considerably over time. The rate of fixation of adaptive parallel mutations decreased by 44% per 300 generations, while their conferred fitness gain decreased by 27% (MT-2) and 18% (MT-4) per every added adaptive mutation in their genetic background. The early and substantial deceleration of adaptation in our HIV-1 populations can, at least in part, be explained by diminishing gains of adaptive mutations. Furthermore, we identified genomic patterns consistent with a hard selective sweep that occurred in one population later in the experiment. Together, our results confirm that HIV-1 genomic evolution is characterized by a swift and substantial deceleration of adaptation, while also revealing that episodes of positive selection can occur beyond the initial adaptive phase.

evolutionary biology↗

bioRxiv2026

eScreen: a deep learning framework for functionally decoding the regulatory genome at single-nucleotide resolution

The human genome is densely populated with cis-regulatory elements (CREs), yet deciphering their functional regulatory syntax and combinational logic remains a fundamental challenge. Here, we integrate 379 genome-scale CRISPR screen experiments, encompassing 21 million perturbations across 23 cell types, to construct a compendium of 41,239 high-confidence functional CREs from 530,527 candidates. Leveraging this resource, we develop eScreen, a deep learning model built on the StripedHyena2 architecture to functionally decode the regulatory genome at single-nucleotide resolution. eScreen achieves three primary functions: (1) predicts genome-wide cell-type-specific CRE functional activity with high accuracy, outperforming existing models; (2) provides mechanistic interpretation of regulatory syntax at single-nucleotide resolution; (3) dissects the functional organization of enhancer clusters through in silico perturbation analysis. We perform multiple independent CRISPR knockout, CRISPR interference (CRISPRi), and base editing screens to validate these functions of eScreen both at scale and on individual cases. Furthermore, we provide an interactive web server (https://escreen.huanglabxmu.com/) for the community to access the integrated CRISPR screen resources and eScreen functions. Collectively, our work establishes a highly precise and convenient tool to decode the causal effects of the regulatory genome.

genomics↗

bioRxiv2026

Synthetic germ granules reveal a direct role of Vasa/DDX4 in RNA localization and translational activation

AbstractCytoplasmic RNA granules, including stress granules, P bodies, neuronal RNA granules, and germ granules, are essential for RNA storage and regulation across a wide range of organisms. However, dissecting the contributions of individual factors to granule function is challenging because of the interdependence of components in vivo. This is especially true for DEAD-box helicases, common regulators of mRNA granules, whose specific contributions remain unclear. In this study, we developed a synthetic approach to de novo generate germ granules, enabling us to identify the minimal machinery needed for RNA localization and translational activation. Using a self-assembling PopTag-based scaffold derived from Caulobacter fused to the RNA-binding domain (RBD) of the germplasm organizer Oskar, we found that the recruitment of endogenous germ granule mRNAs (nanos and pgc) depended on the DDX4 protein Vasa. By employing orthogonal RNA tethering approaches, we demonstrate that Vasa is both necessary and sufficient for localized mRNA translation. Consistent with these findings, acute depletion of Vasa from endogenous germ granules specifically reduced Nanos translation without affecting mRNA localization, confirming Vasa as a core factor linking RNA recruitment to localized translational activation. These in vivo reconstitution experiments reveal a two-component module in which a scaffold RBD and the Vasa helicase, but not other DEAD-box helicases, enable RNP condensates to accumulate specific RNAs and promote their translation. Overall, our study uncovers previously unrecognized functions of an RNA helicase within ribonucleoprotein condensates and demonstrates the power of synthetic biology to analyze complex biomolecular condensates in living organisms.

developmental biology↗

bioRxiv2026

Pathological TDP-43 filaments accumulate at synapses and cause synaptic dysfunction

The assembly of TAR DNA-binding protein 43 (TDP-43) into amyloid filaments within neurons is a hallmark of multiple neurodegenerative diseases, including motor neuron diseases (MND), frontotemporal dementias (FTD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). These diseases result from the deterioration and loss of neurons, with synaptic dysfunction and neuronal hyperexcitability being prominent early events. Pathogenic mutations in the TDP-43 gene, TARDBP, that promote filament formation have established a causal role for TDP-43 assembly in neurodegenerative diseases. However, the molecular mechanisms underlying filament accumulation and their contribution to neurodegeneration are poorly understood. TDP-43 filaments can propagate between neurons in a prion-like manner, which may underlie the progressive spread and accumulation of TDP-43 pathology in disease. Here, we studied early stages of TDP-43 filament accumulation following internalisation of patient-derived TDP-43 filaments by mouse and human cortical neurons. Using proximity labelling, we identified molecular environments and putative interactions of TDP-43 filaments. We found that TDP-43 filaments accumulated at synapses, particularly in proximity to the presynaptic active zone, which we confirmed in FTD patient brain sections. Electron cryo-tomography (cryo-ET) directly visualised abundant TDP-43 filaments spanning the presynaptic cytoplasm in situ, which contacted synaptic vesicles and the plasma membrane. Functional measurements revealed that the accumulation of TDP-43 filaments led to presynaptic dysfunction and subsequent neuronal hyperexcitability. These findings suggest that synapses are a major early site of TDP-43 filament accumulation, relevant to their propagation, and directly link TDP-43 filament gain of function to synaptic dysfunction.

neuroscience↗

bioRxiv2026

Memory erasure by dopamine-gated retrospective learning

Erasing outdated memories is crucial for adaptive behavior. Yet once a cue-outcome association is learned, repeated cue exposure without outcome suppresses conditioned behavior without erasing the underlying memory. This allows rapid behavioral recovery when outcomes are reintroduced. Here, we confirm this limitation for standard "prospective extinction" protocols that present cues without the associated outcome, but show that true memory erasure is achieved by inverting the paradigm: presenting outcomes without associated cues, i.e., "retrospective extinction". We demonstrate that orbitofrontal cortex activity at outcome is necessary for the rapid behavioral recovery following prospective extinction, and that mesolimbic dopamine activity at outcome is necessary for retrospective extinction. These findings reconceptualize extinction mechanisms and suggest complementary strategies to mitigate relapse and erase maladaptive memories.

neuroscience↗

bioRxiv2026

Molecular and Cellular Underpinnings of Spatial Heterogeneity in Fetal Cortical Folding

Cortical folding is a defining feature of human brain development, yet the molecular and cellular mechanisms that produce regionally specific cortical folding remain incompletely understood. Here we combined high-resolution in-utero T2-weighted and diffusion MRI atlases (23-38 weeks of gestational age) with prenatal transcriptomic profiles to map regional macrostructural and microstructural features of cortical folding to underlying gene expression. We found genes whose expression patterns correlated with cortical curvature were enriched for neurogenesis, progenitor proliferation and radial glia guided neuronal migration, and localized to ventricular zone/subventricular zone progenitor cell subtypes, supporting for the protomap hypothesis of areal specification. By contrast, cortical microstructural markers-associated gene sets were implicated in myelination, cell adhesion and extracellular matrix remodeling, and mapped to astrocyte and endothelial cell programs. The microstructure-related gene expression peaked in the early postnatal period and remained high throughout childhood, while the curvature-associated gene expression reduced with age. Several of these cortical folding-related genes overlapped with autism spectrum disorder risk loci (e.g., SCN2A, STXBP1, DVL3). Collectively, these cross-modal findings outline a sequential developmental architecture--early progenitor driven patterning followed by myelin and extracellular matrix consolidation. In addition, we released a high-resolution fetal labeling atlas to facilitate further imaging-genetic studies of early cortical development.

neuroscience↗

bioRxiv2026

CTLA-4 inhibitors drive colitis through metabolic reprogramming-mediated Treg/Th17 imbalance

Immune checkpoint inhibitors (ICIs), especially CTLA-4 inhibitors (CTLA-4), exhibit a high incidence of colitis as an immune-related adverse event (irAE) during cancer treatment, severely limiting patient benefit. Clinically, both treatment interruption and existing intervention drugs for ICI-mediated colitis may compromise antitumor efficacy. However, there is inadequate research on the pathogenesis of ICI-mediated colitis, with findings often conflicting. Here, we first established multiple clinically relevant animal models, including an immuno-humanized ICI-mediated colitis model. Through time-series transcriptomics, we discovered that CTLA-4-induced colonic toxicity exhibits characteristics ranging from early metabolic reprogramming represented by glycolysis to later immune disorders represented by Th17 responses. By targeting colonic CTLA-4+ T cells, CTLA-4 blocked CD80/CD86-CTLA-4 interaction, thereby activating the PI3K-AKT-mTOR pathway. Subsequently, mTOR mediated metabolic reprogramming in T cells, shifting them from Treg-biased oxidative phosphorylation to Th17-biased glycolysis. The colonic toxicity of CTLA-4 has also been demonstrated to depend on the PI3K-AKT-mTOR pathway, glycolysis, and Th17 responses. Notably, metformin significantly relieved ICI-mediated colitis by inhibiting mTOR without impeding antitumor efficacy. Collectively, these findings highlighted the metabolic-immune axis in the colonic toxicity of ICI and provided a clinically superior intervention strategy.

pharmacology and toxicology↗

bioRxiv2026

Monitoring Gene Expression in Retina with Synthetic Serum Markers

Gene expression underlies retinal development, function, and pathogenesis. However, monitoring retinal gene expression in vivo is challenging. In this study, we apply synthetic serum markers, called Released Markers of Activity, or RMAs, to quantify gene expression in an intact retina through a simple blood test. We show that RMAs can quantify transduction of multiple retinal cell-types and monitor cell implantation or cell loss. We found RMAs are sensitive enough to measure transduction of rare cell populations, such as retinal ganglion cells and could detect transplantation of as few as 100 stem cells. Expression of RMAs showed no evidence of cell loss or immune activation in the retina. Overall, these findings demonstrate that RMAs are highly sensitive, tissue non-destructive gene expression reporters that can continuously track gene delivery and cell survival in intact retina through a simple blood test.

bioengineering↗

bioRxiv2026

Striatal Acetylcholine Dip-Rebound Is Induced by Direct Pathway Neurons and Encodes Action-Outcome Contingency

Learning contingencies between actions and outcomes is pivotal in adaptive behavior and requires ongoing flexible sensory, motor, and reward information integration. The striatum is central to the integration. However, it remains unclear how the striatal neurons interact with each other to facilitate learning. Here we show that in the dorsomedial striatum, direct-pathway medium spiny neurons (dMSNs), but not dopamine (DA), inhibit cholinergic interneurons, inducing a characteristic acetylcholine (ACh) dip-rebound, encoding action-outcome (A-O) contingency. Using genetically encoded sensors and in vivo fiber photometry, we find that dMSN activation, ACh dip-rebound, and DA transients emerge only after mice acquire the contingency. dMSN activity and ACh dynamics persist even when rewards are probabilistic, adapt in time as the learned relationship between action and reward evolves, and vanish when the contingency is degraded. Ex vivo recordings and in vivo optogenetics further show that dMSN activity is both sufficient and required to generate the ACh dip-rebound through GABAergic inhibition of cholinergic interneurons. Disrupting the dip-rebound slows acquisition and accelerates extinction. Together, these findings reveal a previously unrecognized dMSN-ACh circuit mechanism that encodes contingency during instrumental learning, operating alongside DA signals that track reward outcomes.

neuroscience↗

bioRxiv2026

Early-Stage Corticostriatal Circuit Hyperactivity Impairs Cholinergic Function and Cognitive Flexibility in an Alzheimer's Model

Cognitive flexibility deficits are a hallmark of early-stage Alzheimers disease (AD), but the underlying circuit mechanisms remain poorly understood. Here, we show that the 5xFAD mouse model of AD neuropathology exhibited early deficits in instrumental reversal learning, indicating cognitive inflexibility preceding spatial memory deficits. This impairment was associated with excessive neuronal reactivation in the medial prefrontal cortex (mPFC) and dorsomedial striatum (DMS), key regions for goal-directed behavior. Electrophysiological recordings revealed that mPFC neurons in young 5xFAD mice were hyperexcitable and received elevated excitatory input. Moreover, the mPFC-to-direct pathway medium spiny neuron (dMSN) circuit and the dMSNs themselves were selectively hyperactive. These hyperactive dMSNs exerted increased inhibitory control over cholinergic interneurons (CINs) in the DMS, coinciding with reduced CIN firing and diminished striatal acetylcholine (ACh) release. Critically, sustained chemogenetic inhibition of the mPFC-to-DMS circuit in 5xFAD mice reduced cortical A{beta} accumulation, normalized glutamatergic transmission in both the mPFC and DMS, restored striatal ACh levels, and rescued reversal learning deficits. Together, these findings identify a hyperactive mPFC-to-DMS circuit that disrupts corticostriatal and cholinergic signaling, contributing to cognitive inflexibility in 5xFAD mice. Targeting this circuit may offer a therapeutic strategy to preserve cognitive function in the early stages of AD. HighlightsO_LI5xFAD mice exhibit early cognitive deficits in instrumental reversal learning. C_LIO_LImPFC neurons and corticostriatal circuits are hyperactive, while cholinergic neurons are hypoactive in 5xFAD mice. C_LIO_LISustained inhibition of mPFC-to-DMS circuit hyperactivity normalizes glutamatergic transmission and slows A{beta} accumulation in 5xFAD mice. C_LIO_LISustained inhibition of the mPFC-to-DMS circuit rescues reversal learning deficits in 5xFAD mice. C_LI

neuroscience↗

bioRxiv2026

Regulation of gastric cancer progression by immune-related transcription factors CEBPB in Helicobacter Pylori infection

Helicobacter pylori infection is a primary risk factor for gastric carcinogenesis, yet the heterogeneity of host cellular responses remains incompletely characterized. This study systematically delineates the transcriptional landscapes of four gastric epithelial cell lines (AGS, GES-1, HGC-27, MKN-45) upon H. pylori infection. Transcriptomic analyses revealed distinct, cell-type-specific dysregulation patterns, with Gene Ontology enrichment highlighting perturbations in processes ranging from epidermal differentiation and metabolism (AGS) to extracellular matrix remodeling (GES-1), cell cycle control (HGC-27), and mitotic regulation (MKN-45). Intersection analysis identified a conserved core signature of 41 upregulated and 11 downregulated genes, implicating unified stress, inflammatory, and metabolic responses. From this signature, nine key transcription factors (TFs) were extracted, with CEBPB and MAFF demonstrating significant prognostic value in gastric adenocarcinoma (STAD) cohorts. Subsequent pan-cancer investigation established CEBPB as a context-dependent regulator, showing upregulation in STAD and other malignancies where its high expression correlated with poor prognosis. Mechanistically, CEBPB expression in STAD was associated with an immunosuppressive microenvironment (correlating with M2 macrophages and dendritic cells), co-expression with chemokine and checkpoint genes, and elevated metrics of genomic instability (MATH, ploidy, LOH). These findings position CEBPB as a central mediator linking H. pylori infection to oncogenic reprogramming, immune evasion, and genomic chaos in gastric cancer, offering a multifaceted target for therapeutic intervention.

bioinformatics↗

bioRxiv2026

Epidural Thin Film Implants of Polymerized Curcumin Downregulate Inflammatory Markers and Improve Functional Recovery After Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a notoriously intractable medical problem for which there is no neuroprotective pharmacotherapy, despite four decades of clinical trials on systemically administered small molecules that target plausible injury mechanisms. The shared failure mode of those trials is delivery: oxidative and inflammatory injury evolves locally at the lesion site over days to weeks, a timescale that far exceeds the half-life of systemically administered drugs. Furthermore, systemically administered drugs often cannot be dosed at high enough levels to achieve efficacious concentrations locally at the lesion site, without off-target effects. In this study, we show the first example of a locally implantable drug depot that exerts long-lasting activity at the lesion site. We demonstrate in vivo efficacy of a poly(pro-drug) thin film deployed locally, to address delivery problem in SCI. The films are composed of a copolyester in which curcumin, a potent natural antioxidant, is incorporated as a backbone repeat unit alongside poly(ethylene glycol) sebacate segments. The soft, thin film ([~]100 {micro}m) is employed as an epidural implant that quenches reactive oxygen species on-contact and can sustain activity for over two months, which is orders of magnitude longer than free curcumin (half-life of [~]10 min in buffer). A single epidural P50 film, placed on the exposed dura immediately after a moderate-to-severe T9 contusion in mice, produced a statistically significant improvement in Basso Mouse Scale locomotor recovery at 14 days post-injury, relative to a non-antioxidant control polymer of same architecture. The film preserved myelinated white matter, attenuated reactive gliosis (GFAP, Iba1), and produced coherent downregulation of neuroinflammatory gene-expression programs. These effects were spatially restricted to tissue rostral to the lesion, consistent with on-contact, locally confined polymer activity. Because curcumin is covalently enchained within the polymer itself, rather than blended as a payload in a carrier matrix, release is governed by backbone hydrolysis and the architecture can generalize to other phenolic bioactives, potentially establishing epidural poly(pro-drug) thin films as a mechanistically grounded, surgical platform for sustained local pharmacotherapy of CNS injury.

bioengineering↗

bioRxiv2026

NORAD orchestrates KLC1-mediated SFPQ transport via liquid-liquid phase separation

Neuronal function requires precise long-distance axonal transport mediated by molecular motors and RNA-binding proteins like SFPQ, though regulatory mechanisms remain poorly defined. We identify long non-coding RNA NORAD as a master regulator of this process through liquid-liquid phase separation (LLPS). While SFPQ and kinesin-1 mediate cargo delivery, the specific RNA coordinating their interaction was unknown. We demonstrate NORAD directly binds kinesin light chain 1 (KLC1) and promotes SFPQ condensation into dynamic LLPS droplets, enabling efficient transport. CRISPR-assisted mapping and functional assays show NORAD depletion disrupts granule dynamics, impairs neuroprotective mRNA localization, and induces axonal degeneration. In vitro reconstitution confirms NORAD-KLC1 synergy enhances SFPQ phase separation, and neuron-specific knockout mice exhibit motor deficits with reduced neuronal density. These findings establish the first evidence of lncRNA NORAD-mediated LLPS in axonal transport, revealing a new paradigm for RNA-guided neuronal maintenance.

molecular biology↗

bioRxiv2026

Microglial Lag3 Drives α-Synuclein--induced Neurotoxic Activated (A1) Astrocytes and Neurodegeneration

BackgroundNeuroinflammation and pathologic -synuclein (-syn) aggregation cooperate to drive dopaminergic neurodegeneration in Parkinsons disease, but the glial receptors that couple extracellular -syn to inflammatory cascades remain incompletely defined. Microglia express higher levels of lymphocyte activation gene 3 (Lag3) than neurons, yet the contribution of microglial Lag3 to -syn recognition, glial crosstalk, and neurodegeneration is unknown. MethodsBiochemical binding assays, live-cell imaging, cytokine profiling, and neuron-microglia-astrocyte co-culture paradigms were used to define Lag3-dependent -syn preformed fibril (PFF) binding, uptake, and microglial activation. To interrogate in vivo function, microglia-specific Lag3 conditional knockout mice (Lag3L/L-Cx3cr1CreER) and littermate controls received unilateral intrastriatal -syn PFF injections, followed by histological, biochemical, and behavioral assessments of -syn pathology, gliosis, nigrostriatal integrity, and motor performance. Results-syn PFFs bound microglial Lag3 with high specificity and nanomolar affinity and required Lag3 for efficient fibril internalization and induction of proinflammatory cytokines. Microglial Lag3 deficiency markedly blunted -syn PFF-evoked microglial activation, prevented cytokine-driven conversion of astrocytes into neurotoxic reactive A1 astrocytes, and abolished astrocyte-dependent neuronal death in vitro. In vivo, microglia-specific Lag3 deletion reduced cortical, striatal, and substantia nigra pS129 -syn pathology, suppressed microgliosis and A1 astrocyte induction, preserved substantia nigra dopaminergic neurons and striatal dopamine transporter/tyrosine hydroxylase expression, and ameliorated -syn PFF-induced motor deficits. ConclusionsThis study identifies microglial Lag3 as a key receptor linking extracellular -syn PFF recognition to inflammatory amplification, neurotoxic reactive A1 astrocyte conversion, and dopaminergic neurodegeneration. Together with prior work on neuronal Lag3, these findings support a cell-type-specific dual-axis model in which neuronal Lag3 mediates -syn propagation while microglial Lag3 drives glia-dependent neurotoxicity, positioning Lag3 as a promising precision therapeutic target in -synucleinopathies.

neuroscience↗

bioRxiv2025

MLL3 and MLL4 sustain hematopoietic stem cell multipotency by opposing a B-cell default state

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) are sustained by networks of transcription factors and epigenetic regulators that prime lineage-specific programs yet maintain multipotency. Two epigenetic regulators, MLL3 and MLL4, play important but opposing roles in maintaining this balance. MLL3 promotes HSC differentiation, whereas MLL4 opposes differentiation. These opposing functions are essential for both normal homeostasis and leukemia suppression, yet it is not clear how MLL3 and MLL4 regulate HSC and MPP gene expression to control HSC/MPP fate decisions. To resolve these mechanisms, we performed an extensive series of single cell genomic studies after conditionally deleting Mll3, Mll4 or both genes together. Mll3 deletion had only limited effects on HSC/MPP enhancer networks at steady state, whereas Mll4 deletion led to precocious activation of myeloid enhancers. Surprisingly, compound Mll3/4 deletion eliminated all myeloid, erythroid and megakaryocytic potential within the hematopoietic hierarchy and caused all progenitors to rapidly default to a B-cell-like identity. These changes were accompanied by widespread inactivation of HSC/MPP enhancers and superenhancers, and ectopic activation of B-cell superenhancers. Disabling MLL3/4 histone methyltransferase activity did not recapitulate the pervasive changes in cell identity that were observed when MLL3 and MLL4 were fully inactivated, indicating that MLL3 and MLL4 activate HSC/MPP enhancers independently from their enzymatic activities. Our findings show that HSC/MPP multipotency requires sustained tension between MLL3/4-dependent enhancers that maintain myeloid, erythroid and megakaryocyte potential, and MLL3/4-independent enhancers that prime B-cell identity. MLL3 and MLL4 therefore serve as critical linchpins of multilineage hematopoiesis. KEY POINTSO_LIMLL3 and MLL4 act redundantly in HSCs to sustain transcription factor and enhancer networks that support multipotency C_LIO_LISimultaneous loss of MLL3 and MLL4 drives hematopoietic progenitors into a uniform B-cell-like default state C_LI

cell biology↗