Microglial P2Y12 Receptor Signaling Governs Epilepsy-Associated Neurogenesis via Bidirectional Regulation of Distinct Microglial Subpopulations

Microglial P2Y12 Receptor Signaling Governs Epilepsy-Associated Neurogenesis via Bidirectional Regulation of Distinct Microglial Subpopulations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Microglial P2Y12 Receptor Signaling Governs Epilepsy-Associated Neurogenesis via Bidirectional Regulation of Distinct Microglial Subpopulations Mingshu Mo, Juan Ling, Lan Wang, Huishan Deng, Yilin Su, Lijian Wei, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6558062/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Following epileptogenesis, a pronounced escalation in microglial activation, neuronal dysfunction, and hippocampal neurogenesis is consistently observed. As principal immune sentinels of the central nervous system, microglia perform multifaceted functions including inflammatory mediator secretion, neurotrophic factor synthesis, synaptic material phagocytosis, and homeostatic regulation. Within the epileptic milieu, microglia exhibit dichotomous regulatory effects, paradoxically influencing both neurodegenerative processes and neurogenesis processes. Despite this critical dual functionality, the mechanistic underpinnings of microglial polarization in epileptogenesis remain incompletely characterized. To address this knowledge gap, we implemented an integrative multi-omics approach combining single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) to delineate distinct epilepsy-associated microglial (EPAM) subpopulations. Complementary conditional knockout murine models were employed to elucidate the molecular determinants of EPAM differentiation. Our analytical pipeline identified two microglial subsets demonstrating reciprocal abundance patterns at 7 days post-epilepsy induction: a diminished P2ry12 high CD74 low H2-Ab1 low population and an expanded P2ry12 low CD74 high H2-Ab1 high subpopulation, temporally correlated with hippocampal neurogenesis onset. Genetic ablation of P2ry12 precipitated a paradoxical expansion of both subpopulations following epileptogenic challenge, concomitant with significant suppression of neurogenesis. Mechanistic investigations revealed that P2ry12 deficiency upregulated CD74 and H2-Ab1 expression within microglia, enhanced hippocampal TNF-α release, and disrupted neurogenesis processes. These findings collectively demonstrate that P2Y12 receptor-mediated signaling governs the dynamic equilibrium of EPAM subpopulations, with perturbation of this regulatory axis impairing compensatory neurogenesis during epileptogenesis. P2ry12 Neurogenesis Epilepsy Microglia subpopulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Introduction Epilepsy (EP), a prevalent neurological disorder characterized by recurrent seizures, manifests electrophysiologically through neuronal synchronization and hyperexcitability 1 . Postictal neurogenic activity in the dentate gyrus (DG) and aberrant mossy fiber sprouting into the CA3 region are hallmark features observed in both clinical populations and experimental models 2 . These structural rearrangements contribute to maladaptive circuitry formation, a critical driver of epileptogenesis 3 . Deciphering the molecular underpinnings of these processes may advance our understanding of adult neurogenesis and catalyze the development of targeted antiepileptogenic therapies 4 . As central nervous system (CNS) immune sentinels, microglia regulate neural homeostasis through multifaceted roles, including inflammatory mediator secretion, neurotrophic factor production, synaptic pruning, and microenvironmental modulation 5 . Their bidirectional influence on neurogenesis and axonal remodeling positions them as critical arbiters of epileptic pathophysiology 6 . While microglial involvement in epilepsy is undisputed, their functional duality remains contentious 7 . Early studies emphasized their detrimental role via pro-inflammatory cytokine release (e.g., TNF-α, IFN-γ, IL-1β), exacerbating neuroinflammation and neuronal injury 8 . Conversely, emerging evidence highlights their neuroprotective capacity through neurotrophic factor synthesis (NGF, NT3, FGF), which supports neuronal survival and neurogenic niches 9 , 10 . This functional dichotomy extends to microglia-neuron crosstalk, where they dynamically sculpt synaptic architecture, suppress hyperexcitability, and eliminate damaged neurons—processes that may paradoxically mitigate or potentiate epileptogenesis 5 , 11 . Resolving this paradox necessitates interrogation of microglial heterogeneity. Transcriptomic profiling has identified four principal microglial subsets in homeostasis: origin-, proliferation-, immune response-, and neuronally enriched populations 12 , 13 . Disease contexts reveal specialized subpopulations, such as Alzheimer’s-associated DAM ( P2ry12 + Trem2 + ApoE + Tyrobp + ), which progress from phagocytic DAM-1 (early Aβ clearance) to pro-inflammatory DAM-2 (late-stage pathology) 14 – 17 . Analogous subsets—including neurodegenerative microglia (MGnD) in ALS/MSA—highlight context-dependent polarization 18 , 19 . Despite these advances, epilepsy-associated microglia (EPAM) remain poorly characterized, obscuring their role in seizure-related neurogenesis 7 , 9 . Here, we define EPAM subpopulations and their functional trajectory during epileptogenesis. Using multimodal sequencing and genetic perturbation, we demonstrate that P2Y12 receptor signaling governs EPAM dynamics, with disruption impairing compensatory neurogenesis. These findings resolve longstanding controversies in microglial duality and establish EPAM as therapeutic targets for precision epilepsy interventions. Materials and methods Animals All experimental procedures were conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Guangzhou Medical University (Approval No. 2020341). Male C57BL/6J mice (8–12 weeks old) were housed under standard conditions (12-hour light/dark cycle, 22°C, 50% humidity). CX3CR1CreER +/+ mice and P2ry12 fl/fl mice (generated via CRISPR/Cas9 as described previously 6 ) were obtained from Cyagen Biosciences (Guangzhou, China). To generate microglia-specific P2ry12 knockout mice (CX3CR1CreER /+ :P2ry12 fl/fl ), heterozygous crosses were performed. The P2ry12floxed allele was engineered by flanking exons 4–3′UTR with loxP sites via homologous recombination. Single-guide RNAs and Cas9 mRNA were synthesized in vitro using T7 RNA polymerase (NEB). A donor vector containing 1 kb homology arms was co-injected with sgRNAs/Cas9 mRNA into fertilized zygotes. Founder (F0) mice were genotyped by PCR (Supplement 1), Sanger sequencing, and Southern blot. For P2ry12 deletion, adult mice received intraperitoneal (IP) tamoxifen (20 mg/mL in corn oil; Sigma T5648) at 75 mg/kg/dose ×4 (48-hour intervals) 6 . Controls received vehicle-only injections. Littermates were randomized across experimental cohorts to minimize genetic bias. Kainic Acid-induced epilepsy model Kainic acid (KA; Tocris 0222) was dissolved in artificial cerebrospinal fluid (ACSF: 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose; pH 7.4) to 0.032 mg/mL. Mice underwent stereotaxic surgery (David Kopf Instruments) under isoflurane anesthesia (2% in O2) for guide cannula (8 mm length; PlasticsOne) implantation into the right lateral ventricle (coordinates: −0.2 mm posterior to bregma, + 0.9 mm lateral, − 2.3 mm ventral). After 4-week recovery, 5 µL KA was infused over 5 minutes via microsyringe (Hamilton). Seizure severity was scored over 120 minutes post-injection using a modified Racine scale 20 . The seizure scoring criteria were as follows: score 1, freezing behavior; score 2, rigid posture with raised tail; score 3, continuous head bobbing and forepaw shaking; score 4, rearing, falling, and jumping; score 5, continuous level 4 seizures; score 6, loss of posture and generalized convulsive activity; and score 7, death. Electroencephalographic (EEG) recording Mice were anesthetized via intraperitoneal (IP) injection of pentobarbital (50 mg/kg) and secured in a stereotaxic frame (Model 940, David Kopf Instruments, CA, USA). After shaving the cranial fur, burr holes (0.7 mm diameter; InterFocus Ltd, Cambridge, UK) were drilled at stereotaxic coordinates relative to bregma: frontal electrode at + 2.0 mm anteroposterior (AP), ± 2.0 mm mediolateral (ML); parietal electrode at + 2.0 mm AP, ± 4.0 mm ML; occipital electrode at + 2.0 mm AP, ± 2.5 mm ML. Stainless steel screw electrodes (0.8 mm diameter, 10–50 kΩ impedance at 1 kHz; Nitto Seiko, Japan) were implanted and affixed with dental cement (Vertex-Dental, Netherlands). A subcutaneous reference electrode was positioned over the nasal bone. A 6-pin surface mount connector (8415-SM, Pinnacle Technology, KS, USA) was integrated for signal transmission. Postoperative care included: thermal support (37°C heating pad) and hydration were maintained intraoperatively; prophylactic ampicillin (100 mg/kg, SC) and analgesic meloxicam (1 mg/kg, SC) were administered postsurgery; mice recovered for ≥ 14 days before EEG recordings. EEG signals were amplified (Grass QP511, RI, USA), digitized at 128 Hz (Digidata 1440A, Molecular Devices, CA, USA), and recorded using Pinnacle Sirenia Acquisition Software (v2.5.0). Mean amplitude analysis focused on a 440-second epoch post-kainic acid (KA) injection, with cross-group comparisons performed using Clampfit 10.7 (Molecular Devices). Tissue processing and histological staining Mice were deeply anesthetized with 5% isoflurane and transcardially perfused with ice-cold phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA; Sigma-Aldrich). Brains were post-fixed in 4% PFA for 6 hours at 4°C, cryoprotected in 30% sucrose/PBS overnight, and sectioned coronally at 14 µm thickness using a cryostat (CM3050S, Leica Microsystems). For Fluoro-Jade B (FJB) staining, Sections were sequentially incubatied in 1% sodium hydroxide dissolved in 80% ethanol for 5 minutes, followed by 70% ethanol for 2 minutes, rinsed with distilled water, treated with 0.06% potassium permanganate for 10 minutes and rinsed again with distilled water, and then immersed in 0.01% FJB (Histo-Chem, Jefferson, AR, U.S.A.) solution containing 0.1% acetic acid for 20 minutes. After PBS rinses, slides were air-dried and coverslipped with DPX mounting medium. For immunofluorescence staining, sections were permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) and blocked in 5% normal goat serum/PBS. The following primary antibodies were applied overnight at 4°C: rabbit anti-Iba1 (1:1000, Wako Chemicals, catalog #019-19741), rabbit anti-Ki67 (1:500, Abcam, catalog #16667), goat anti-doublecortin (DCX, 1:500, Sigma-Aldrich, catalog #SAB2501666), rabbit anti-CD74 (1:500, Sigma-Aldrich, catalog # 3352229), and rabbit anti-GFAP (1:200, Sigma-Aldrich, catalog #ZRB2383). After TBST washes, sections were incubated with AlexaFluor-488/594-conjugated secondary antibodies (1:500, Invitrogen) for 40 min at 25°C and mounted with DAPI Fluoromount-G™ (SouthernBiotech). For BrdU labeling and detection, mice received daily BrdU (100 mg/kg, i.p.; Sigma) for 3 days post-KA 6 . Sections were incubated in 2× SSC/50% formamide for 2 hours at 65°C, 2 N HCl at 37°C for 1 hour, and 0.05 M borate buffer (pH 8.5) for 10 minutes. After blocking with 10% BSA, sections were co-stained with mouse anti-BrdU (1:500, Sigma #B8434) and other primary antibodies as described above. Quantitative morphometric analysis Fluorescent images were captured using an EVOS FL Auto 2.0 microscope (40× objective; Life Technologies) 6 . Six evenly spaced sections (240 µm intervals) per brain were analyzed. Iba1 + /BrdU + /DCX + /Ki67 + cells were manually counted across hippocampal subfields (CA1, CA3, DG) using ImageJ FIJI (v2.3.0). Data represent means ± SEM from ≥ 3 animals per group. DCX + projections were traced in ImageJ to measure the area above the granular cell layer as a suprapyramidal blade (SMF); and the area below the granular cell layer as an infrapyramidal blade (IMF). Volumes were calculated as follows: Volume=∑(Area×14 µm )×6 (section interval factor). RNA Sequencing and qRT-PCR Analysis Mice were transcardially perfused with ice-cold PBS under deep anesthesia. Whole brains were rapidly dissected, snap-frozen in liquid nitrogen, and homogenized in TRIzol® Reagent (15596026, Invitrogen) for total RNA isolation via the guanidinium thiocyanate-phenol-chloroform method. RNA purity (A260/A280 > 1.8) and integrity (RIN ≥ 8.0) were verified using a NanoDrop OneC spectrophotometer (Thermo Scientific) and Experion™ Automated Electrophoresis System (Bio-Rad). Ribosomal RNA was depleted from 2 µg total RNA using the Ribo-Zero™ Gold rRNA Removal Kit (MRZG12324, Illumina). Libraries were constructed with the NEBNext® Ultra™ II Directional RNA Library Prep Kit (E7760S, NEB) following manufacturer protocols: (1) Fragmentation: RNA shearing to 300–400 bp in Illumina fragmentation buffer (94°C, 8 min); (2) cDNA synthesis: first-strand synthesis with random hexamers; second-strand synthesis incorporating dUTP; (3) library construction: end repair, adenylation, and adapter ligation using NEBNext® Multiplex Oligos; (4) enzyme treatment: degradation of uracil-containing second strands (37°C, 15 min), size-selected libraries (400–500 bp) were PCR-amplified (15 cycles) and quantified via Agilent 2100 Bioanalyzer, and paired-end sequencing (2×150 bp) was performed on Illumina NovaSeq 6000 (Shanghai Personal Biotechnology). For bioinformatic analysis, raw reads were aligned to the GRCm38 mouse genome using HISAT2 (v2.2.1). Gene expression was quantified as FPKM (fragments per kilobase per million) with Cufflinks (v2.2.1). Differentially expressed genes (DEGs) were defined by |fold change| >1.5 and Benjamini-Hochberg adjusted p < 0.05. In qRT-PCR validation, primers for P2ry12 (NM_027571.4), Aif1 (NM_009797.3), CD74 (NM_010545.3), H2-Eb1 (NM_207105.2), and Tnf (NM_013693.3) were designed using Oligo 7.6 (Molecular Biology Insights) and synthesized by TIANGEN Biotech (Beijing, China; sequences in Supplement 2). The reactions (25 µL) contained: 6.25 µL of cDNA; 12.5 µL of 2× TaqMan™ PreAmp Master Mix (4440043, Applied Biosystems); 6.25 µL of 0.2× pooled assay mix; 5 pmol each primer. Amplification was performed on a QuantStudio™ 6 Flex System (Applied Biosystems) at 95°C for 10 min for initial denaturation, followed by 40 cycles of 95°C/15 s, 65°C/30 s, and 72°C/30 s. U6 snRNA (NR_003280.1) served as the endogenous control. Relative expression was calculated via the 2 −ΔΔCT method: ΔΔCT = (CT target – CT GAPDH ) experimental − (CT target − CT GAPDH ) control . Single-cell RNA sequencing and bioinformatics analysis Mice were transcardially perfused with ice-cold PBS under deep anesthesia. Brains were rapidly dissected, mechanically dissociated, and processed for single-cell RNA sequencing (scRNA-seq) using the BD Rhapsody™ Whole Transcriptome Analysis (WTA) Pipeline (v1.8; BD Biosciences). Single-cell suspensions were captured on a BD Rhapsody™ Cartridge, and cDNA libraries were constructed following manufacturer protocols (Shanghai Genechem Co., Ltd., China). Sequencing was performed on an Illumina NovaSeq 6000 platform (2×150 bp). The raw FASTQ files were aligned to the GRCm38 mouse reference genome (Ensembl v103) using the BD Rhapsody™ Analysis Pipeline. A unique molecular identifier (UMI) count matrix was generated and imported into Scanpy (v1.8) for downstream analysis 21 . Low-quality cells were filtered using thresholds: UMI counts ≤ 100,000; detected genes ≤ 8,000; and mitochondrial gene content > 10%. After quality control, 44,810 high-quality cells were retained. Counts were normalized via total expression scaling (factor = 10,000) and log-transformed (log1p) using pp.normalize_total and pp.log1p in Scanpy. For dimensionality reduction and clustering, highly variable genes were identified using the repored method 22 , implemented via pp.highly_variable_genes. Principal component analysis (PCA; 50 components) was performed (tl.pca), followed by graph-based clustering (pp.neighbors, resolution = 0.5). The cell populations were visualized in 2D/3D UMAP space (tl.umap). For marker gene identification and functional enrichment, cluster-specific marker genes were identified using Seurat’s FindAllMarkers (Wilcoxon rank-sum test; adjusted p 1) 23 . The enriched pathways were analyzed via g: Profiler2 (v0.2.1) with hypergeometric testing (FDR < 0.05) across Gene Ontology (GO), KEGG, and Reactome databases 24 . For validation and Cell-Cell Communication Analysis, Volcano plots (ggplot2 v3.4.0), heatmaps (EnhancedVolcano v1.14.0), and cell-cell interaction networks (CellChat v1.6.0) were generated in R Studio (v4.2.2). Statistical analysis The sample sizes are detailed in figure legends. Data are presented as mean ± SD (normal distribution) or median ± IQR (non-normal distribution). Between-group comparisons used: Student’s t -test for two groups; One-way ANOVA with Tukey’s post hoc for more than 3 groups; and Wilcoxon rank-sum test for non-parametric data. Analyses were performed in SPSS (v19.0), Prism 9 (v9.0.2), and R Studio. Significance was defined as p < 0.05. Results Characterization of Microglial Activation and Neurogenic Responses in KA-Induced Epileptogenesis To delineate microglial dynamics during epileptogenesis, we established a kainic acid (KA)-induced seizure model validated through multimodal assessment. Behavioral seizure scoring confirmed progressive ictogenesis (Fig. 1 A), while FJB histochemistry revealed significant hippocampal neurodegeneration at 3 days post-seizure (**p < 0.01) and 7 days post-seizure (**p < 0.01) versus controls (Fig. 1 B, E). Electrocorticographic (EEG) analysis demonstrated sustained epileptiform activity post-KA, with mean amplitude increases in frontal (**p < 0.01), parietal (*p = 0.01), and occipital (**p < 0.01) cortices (Fig. 1 C). Neurogenic remodeling was quantified via BrdU pulse-chase labeling. Proliferative BrdU + cells localized predominantly to CA3 subfields at 3 and 7 days post-seizure (Fig. 2 A), paralleled by microgliosis (Iba1 + cell density: **p < 0.01; Fig. 2 B, C). Co-staining revealed selective expansion of proliferating microglial subsets in CA3:Iba1 + BrdU + (**p < 0.01); Iba1 + Ki67 + (**p < 0.01). No significant changes occurred in NeuN + BrdU + (neuronal) or GFAP + BrdU + (astroglial) populations, indicating microglia-specific proliferative engagement (Fig. 2 B, E). RNA-seq analysis of whole-brain lysates identified 1,057 differentially expressed genes (DEGs) at 3 days post-seizure, with 89 persistent changes at 7 days post-seizure (Fig. 3 A). Three-dimensional volcano plots and hierarchical clustering highlighted biphasic expression trajectories that peaking at 3 days post-seizure before partial normalization (Fig. 3 B, C). Microglial signature genes ( C1qb , C1qc , C3 , Cx3cr1 , H2-DMb1 ) followed this temporal pattern (Fig. 3 D). KEGG pathway analysis implicated stage-specific mechanisms: TNF signaling (neuroinflammation; FDR = 1.2×10⁻⁵) at 3 days post-seizure, Chemokine signaling (microglial migration; FDR = 3.8×10⁻⁴) at 7 days post-seizure (Fig. 3 E). Unsupervised clustering partitioned microglial DEGs into three functional modules: Cluster 1: Extracellular matrix regulators ( Adamts1 , Aif1 ); Cluster 2: Immunomodulators ( Cx3cr1 , CD74 ); Cluster 3: MHC-II complex ( H2-Eb1 ) and calcium-binding proteins ( S100a9 , S100a8 ), and others (Fig. 3 F). Notably, P2ry12, as a genetic risk factor for seizures 6 , showed stable transcript levels at both timepoints that was confirmed by qRT-PCR (Fig. 3 G), despite upregulation of P2RY12 + microglia in CA3 at 3 and 7 days post-seizure in immunohistochemical staining (both **p < 0.01) (Fig. 3 H,I). This discordance seems to suggest post-transcriptional regulation of P2RY12 during epileptogenesis. Single-Cell Resolution Reveals Seizure-Induced Microglial Reconfiguration despite Stable P2ry12 Transcript Levels To reconcile the paradox between unchanged whole-brain P2ry12 mRNA levels (Fig. 3 G) and CA3-specific P2RY12 + microgliosis (Fig. 3 H, I), we performed single-cell RNA sequencing (scRNA-seq) at 7 days post-seizure. Unsupervised clustering of 44,810 cells identified 30 transcriptomically distinct populations (Fig. 4 A), annotated as microglia (Clusters 6, 8, 20), neurons, astrocytes, and oligodendrocytes (Fig. 4 B; Supplement 2). Unique molecular identifier (UMI) distributions confirmed cluster-specific transcriptional identities (Supplement 3, 4). Microglial clusters (6/8/20) exhibited elevated Aif1 (Iba1) expression versus other lineages (Fig. 4 C), with kernel density estimates validating inter-cluster heterogeneity (Fig. 4 D). Seizures induced significant microglial subset redistribution (decreased in Cluster 6, increased in Cluster 8; both p < 0.001; Fig. 4 E, F) without altering total microglial abundance (proportion unchanged: p = 0.45; Fig. 4 G, H; Supplement 5). Differential expression analysis of microglia revealed the following: upregulated: Csf1r (M-CSFR), Ccl4 (chemokine), Ctss (cathepsin S); downregulated: Trt (telomerase), EnPP2 (ectonucleotide pyrophosphatase), and Ly6c1 (stem cell marker). Notably, Aif1 expression spiked transiently at 3 days post-seizure (qRT-PCR: **p < 0.01; RNA-seq: p < 0.01) but normalized by 7 days post-seizure (Fig. 4 I; Supplement 6), underscoring its limitations as a standalone microglial activation marker given pathway-specific dynamics (Fig. 3 F). Subpopulation trajectory analysis uncovered divergent responses: decreased Cluster 6 (homeostatic), marked with down-regulated P2ry12 / Csf1r / Cx3cr1 and up-regulated H2-Ab1 / CD74 ; increased Cluster 8 (immunomodulatory, p < 0.001), marked with up-regulated Ms4a7 / Rab7b / Slamt7 and down-regulated Tmem119 / Siglech ; and stable Cluster 20 (transitional; Fig. 5 A-F). KEGG pathway enrichment implicated cytokine-cytokine receptor interactions across all subsets, with Cluster 8 showing dominant involvement (Fig. 5 G). These data demonstrate the seizure-driven microglial repolarization via subset-specific transcriptional rewiring. Volcano plot analysis confirmed the coordinated upregulation of P2ry12 , Trem2 , Cx3cr1 , and CD74 in microglia (Fig. 6 A). UMAP visualization revealed selective P2ry12 downregulation within Cluster 8 (Fig. 6 B), corroborated by scatter/violin plots showing global microglial P2ry12 reduction post-seizure ( Fig. 6 C, E). Co-expression analysis identified a diminished Cx3cr1 high P2ry12 high subpopulation (Fig. 6 D), indicating that P2Y12R loss accompanies immunomodulatory polarization. Microglia-Specific P2ry12 Ablation Perturbs Neural Cell Homeostasis While Preserving CD74highH2-Ab1high Subpopulation Stability Prior studies established that conventional P2ry12 knockout (KO) exacerbates kainic acid (KA)-induced neurotoxicity 6 , 25 . To isolate microglial-specific mechanisms, we employed tamoxifen-inducible CX3CR1CreER /+ : P2ry12 fl/fl mice (hereafter P2ry12 cKO), and administering four tamoxifen doses (75 mg/kg, IP) at 48-hour intervals post-seizure (Fig. 7 A). Pre-KO validation confirmed comparable baseline EEG profiles ( p = 0.70 ; Fig. 7 B, E) and equivalent KA-induced outcomes between genotypes: hippocampal neurodegeneration (FJB + cells: p = 0.95 ; Fig. 7 C,F); and seizure severity (Racine scores: NS; Fig. 7 D). This protocol ensured equivalent epileptogenic priming prior to microglial P2ry12 deletion. UMAP analysis confirmed efficient P2ry12 knockdown with pan-cellular repercussions (Fig. 8 A): increased populations contained microglia ( p = 0.03 ), endothelial cells ( p = 0.01 ), and oligodendrocytes ( p < 0.01); and decreased populations contained choroid plexus epithelium ( p < 0.01), and neurons ( p = 0.01 ) (Fig. 8 B, E, F). Microglial subpopulations exhibited divergent responses: expanded Cluster 6 ( P2ry12 high homeostatic; p = 0.01 ), increased Cluster 20 ( P2ry12 int transitional; p = 0.02 ), and stable Cluster 8 ( P2ry12 low CD74 high H2-Ab1 high , p = 0.80 ) (Fig. 8 C,F). Violin plots revealed global P2ry12 suppression (Fig. 8 D), predominantly driven by Cluster 6 attenuation (Fig. 8 G). Transcriptional profiling confirmed that P2ry12 cKO promoted Cluster 6-specific downregulation of homeostatic markers ( Hexb , Tmem119 , P2ry12 ), with minimal impact on Clusters 8/20 (Fig. 8 H). P2Y12 Receptor Dysfunction Amplifies CD74 high H2-Ab1 high Microglial Subsets in Postictal Remodeling To interrogate P2Y12R’s role in seizure-associated microglial plasticity, we employed a KA-induced epilepsy model in P2ry12 cKO mice. scRNA-seq at 7 days post-seizure revealed profound cellular reorganization in P2ry12 cKO brains, with UMAP visualization showing distinct global transcriptomic shifts versus wildtype (WT) controls (Fig. 9 A). Microglial expansion (p < 0.01) dominated the P2ry12 cKO response, accompanied by neuronal ( p < 0.01), endothelial (p < 0.01), and oligodendrocytic ( p = 0.02 ) depletion, while choroid plexus epithelium remained stable ( p = 0.24 ; Fig. 9 B, C, E). Differential gene expression analysis revealed the following: microglia with upregulated Lyz2 (lysozyme), Ctss (cathepsin S), C1qb (complement), and downregulated CD74 , Ttr (transthyretin); neurons with Upregulated Sox11 , Tubb2b , Sox4 , and downregulated Itm2a , Flt1 (Fig. 9 D). Immunofluorescence staining confirmed sustained P2Y12R suppression in P2ry12 cKO microglia post-seizure (p < 0.01; Fig. 10 A, D), concomitant with expanded proliferative microglial pools (p = 0.02; Fig. 10 B, C). Strikingly, all microglial clusters (6/8/20) exhibited aberrant expansion in P2ry12 cKO mice versus WT (enlarged cluster 8, decreased cluster 6, stable cluster 20; all p < 0.01; Fig. 11 A-D), establishing Cluster 8 with CD74 high H2-Ab1 high as the dominant pathological subset. After P2Y12R ablation, the marker genes of homeostatic Cluster 6, including Tmem119 and Siglech , had partial recoveries and were dramatically down-regulated in WT after seizures, and Ms4a7 , Rab7b , Slamt7 in Cluster 8 were hyperactivated (Fig. 12 A). Special transcriptional reprogramming included up-regulated Sparc / CD81 / Hexb , and down-regulated Gpnmb/Lyz2 in Cluster 6; up-regulated Thbs1/Gpnmb/CD74 , and down-regulated Hexb/Sparc in Cluster 8; down-regualted Gpnmb/Thbs1/Lgals3 in Cluster 20 (Fig. 12 B-D). UMAP spatial mapping localized CD74 / H2-Eb1 co-expression to Cluster 8, which inversely correlated with P2Y12R + microglia (Fig. 12 E, F). Sub-clustering of 4,463 microglia identified 13 transcriptomic states (Fig. 13 A), with three principal subsets: Subcluster 1 expressing H2-Eb1 high CD74 high Thbs1 high Saa3 high , and Cx3cr1 low P2ry12 low ; Subcluster 2 expressing Cx3cr1 high Gpr34 high Tmem119 high , and H2-Eb1 low CD74 low ; and Others in transitional states (Fig. 13 B, C, F). Postictal P2ry12 cKO selectively amplified Subcluster 1 after seizure (p < 0.01; Fig. 13 H), validated by H2-Eb1 (p < 0.001) and CD74 (p < 0.001) upregulation (Fig. 13 I). UMAP spatial mapping localized CD74 / H2-Eb1 co-expression to Subcluster 1, which inversely correlated with P2Y12R + microglia (Fig. 12 E). Microglial P2Y12 Receptor Deficiency Suppresses Postictal Neurogenesis via Neuronal-Glial Crosstalk As demonstrated in Fig. 9 E, microglial P2Y12 receptor deficiency (P2ry12 cKO) abolished seizure-induced neurogenic augmentation. To mechanistically dissect this phenotype, we performed immunofluorescence analysis of the hippocampal dentate gyrus (DG) using DCX as an immature neuronal marker, BrdU as a proliferative cell tracer, and Ki67 as a cell cycle progression indicator. Both DCX + Ki67 + (p < 0.01) and DCX + BrdU + (p < 0.01) double-positive cells, as hallmarks of active neurogenesis, were elevated post-seizure in WT but suppressed in P2ry12 cKO mice (Fig. 14 A-C). scRNA-seq volcano plots revealed concomitant downregulation of neurogenic regulators ( Sox11 , Sox4 , and Rgs5 ) and upregulation of microglial activation markers ( CD74 , Thbs2 , and Clec4e ) in P2ry12 cKO (Fig. 14 D), suggesting glial-mediated neurogenic inhibition. UMAP-based cell cycle analysis identified neurons as the predominant proliferative population post-seizure (Fig. 15 A). The key cell cycle drivers, Top2a (DNA topoisomerase IIα) and Mki67 (proliferation marker), presented neuronal-specific expression, with DCX + cells peaking in G1 phase (Fig. 15 B). P2ry12 cKO attenuated the seizure-induced upregulation of Top2a (S phase; p < 0.01), Mki67 (M phase; p < 0.01), and DCX (p < 0.01) (Fig. 15 C, D; Supplement 7). While global cell cycle distribution remained stable, P2ry12 cKO reduced the G1 neuronal proportion from 64% in WT to 58% in P2ry12 cKO (Fig. 15 E), indicating subtle cell cycle dysregulation. CD74-TNF-α Axis Mediates P2Y12-Regulated Neurogenic Suppression in Postictal Hippocampus The mechanistic link between CD74 high P2ry12 low microglia and impaired neurogenesis post-seizures remains undefined. Given TNF-α’s role as a downstream effector of CD74 signaling in neuroinflammation 26 , we performed hippocampal immunofluorescence mapping. Quantitative analysis revealed seizure-induced elongation of both supra- and infra-pyramidal blades—structural correlates of neuronal maturation—in WT mice (p < 0.01; Fig. 16 A, B; Supplement 8). This neurogenic plasticity was abolished in P2ry12 cKO mice (supra-blade: p = 0.01 ; infra-blade: p = 0.03 ; Fig. 16 A, B). Concomitantly, CD74 + and TNF-α + cell densities surged post-seizure in WT (both p < 0.01), with further amplification in P2ry12 cKO hippocampi ( CD74 + : p = 0.01 ; TNF-α + : p < 0.01) and CA3 subfields (Fig. 16 C, D). CellChat interrogation of scRNA-seq data identified CD74 high Cluster 8 microglia as central interactors, forming putative signaling networks with endothelial cells (angiocrine factors), astrocytes (C1q/C3 complement), erythrocytes (iron homeostasis), and neurons (semaphorin/plexin axes). Discussion In this investigation, we delineated the dual regulatory role of microglia in postictal neuronal impairment and compensatory neurogenesis following epileptogenesis. Quantitative analysis revealed a transient microglial expansion peaking at 3 days post-epilepsy, followed by population normalization by 7 days post-epilepsy, as a timeframe coinciding with sustained hippocampal neurogenic activity. Through integrated single-cell transcriptomics and flow cytometric validation, we identified two reciprocally regulated microglial subsets: a diminished P2ry12 high CD74 − H2-Ab1 low subpopulation and an expanded CD74 high H2-Ab1 high P2ry12 low subpopulation, collectively designated as epilepsy-associated microglia (EPAM). Conditional ablation of microglial P2Y12 receptors precipitated paradoxical expansion of both subsets post-epileptogenesis, concomitant with significant suppression of neurogenic activity (Fig. 17 ). Mechanistically, P2Y12R deficiency upregulated CD74 expression, a MHC-II chaperone, and potentiated hippocampal TNF-α release, establishing a pro-inflammatory milieu inhibitory to neurogenesis. Our proposed model implicates neuronal ADP release as the primary activator of microglial P2Y12R signaling, which normally constrains EPAM expansion while promoting subpopulation equilibrium. Disruption of this pathway destabilizes microglial homeostasis, driving CD74-mediated neuroinflammatory cascades that impair neurogenesis in hippocampus. Traditional neuroimmunology classifies microglia into three broad subtypes (M0 resting, M1 pro-inflammatory, and M2 anti-inflammatory states) through analogy with macrophage polarization 27 . However, emerging evidence underscores fundamental distinctions between microglia and peripheral macrophages in their ontogenetic origins, niche-specific adaptations, ultrastructural organization, and disease-modulatory functions 28 . This M1/M2 dichotomy, while heuristically useful, fails to capture the context-dependent plasticity of microglia in neurological pathologies or delineate disease-etiological subsets 29 , 30 . The resolution revolution brought by scRNA-seq has enabled high-dimensional deconstruction of microglial heterogeneity 31 . In AD, temporally dynamic disease- DAM subsets were identified: DAM-1 (early-stage, phagocytic; Trem2 high ApoE high Tyrobp high ) and DAM-2 (late-stage, inflammatory; Cxcr4 high ) 31 . Similarly, aging-associated subsets marked by P2ry12 , Cx3cr1 (chemokine receptor), and Cd11b (integrin) expression demonstrate chronological functional specialization 14 . In epilepsy research, while lipid-laden reactive astrocytes and pan-glial upregulation of Spp1 (osteopontin), Trem2 , and Cd68 (lysosomal marker) have been documented 32 , 33 , the transcriptomic identity and functional hierarchy of EPAM remained unresolved. Our scRNA-seq analysis revealed two candidate EPAM subsets: Cluster 6: P2ry12 high CD74 low H2-Ab1 low and Cluster 8: CD74 high H2-Ab1 high P2ry12 low . These subsets exhibited reciprocal abundance shifts post-epileptogenesis (increased Cluster 6/decreased Cluster 8), potentially explaining the net microglial population stability despite subset reorganization. The decline in P2RY12 expression, as a homeostatic marker, aligns with this phenotypic transition, suggesting functional repolarization toward neuroinflammatory states. While inter-subset plasticity is hypothesized (e.g., Cluster 6 transforms to Cluster 8), alternative mechanisms like proliferative asymmetry require exclusion. This bimodal EPAM model provides a mechanistic scaffold to reconcile microglia’s dual roles in postictal neurodegeneration (via CD74 high / TNF-α high subsets) and neurogenesis (via P2ry12 high surveillance subsets). By mapping these state transitions, we establish a framework for targeting pathology-driving subpopulations while preserving homeostatic functions. Microglia exhibit CNS-selective overexpression of the purinergic receptor P2RY12, a Gi-coupled sensor for extracellular ADP that governs surveillance motility and homeostatic maintenance 25 , 34 . In AD, coordinated signaling through Trem2, P2RY12, and TAM receptors (Tyro3/Axl/Mer) enables microglial detection of neurodegeneration-associated molecular patterns, including apoptotic neurons, lipid droplets, and Aβ aggregates, triggering their transition into DAM 35 , 36 . Parallel mechanisms operate in epilepsy, where hippocampal neuronal apoptosis releases ADP, oxidized lipids, and myelin debris that activate microglial P2Y12R 6 , 25 , 37 . As a triad biomarker of homeostatic microglia alongside CX3CR1 and CSF-1R, P2Y12R expression inversely correlates with activation states 38 . Its functional antagonism against the pro-inflammatory P2X7 receptor mirrors CX3CR1’s role in suppressing microglial hyperactivation 28 , 39 . Our seizure model revealed P2Y12R downregulation concurrent with EPAM emergence, suggesting receptor loss destabilizes microglial quiescence—a hypothesis supported by exacerbated KA-induced neurotoxicity in P2ry12 −/− models 6 , 40 . Notably, human genetic evidence links P2RY12 enhancer variants (e.g., rs11707416) to Parkinson’s disease risk 41 , implying conserved roles in neurological disorder susceptibility. Microglia exhibits specific overexpression of P2RY12 in the brain 25 . As a G protein-coupled receptor, P2RY12 recognizes ADP and plays a critical role in regulating microglial functions 34 . In AD, microglia detect apoptotic neurons, lipid metabolites, myelin debris, and extracellular protein aggregates through receptors such as Trem2, P2RY12, and TAM (Tyro3/Axl/Mer), leading to their transformation into DAM 35 , 36 . Similarly, EP induces neuronal apoptosis in the hippocampus, accompanied by the release of ADP, lipid metabolites, and myelin debris, which activate microglial P2RY12 receptors 6 , 25 , 37 . P2RY12, along with CX3CR1 and CSF-1R, is classified as a biomarker of homeostatic microglia, with its expression downregulated in activated microglial subpopulations 38 . Upon ligand binding, CX3CR1 facilitates the transition of microglia from an activated state back to a homeostatic state 28 . P2RY12 is hypothesized to counteract the function of another purinergic receptor, P2X7R (Purinergic receptor 2X7), thereby inhibiting microglial activation and the formation of activated subpopulations, similar to the role of CX3CR1 39 . In this study, we observed alterations in the composition of microglial subpopulations following seizures, accompanied by a reduction in the average expression of P2RY12, and identified candidate EPAM. The decreased expression of P2RY12 may contribute to the instability of the resting state of microglia post-seizure 40 . In our previous work, we demonstrated that complete knockout of P2RY12 exacerbates KA-induced neuronal damage 6 . To avoid the confounding effects of KA, a conditional P2RY12 KO approach was employed in this study. A quantitative trait loci (QTLs) study revealed that mutations at the rs11707416 enhancer of P2ry12 lead to its downregulation, increasing the risk of Parkinson’s disease 41 . Based on these findings, we hypothesize that low expression of P2RY12 in the brain may also influence the risk of epileptic seizures. These insights underscore the potential role of P2RY12 in maintaining microglial homeostasis and its implications in neurological disorders. The P2Y12R-CSF1R-CX3CL1 axis orchestrates dichotomous regulation of microglial polarization 42 . In AD, DAM evolution occurs via two phases: (1) the first stage, TREM2-Independent Priming: P2RY12 downregulation initiates phenotypic shift 43 , 44 ; (2) The second stage, TREM2-Dependent Maturation: Coordinated TREM2 upregulation stabilizes DAM 45 . P2RY12 is hypothesized to regulate the initial transition to DAM in the first stage and collaborate with TREM2 in the second stage to facilitate DAM formation. Our epilepsy models recapitulate this biphasic logic. Conditional P2ry12 ablation precipitated disproportionate EPAM subset expansion—particularly Cluster 8 ( CD74 high H2-Ab1 high P2ry12 low )—while suppressing homeostatic Cluster 6 ( P2ry12 high CD74 low H2-Ab1 low ). This skewed amplification suggests P2Y12R normally constrains neuroinflammatory EPAM differentiation, with receptor deficiency permitting uncontrolled Cluster 8 dominance. Such subset reconfiguration likely drives postictal microglial dysfunction, mirroring DAM-mediated neurodegeneration in AD. The dualistic influence of microglia on neurogenesis, as context-dependent facilitation versus inhibition, reflects functional specialization across subpopulations 9 . Emerging evidence implicates microglia in neurogenic suppression through pro-inflammatory activation 9 . For instance, intracerebroventricular (I.C.V.) lipopolysaccharide (LPS) administration induces microglial hyperactivation, abolishing basal dentate gyrus (DG) neurogenesis, shown an effect reversible via microglial depletion 46 . Conversely, genetic disruption of specific receptors (e.g., P2ry13 ) enhances DG neurogenesis, suggesting tonic inhibitory regulation 47 . In epilepsy, microglial TLR9 signaling drives aberrant hippocampal neurogenesis, potentially exacerbating circuit hyperexcitability 48 . Paradoxically, microglia also exhibit neurogenic support: CX3CR1 deficiency impairs basal neurogenesis 49 , while global P2ry12 knockout suppresses both basal and epilepsy-related neurogenesis—a phenotype recapitulated by microglial ablation 6 . Notably, we identified seizure-induced proliferation of microglial "process pouches" at injured dendrites, structures attenuated by P2ry12 deletion, hinting at unresolved neuroprotective interactions 50 . This functional paradox is resolvable through a subpopulation lens. Our single-cell analysis revealed polarized expression of neurogenesis-modulating receptors (P2ry13, TLR9, Cx3cr1, P2ry12) across microglial subsets. P2ry12 ablation skewed EPAM composition toward Cluster 8 dominance ( CD74 high H2-Ab1 high P2ry12 low ), concomitant with hippocampal CD74 upregulation. As an MHC-II chaperone and macrophage migration inhibitory factor (MIF) receptor, CD74 propagates neuroinflammatory cascades via TNF-α release—a mechanism implicated in neurogenic suppression 50 – 53 . The mechanistic hierarchy was summarized as: P2Y12R loss induced EPAM subset imbalance, such increased Cluster 8; CD74 overexpression in Cluster promoted TNF-α-driven neuroinflammation; and inflammatory milieu inhibited the neurogenesis in hippocampus. This axis positions CD74 + EPAM as neurogenic gatekeepers, contrasting with P2ry12 + subsets that may sustain permissive microenvironments. Such bimodal regulation underscores the therapeutic potential of subset-specific modulation over global microglial targeting. Conclusions This study establishes EPAM as a dynamic bimodal entity regulated by P2Y12R-CD74 signaling. We demonstrated that the transcriptomic profiling of EPAM subpopulations, mechanistic link between P2Y12R signaling and CD74-mediated neuroinflammation, and subpopulation-specific explanation for microglia’s dual neurogenic roles. These findings advocate for precision therapeutics targeting EPAM subsets rather than global microglial modulation. For instance, CD74 inhibition could mitigate neuroinflammatory EPAM while preserving homeostatic subsets, as a strategy superior to broad-spectrum anti-microglial agents. Declarations Data availability Data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data for each of the graphs presented are provided in this paper and stored in files created using Microsoft® Excel for Mac (Version 16.78.3; Microsoft Corporation) and GraphPad Prism 10 (GraphPad Software Company). The single cell sequencing data generated in this study have been deposited in the SAR database under accession code 9PL6pbW21yR64480, and annotated data provided in attachment. Acknowledgements The authors would like to thank Prof. Pingyi Xu, and Dr. Longjun Wu for their assistance in the research. Funding This work was supported by the National Natural Science Foundation of China (81701254), General Project of Basic and Applied Basic Research of Guangzhou Bureau of Science and Technology (2060206), Yang-cheng Scholar Project of Guangzhou Municipal Bureau of Education (202032790), General Project of Natural Science Foundation of Guangdong Province (2021A1515011043) and Guangzhou key medical discipline grant (2021-2023). Author information Mingshu Mo and Juan Ling contributed equally to this work. Authors and Affiliations Department of Neurology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China Mingshu Mo, Lan Wang, Huishan Deng, Yilin Su, Lijian Wei, Yuting Tang Guangzhou Key Laboratory for Research and Development of Crop Germplasm Resources, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China Juan Ling Contributions M.S.M, J.L., W.L., Y.L.S, L.J.W, and Y.T.T. conceived of the experiments and developed the methods. M.S.M, J.L., and W.L. performed the experiments. J.L., W.L., Y.L.S, and M.S.M. analyzed the data. M.S.M and J.L. acquired financial support for this study. M.S.M and J.L. wrote the first draft of the manuscript. J S, L.J.W, and Y.T.T. reviewed and edited the manuscript. Corresponding authors Correspondence to Mingshu Mo. Ethics declarations Ethics approval and consent to participate All experiments were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Guangzhou Medical University (Approval No. 2020341) and reported in accordance with the ARRIVE 2.0 guidelines. Consent for publication Not applicable. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Thijs RD, Surges R, O'Brien TJ, Sander JW. Epilepsy in adults. lancet. 2019;393:689–701. Vaidya V, Siuciak J, Du F, Duman R. Hippocampal mossy fiber sprouting induced by chronic electroconvulsive seizures. Neuroscience. 1999;89:157–66. Butler CR, Westbrook GL, Schnell E. Adaptive mossy cell circuit plasticity after status epilepticus. J Neurosci. 2022;42:3025–36. Cavarsan CF, Malheiros J, Hamani C, Najm I, Covolan L. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6558062","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456649716,"identity":"21b995be-4d04-4039-bfb2-de2dd6c76a4c","order_by":0,"name":"Mingshu Mo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYJCCA0CcwMDMfPjBBwMbOxK0sLOlGc4oSEsm2qYEBn4eA2meD4cYGwgp1W3vTjxc8Ksuz+Awg4GxjcEBZgb2w0c34NNidubshsMz+w4XA7UkPM4xuMPHwJOWdgOvlhu5Gw7z9hxI3HCY4YBxjsEzZgYJHjNitNQBtTA2SFsYAEmitPD8YAZqYWaQZiBKC8gvvA2HE2ceZmMz7DFIS2Yj6JfjvZs/8/ypS+w7f/7zgx9/bOz42Q8fw6sFDBjbkDhsBJWDwR/ilI2CUTAKRsEIBQCRPlR2gzTGzwAAAABJRU5ErkJggg==","orcid":"","institution":"First Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Mingshu","middleName":"","lastName":"Mo","suffix":""},{"id":456649717,"identity":"ea379617-94fe-419a-a542-a42ecd05f889","order_by":1,"name":"Juan Ling","email":"","orcid":"","institution":"Zhongkai University of Agriculture and Engineering","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Ling","suffix":""},{"id":456649719,"identity":"eb64fd66-8135-48b0-a8d9-9643983ea87e","order_by":2,"name":"Lan Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lan","middleName":"","lastName":"Wang","suffix":""},{"id":456649722,"identity":"192063bd-697d-4da6-856a-3bf732babc39","order_by":3,"name":"Huishan Deng","email":"","orcid":"","institution":"First Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huishan","middleName":"","lastName":"Deng","suffix":""},{"id":456649723,"identity":"7bdc6463-1d13-4d0a-bf69-f5168df75c8e","order_by":4,"name":"Yilin Su","email":"","orcid":"","institution":"First Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yilin","middleName":"","lastName":"Su","suffix":""},{"id":456649724,"identity":"4f22c96a-2c5e-4837-92b2-023edc03e9e0","order_by":5,"name":"Lijian Wei","email":"","orcid":"","institution":"First Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lijian","middleName":"","lastName":"Wei","suffix":""},{"id":456649725,"identity":"916e1072-a9c2-4477-88bf-69e1d66a9aaf","order_by":6,"name":"Yuting Tang","email":"","orcid":"","institution":"First Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2025-04-29 15:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6558062/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6558062/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82923952,"identity":"35c7e045-66f7-4dcc-9238-567015919d5c","added_by":"auto","created_at":"2025-05-16 18:48:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":599109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEpilepsy induces hippocampal neuronal damage. \u003c/strong\u003e(A) Seizure severity scores over 120 minutes post-KA injection (N = 7). (B) Fluoro-Jade B (FJB) staining of hippocampal neuronal impairment; quantitative FJB\u003csup\u003e+\u003c/sup\u003e cell counts per section (E; N = 7). (C) EEG traces from frontal, parietal, and occipital cortices post-KA. (D) Mean EEG amplitudes across cortical regions (N = 4). Data are presented as mean ± SD. *P＜0.05, **P＜0.01, by two-tailed Student’s test (D), by 2-way ANOVA with Tukey’s multiple comparison test (E).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/e3fc185c842c099ac11a497a.png"},{"id":82923954,"identity":"171fe9f2-db61-45cb-b836-bb72e8826cce","added_by":"auto","created_at":"2025-05-16 18:48:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1428856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransient microglial proliferation near injured neurons in CA3 post-seizure. (\u003c/strong\u003eA) Representative images of Iba1\u003csup\u003e+\u003c/sup\u003e microglia and BrdU\u003csup\u003e+\u003c/sup\u003e proliferating cells in hippocampus. (B) Co-staining of Iba1\u003csup\u003e+\u003c/sup\u003e microglia, NeuN\u003csup\u003e+\u003c/sup\u003e neurons, GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes, and BrdU\u003csup\u003e+\u003c/sup\u003e/Ki67\u003csup\u003e+\u003c/sup\u003e proliferating cells in CA3. (C) Quantification of hippocampal Iba1\u003csup\u003e+\u003c/sup\u003e cells (N = 6). (D) Iba1\u003csup\u003e+\u003c/sup\u003eBrdU\u003csup\u003e+\u003c/sup\u003e cells in CA3 (N = 6). (E) Iba1\u003csup\u003e+\u003c/sup\u003eKi67\u003csup\u003e+\u003c/sup\u003e cells in CA3 (N = 6). Data are presented as mean ± SD. *P＜0.05, **P＜0.01, by 2-way ANOVA with Tukey’s multiple comparison test (C, D, E).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/9a0b10484e24bcb0de0ba549.png"},{"id":82924384,"identity":"9fded088-5655-4a4e-971e-f301a914a1e8","added_by":"auto","created_at":"2025-05-16 18:56:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1142196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiscrepancy between P2ry12 mRNA levels and P2ry12\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cell density in postictal hippocampus. \u003c/strong\u003e(A) Venn diagram of differentially expressed genes (DEGs) at 3/7 days post-seizure (N = 3). (B) 3D volcano plot and (C) heatmap of temporal DEG patterns. (D) Time-resolved expression trajectories of microglial genes. (E) KEGG pathway enrichment. (F) Clustered microglial gene modules. (G) P2ry12 mRNA levels by RNA-seq (N = 3)/qRT-PCR (N = 4), Data are presented as mean ± S.E.M. (H) P2ry12\u003csup\u003e+\u003c/sup\u003e cell distribution in CA3. (I) Quantification of CA3 P2ry12\u003csup\u003e+\u003c/sup\u003e cells (N = 6). Data are presented as mean ± SD. *P＜0.05, **P＜0.01, by 2-way ANOVA with Tukey’s multiple comparison test (G, I).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/8b85108d0923e3420c4e6469.png"},{"id":82924385,"identity":"bd9973a7-9c29-4293-94a7-a63aa0708b88","added_by":"auto","created_at":"2025-05-16 18:56:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":682523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglial population restoration at 7 days post-seizure. \u003c/strong\u003e(A) UMAP of 30 brain cell clusters. (B) Annotation of major lineages. (C) Aif1 (Iba1) expression in microglia. (D) Kernel density plots of lineage-specific markers. (E) UMAP comparison of seizure vs. control. (F) Volcano plots of DEGs across lineages. (G) Compositional ratios of 15 cell types. (H) Microglial proportion analysis. (I) Iba1 expression validation by RNA-seq (N = 3)/qRT-PCR (N = 4). Data are presented as mean ±S.E.M. *P＜0.05, **P＜0.01, by two-tailed Student’s test (H), by 2-way ANOVA with Tukey’s multiple comparison test (I).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/1c078eb15d5692a9768b41b7.png"},{"id":82923958,"identity":"4ebb6c04-0543-47d7-a64f-ebe249604e53","added_by":"auto","created_at":"2025-05-16 18:48:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":595764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDivergent dynamics of P2ry12-expressing microglial subsets post-seizure. \u003c/strong\u003e(A) UMAP of microglial clusters 6/8/20. (B-D) Volcano plots of subset-specific DEGs. (E) Dot plot of subset markers. (F) Proportional changes in microglial clusters. (G) Subset-enriched KEGG pathways. Data are presented as mean ± SEM. *P＜0.05, **P＜0.01, by two-tailed Student’s test (F).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/d13dec123922bda1a0489067.png"},{"id":82924528,"identity":"9eec5918-d071-4576-83bd-78ff2523476e","added_by":"auto","created_at":"2025-05-16 19:04:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLate-stage P2ry12 downregulation in hippocampal microglia. \u003c/strong\u003e(A) Volcano plot of microglial DEGs, highlighting P2ry12. (B) UMAP of P2ry12 expression. (C) Log(avg.expr) scatter plots. (D) P2ry12 vs. Cx3cr1 co-expression. (E) Violin plot of P2ry12 expression in microglia.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/f2e5e0839aaa48ca267dd5fa.png"},{"id":82924742,"identity":"db95e7a3-1d16-45d1-9de5-4ee26f5613cc","added_by":"auto","created_at":"2025-05-16 19:12:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":421615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConditional P2ry12 knockout minimally affects acute seizure pathology. \u003c/strong\u003e(A) Experimental timeline for postictal P2ry12 deletion. (B) Parietal EEG traces and mean amplitudes (F; N = 5). (C) FJB staining of neuronal injury; quantification (H; N = 7). (D) Seizure scores (N = 7). (E) EEG amplitude comparison (N = 5). (F) FJB\u003csup\u003e+\u003c/sup\u003e cell counts (N = 7). Data are presented as mean ± SD. *P＜0.05, **P＜0.01, by 2-way ANOVA with Tukey’s multiple comparison test (E), by two-tailed Student’s test (F).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/22a93fa061e06d99bc61305f.png"},{"id":82923963,"identity":"4a733b28-c1bf-405f-ac1b-cd6a58518e22","added_by":"auto","created_at":"2025-05-16 18:48:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":763400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConditional P2ry12 knockout alters brain cell composition. \u003c/strong\u003e(A) UMAP of P2ry12 expression post-cKO. (B) Annotated cell distributions. (C) Microglial cluster dynamics. (D) Normalized P2ry12 expression. (E) Cell type ratios. (F) Proportional changes in clusters 6/8/20. (G) Subset marker dot plots. (H) Cluster-specific DEGs. Data are presented as mean ± SEM. *P＜0.05, **P＜0.01, by two-tailed Student’s test (F).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/8d3efc95afbdd73dca230017.png"},{"id":82923967,"identity":"99389a51-daaa-4aa9-ad7e-04e7fe5fcb99","added_by":"auto","created_at":"2025-05-16 18:48:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":655775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2ry12 deficiency drives microglial expansion post-seizure. \u003c/strong\u003e(A) UMAP of scRNA-seq data. (B) Annotated cell populations. (C) Cell type ratios in cKO mice. (D) Volcano plots of pan-cellular DEGs. (E) Microglial proportion changes. Data are presented as mean ± SEM. *P＜0.05, **P＜0.01, by 2-way ANOVA with Tukey’s multiple comparison test (E).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/7cb438b83e680f79de939f69.png"},{"id":82924740,"identity":"c3182457-6f6a-429d-b51f-341b70762b71","added_by":"auto","created_at":"2025-05-16 19:12:44","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":75376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2ry12 ablation enhances microglial proliferation in CA3.\u003c/strong\u003e (A) Iba1\u003csup\u003e+\u003c/sup\u003eP2ry12\u003csup\u003e+\u003c/sup\u003e cells in CA3. (B) Iba1\u003csup\u003e+\u003c/sup\u003eBrdU\u003csup\u003e+\u003c/sup\u003e proliferating microglia. (C) Quantification of Iba1\u003csup\u003e+\u003c/sup\u003eBrdU\u003csup\u003e+\u003c/sup\u003e cells (N = 5). (D) Iba1\u003csup\u003e+\u003c/sup\u003eP2ry12\u003csup\u003e+\u003c/sup\u003e cell counts (N = 5). Data are presented as mean ± SD. *P＜0.05, **P＜0.01, by 2-way ANOVA with Tukey’s multiple comparison test (C, D).\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/afcaf6b641ba85a8afa0920c.png"},{"id":82924531,"identity":"d31d4348-fc87-4ee4-bd08-f1f6ddd921db","added_by":"auto","created_at":"2025-05-16 19:04:44","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":323487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2ry12 deficiency amplifies CD74highH2-Ab1high microglial subsets. \u003c/strong\u003e(A) 2D UMAP of clusters 6/8/20 in cKO mice; 3D projections (C). (B) Proportional changes. (D) Temporal subset dynamics. Data are presented as mean ± SEM. *P＜0.05, **P＜0.01, by 2-way ANOVA with Tukey’s multiple comparison test (B).\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/de10251f69dfccf9523fd25d.png"},{"id":82924534,"identity":"6cd5ed75-3225-4289-a797-89ca40f06493","added_by":"auto","created_at":"2025-05-16 19:04:44","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":565805,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD74 upregulation in P2ry12-deficient microglia.\u003c/strong\u003e (A) Dot plot of subset markers. (B-D) Subset-specific KEGG pathways. (E) Volcano plots of cluster DEGs. (F) P2ry12 vs. CD74 co-expression.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/76a005f219c0bdf74b6906e4.png"},{"id":82923973,"identity":"30953df9-bb6f-49e3-ae0e-c8ffdd7fe03f","added_by":"auto","created_at":"2025-05-16 18:48:44","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":530045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubclustering identifies \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCD74\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003ehigh\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eH2-Eb1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003ehigh\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e microglia in cKO mice.\u003c/strong\u003e (A) UMAP of 13 microglial subclusters. (B) Subcluster annotation. (C) Combined microglial UMAP. (D) Marker gene heatmap. (E) CD74/H2-Eb1 expression. (F) Subcluster markers. (H) Subcluster proportions. (I) qRT-PCR validation (N = 4). Data are presented as mean ±S.E.M. *P＜0.05, **P＜0.01, by 2-way ANOVA with Tukey’s multiple comparison test (H, I).\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/7551319f5b460192c088be54.png"},{"id":82924831,"identity":"0b6e0c6a-249e-4ed9-a13d-90ad38d7a5aa","added_by":"auto","created_at":"2025-05-16 19:20:44","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":651446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2ry12 deficiency suppresses seizure-induced neurogenesis. \u003c/strong\u003e(A) Cell cycle phase UMAP. (B) Top2a/Mki67/DCX expression. (C) Violin plots of gene expression. (D) Dot plot validation. (E) Neuronal cell cycle shifts.\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/ec8c627f9f08741e2a589555.png"},{"id":82924392,"identity":"95a7f174-6ba2-4e60-914b-7287b3d09c6e","added_by":"auto","created_at":"2025-05-16 18:56:44","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":834762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2ry12 dysfunction inhibits dentate gyrus neurogenesis. \u003c/strong\u003e(A) DCX\u003csup\u003e+\u003c/sup\u003eBrdU\u003csup\u003e+\u003c/sup\u003e cells in DG. (B) DCX\u003csup\u003e+\u003c/sup\u003eKi67\u003csup\u003e+\u003c/sup\u003e cells in DG. (C) Quantification (N = 5). (D) scRNA-seq volcano plot. Data are presented as mean ± SD. *P＜0.05, **P＜0.01, by 2-way ANOVA with Tukey’s multiple comparison test (C).\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/5b4a64a94b5eb3bf891c566f.png"},{"id":82924389,"identity":"c983d997-243f-4a5f-a839-184bcb874de1","added_by":"auto","created_at":"2025-05-16 18:56:44","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":923302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD74-TNF-α axis mediates P2ry12-regulated neurogenic suppression.\u003c/strong\u003e (A) CD74/DCX co-staining in hippocampus; suprapyramidal/infrapyramidal blade quantification (B; N = 5, 100μm). (C) CD74/TNF-αco-staining in in CA3 (D; N = 5). (E) CellChat-predicted interactions between microglial clusters and other lineages. Data are presented as mean ± SD. *P＜0.05, **P＜0.01, by 2-way ANOVA with Tukey’s multiple comparison test (B, D).\u003c/p\u003e","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/546425e7c169fc98358c8179.png"},{"id":82923982,"identity":"c6e8f66c-14da-4eac-8e4a-5dc1cc8dad8c","added_by":"auto","created_at":"2025-05-16 18:48:44","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":345099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulatory Model of Microglial P2Y12 Receptor Signaling in Epilepsy-Associated Neurogenesis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/57dda7ab6f243f9fc66e6408.png"},{"id":86045733,"identity":"f4ef21db-3f7c-4b45-ba79-d45ecd7a4ec2","added_by":"auto","created_at":"2025-07-04 20:16:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12324933,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/abff766e-c573-4eec-bd19-4497006c61f0.pdf"},{"id":82924530,"identity":"cffc9bc6-fb56-4e9b-a34e-630ee4bc8fb7","added_by":"auto","created_at":"2025-05-16 19:04:44","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1772622,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-6558062/v1/5feabe3f66206fc0fb91da14.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Microglial P2Y12 Receptor Signaling Governs Epilepsy-Associated Neurogenesis via Bidirectional Regulation of Distinct Microglial Subpopulations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpilepsy (EP), a prevalent neurological disorder characterized by recurrent seizures, manifests electrophysiologically through neuronal synchronization and hyperexcitability \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Postictal neurogenic activity in the dentate gyrus (DG) and aberrant mossy fiber sprouting into the CA3 region are hallmark features observed in both clinical populations and experimental models \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These structural rearrangements contribute to maladaptive circuitry formation, a critical driver of epileptogenesis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Deciphering the molecular underpinnings of these processes may advance our understanding of adult neurogenesis and catalyze the development of targeted antiepileptogenic therapies \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs central nervous system (CNS) immune sentinels, microglia regulate neural homeostasis through multifaceted roles, including inflammatory mediator secretion, neurotrophic factor production, synaptic pruning, and microenvironmental modulation \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Their bidirectional influence on neurogenesis and axonal remodeling positions them as critical arbiters of epileptic pathophysiology \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. While microglial involvement in epilepsy is undisputed, their functional duality remains contentious \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Early studies emphasized their detrimental role via pro-inflammatory cytokine release (e.g., TNF-α, IFN-γ, IL-1β), exacerbating neuroinflammation and neuronal injury \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Conversely, emerging evidence highlights their neuroprotective capacity through neurotrophic factor synthesis (NGF, NT3, FGF), which supports neuronal survival and neurogenic niches \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This functional dichotomy extends to microglia-neuron crosstalk, where they dynamically sculpt synaptic architecture, suppress hyperexcitability, and eliminate damaged neurons\u0026mdash;processes that may paradoxically mitigate or potentiate epileptogenesis \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eResolving this paradox necessitates interrogation of microglial heterogeneity. Transcriptomic profiling has identified four principal microglial subsets in homeostasis: origin-, proliferation-, immune response-, and neuronally enriched populations \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Disease contexts reveal specialized subpopulations, such as Alzheimer\u0026rsquo;s-associated DAM (\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e\u003cem\u003eTrem2\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e\u003cem\u003eApoE\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e\u003cem\u003eTyrobp\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e), which progress from phagocytic DAM-1 (early Aβ clearance) to pro-inflammatory DAM-2 (late-stage pathology) \u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Analogous subsets\u0026mdash;including neurodegenerative microglia (MGnD) in ALS/MSA\u0026mdash;highlight context-dependent polarization \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Despite these advances, epilepsy-associated microglia (EPAM) remain poorly characterized, obscuring their role in seizure-related neurogenesis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we define EPAM subpopulations and their functional trajectory during epileptogenesis. Using multimodal sequencing and genetic perturbation, we demonstrate that P2Y12 receptor signaling governs EPAM dynamics, with disruption impairing compensatory neurogenesis. These findings resolve longstanding controversies in microglial duality and establish EPAM as therapeutic targets for precision epilepsy interventions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All experimental procedures were conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Guangzhou Medical University (Approval No. 2020341). Male C57BL/6J mice (8\u0026ndash;12 weeks old) were housed under standard conditions (12-hour light/dark cycle, 22\u0026deg;C, 50% humidity). CX3CR1CreER\u003csup\u003e+/+\u003c/sup\u003e mice and P2ry12\u003csup\u003efl/fl\u003c/sup\u003e mice (generated via CRISPR/Cas9 as described previously \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e) were obtained from Cyagen Biosciences (Guangzhou, China). To generate microglia-specific P2ry12 knockout mice (CX3CR1CreER\u003csup\u003e/+\u003c/sup\u003e:P2ry12\u003csup\u003efl/fl\u003c/sup\u003e), heterozygous crosses were performed. The P2ry12floxed allele was engineered by flanking exons 4\u0026ndash;3\u0026prime;UTR with loxP sites via homologous recombination. Single-guide RNAs and Cas9 mRNA were synthesized in vitro using T7 RNA polymerase (NEB). A donor vector containing 1 kb homology arms was co-injected with sgRNAs/Cas9 mRNA into fertilized zygotes. Founder (F0) mice were genotyped by PCR (Supplement 1), Sanger sequencing, and Southern blot. For P2ry12 deletion, adult mice received intraperitoneal (IP) tamoxifen (20 mg/mL in corn oil; Sigma T5648) at 75 mg/kg/dose \u0026times;4 (48-hour intervals) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Controls received vehicle-only injections. Littermates were randomized across experimental cohorts to minimize genetic bias.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKainic Acid-induced epilepsy model\u003c/h3\u003e\n\u003cp\u003eKainic acid (KA; Tocris 0222) was dissolved in artificial cerebrospinal fluid (ACSF: 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose; pH 7.4) to 0.032 mg/mL. Mice underwent stereotaxic surgery (David Kopf Instruments) under isoflurane anesthesia (2% in O2) for guide cannula (8 mm length; PlasticsOne) implantation into the right lateral ventricle (coordinates: \u0026minus;0.2 mm posterior to bregma, +\u0026thinsp;0.9 mm lateral, \u0026minus;\u0026thinsp;2.3 mm ventral). After 4-week recovery, 5 \u0026micro;L KA was infused over 5 minutes via microsyringe (Hamilton). Seizure severity was scored over 120 minutes post-injection using a modified Racine scale\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The seizure scoring criteria were as follows: score 1, freezing behavior; score 2, rigid posture with raised tail; score 3, continuous head bobbing and forepaw shaking; score 4, rearing, falling, and jumping; score 5, continuous level 4 seizures; score 6, loss of posture and generalized convulsive activity; and score 7, death.\u003c/p\u003e\n\u003ch3\u003eElectroencephalographic (EEG) recording\u003c/h3\u003e\n\u003cp\u003eMice were anesthetized via intraperitoneal (IP) injection of pentobarbital (50 mg/kg) and secured in a stereotaxic frame (Model 940, David Kopf Instruments, CA, USA). After shaving the cranial fur, burr holes (0.7 mm diameter; InterFocus Ltd, Cambridge, UK) were drilled at stereotaxic coordinates relative to bregma: frontal electrode at +\u0026thinsp;2.0 mm anteroposterior (AP), \u0026plusmn;\u0026thinsp;2.0 mm mediolateral (ML); parietal electrode at +\u0026thinsp;2.0 mm AP, \u0026plusmn;\u0026thinsp;4.0 mm ML; occipital electrode at +\u0026thinsp;2.0 mm AP, \u0026plusmn;\u0026thinsp;2.5 mm ML. Stainless steel screw electrodes (0.8 mm diameter, 10\u0026ndash;50 kΩ impedance at 1 kHz; Nitto Seiko, Japan) were implanted and affixed with dental cement (Vertex-Dental, Netherlands). A subcutaneous reference electrode was positioned over the nasal bone. A 6-pin surface mount connector (8415-SM, Pinnacle Technology, KS, USA) was integrated for signal transmission. Postoperative care included: thermal support (37\u0026deg;C heating pad) and hydration were maintained intraoperatively; prophylactic ampicillin (100 mg/kg, SC) and analgesic meloxicam (1 mg/kg, SC) were administered postsurgery; mice recovered for \u0026ge;\u0026thinsp;14 days before EEG recordings. EEG signals were amplified (Grass QP511, RI, USA), digitized at 128 Hz (Digidata 1440A, Molecular Devices, CA, USA), and recorded using Pinnacle Sirenia Acquisition Software (v2.5.0). Mean amplitude analysis focused on a 440-second epoch post-kainic acid (KA) injection, with cross-group comparisons performed using Clampfit 10.7 (Molecular Devices).\u003c/p\u003e\n\u003ch3\u003eTissue processing and histological staining\u003c/h3\u003e\n\u003cp\u003eMice were deeply anesthetized with 5% isoflurane and transcardially perfused with ice-cold phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA; Sigma-Aldrich). Brains were post-fixed in 4% PFA for 6 hours at 4\u0026deg;C, cryoprotected in 30% sucrose/PBS overnight, and sectioned coronally at 14 \u0026micro;m thickness using a cryostat (CM3050S, Leica Microsystems).\u003c/p\u003e \u003cp\u003eFor Fluoro-Jade B (FJB) staining, Sections were sequentially incubatied in 1% sodium hydroxide dissolved in 80% ethanol for 5 minutes, followed by 70% ethanol for 2 minutes, rinsed with distilled water, treated with 0.06% potassium permanganate for 10 minutes and rinsed again with distilled water, and then immersed in 0.01% FJB (Histo-Chem, Jefferson, AR, U.S.A.) solution containing 0.1% acetic acid for 20 minutes. After PBS rinses, slides were air-dried and coverslipped with DPX mounting medium.\u003c/p\u003e \u003cp\u003eFor immunofluorescence staining, sections were permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) and blocked in 5% normal goat serum/PBS. The following primary antibodies were applied overnight at 4\u0026deg;C: rabbit anti-Iba1 (1:1000, Wako Chemicals, catalog #019-19741), rabbit anti-Ki67 (1:500, Abcam, catalog #16667), goat anti-doublecortin (DCX, 1:500, Sigma-Aldrich, catalog #SAB2501666), rabbit anti-CD74 (1:500, Sigma-Aldrich, catalog # 3352229), and rabbit anti-GFAP (1:200, Sigma-Aldrich, catalog #ZRB2383). After TBST washes, sections were incubated with AlexaFluor-488/594-conjugated secondary antibodies (1:500, Invitrogen) for 40 min at 25\u0026deg;C and mounted with DAPI Fluoromount-G\u0026trade; (SouthernBiotech).\u003c/p\u003e \u003cp\u003eFor BrdU labeling and detection, mice received daily BrdU (100 mg/kg, i.p.; Sigma) for 3 days post-KA \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Sections were incubated in 2\u0026times; SSC/50% formamide for 2 hours at 65\u0026deg;C, 2 N HCl at 37\u0026deg;C for 1 hour, and 0.05 M borate buffer (pH 8.5) for 10 minutes. After blocking with 10% BSA, sections were co-stained with mouse anti-BrdU (1:500, Sigma #B8434) and other primary antibodies as described above.\u003c/p\u003e\n\u003ch3\u003eQuantitative morphometric analysis\u003c/h3\u003e\n\u003cp\u003eFluorescent images were captured using an EVOS FL Auto 2.0 microscope (40\u0026times; objective; Life Technologies) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Six evenly spaced sections (240 \u0026micro;m intervals) per brain were analyzed. Iba1\u003csup\u003e+\u003c/sup\u003e/BrdU\u003csup\u003e+\u003c/sup\u003e/DCX\u003csup\u003e+\u003c/sup\u003e/Ki67\u003csup\u003e+\u003c/sup\u003e cells were manually counted across hippocampal subfields (CA1, CA3, DG) using ImageJ FIJI (v2.3.0). Data represent means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from \u0026ge;\u0026thinsp;3 animals per group. DCX\u003csup\u003e+\u003c/sup\u003e projections were traced in ImageJ to measure the area above the granular cell layer as a suprapyramidal blade (SMF); and the area below the granular cell layer as an infrapyramidal blade (IMF). Volumes were calculated as follows: Volume=\u0026sum;(Area\u0026times;14 \u003cem\u003e\u0026micro;m\u003c/em\u003e)\u0026times;6 (section interval factor).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA Sequencing and qRT-PCR Analysis\u003c/h2\u003e \u003cp\u003eMice were transcardially perfused with ice-cold PBS under deep anesthesia. Whole brains were rapidly dissected, snap-frozen in liquid nitrogen, and homogenized in TRIzol\u0026reg; Reagent (15596026, Invitrogen) for total RNA isolation via the guanidinium thiocyanate-phenol-chloroform method. RNA purity (A260/A280\u0026thinsp;\u0026gt;\u0026thinsp;1.8) and integrity (RIN\u0026thinsp;\u0026ge;\u0026thinsp;8.0) were verified using a NanoDrop OneC spectrophotometer (Thermo Scientific) and Experion\u0026trade; Automated Electrophoresis System (Bio-Rad). Ribosomal RNA was depleted from 2 \u0026micro;g total RNA using the Ribo-Zero\u0026trade; Gold rRNA Removal Kit (MRZG12324, Illumina). Libraries were constructed with the NEBNext\u0026reg; Ultra\u0026trade; II Directional RNA Library Prep Kit (E7760S, NEB) following manufacturer protocols: (1) Fragmentation: RNA shearing to 300\u0026ndash;400 bp in Illumina fragmentation buffer (94\u0026deg;C, 8 min); (2) cDNA synthesis: first-strand synthesis with random hexamers; second-strand synthesis incorporating dUTP; (3) library construction: end repair, adenylation, and adapter ligation using NEBNext\u0026reg; Multiplex Oligos; (4) enzyme treatment: degradation of uracil-containing second strands (37\u0026deg;C, 15 min), size-selected libraries (400\u0026ndash;500 bp) were PCR-amplified (15 cycles) and quantified via Agilent 2100 Bioanalyzer, and paired-end sequencing (2\u0026times;150 bp) was performed on Illumina NovaSeq 6000 (Shanghai Personal Biotechnology).\u003c/p\u003e \u003cp\u003eFor bioinformatic analysis, raw reads were aligned to the GRCm38 mouse genome using HISAT2 (v2.2.1). Gene expression was quantified as FPKM (fragments per kilobase per million) with Cufflinks (v2.2.1). Differentially expressed genes (DEGs) were defined by |fold change| \u0026gt;1.5 and Benjamini-Hochberg adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. In qRT-PCR validation, primers for \u003cem\u003eP2ry12\u003c/em\u003e (NM_027571.4), \u003cem\u003eAif1\u003c/em\u003e (NM_009797.3), \u003cem\u003eCD74\u003c/em\u003e (NM_010545.3), \u003cem\u003eH2-Eb1\u003c/em\u003e (NM_207105.2), and \u003cem\u003eTnf\u003c/em\u003e (NM_013693.3) were designed using Oligo 7.6 (Molecular Biology Insights) and synthesized by TIANGEN Biotech (Beijing, China; sequences in Supplement 2). The reactions (25 \u0026micro;L) contained: 6.25 \u0026micro;L of cDNA; 12.5 \u0026micro;L of 2\u0026times; TaqMan\u0026trade; PreAmp Master Mix (4440043, Applied Biosystems); 6.25 \u0026micro;L of 0.2\u0026times; pooled assay mix; 5 pmol each primer. Amplification was performed on a QuantStudio\u0026trade; 6 Flex System (Applied Biosystems) at 95\u0026deg;C for 10 min for initial denaturation, followed by 40 cycles of 95\u0026deg;C/15 s, 65\u0026deg;C/30 s, and 72\u0026deg;C/30 s. \u003cem\u003eU6\u003c/em\u003e snRNA (NR_003280.1) served as the endogenous control. Relative expression was calculated via the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method: ΔΔCT = (CT\u003csub\u003etarget\u003c/sub\u003e \u0026ndash; CT\u003csub\u003eGAPDH\u003c/sub\u003e) \u003csub\u003eexperimental\u003c/sub\u003e \u0026minus; (CT\u003csub\u003etarget\u003c/sub\u003e \u0026minus; CT\u003csub\u003eGAPDH\u003c/sub\u003e) \u003csub\u003econtrol\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSingle-cell RNA sequencing and bioinformatics analysis\u003c/h3\u003e\n\u003cp\u003eMice were transcardially perfused with ice-cold PBS under deep anesthesia. Brains were rapidly dissected, mechanically dissociated, and processed for single-cell RNA sequencing (scRNA-seq) using the BD Rhapsody\u0026trade; Whole Transcriptome Analysis (WTA) Pipeline (v1.8; BD Biosciences). Single-cell suspensions were captured on a BD Rhapsody\u0026trade; Cartridge, and cDNA libraries were constructed following manufacturer protocols (Shanghai Genechem Co., Ltd., China). Sequencing was performed on an Illumina NovaSeq 6000 platform (2\u0026times;150 bp). The raw FASTQ files were aligned to the GRCm38 mouse reference genome (Ensembl v103) using the BD Rhapsody\u0026trade; Analysis Pipeline. A unique molecular identifier (UMI) count matrix was generated and imported into Scanpy (v1.8) for downstream analysis \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Low-quality cells were filtered using thresholds: UMI counts\u0026thinsp;\u0026le;\u0026thinsp;100,000; detected genes\u0026thinsp;\u0026le;\u0026thinsp;8,000; and mitochondrial gene content\u0026thinsp;\u0026gt;\u0026thinsp;10%. After quality control, 44,810 high-quality cells were retained. Counts were normalized via total expression scaling (factor\u0026thinsp;=\u0026thinsp;10,000) and log-transformed (log1p) using pp.normalize_total and pp.log1p in Scanpy. For dimensionality reduction and clustering, highly variable genes were identified using the repored method \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, implemented via pp.highly_variable_genes. Principal component analysis (PCA; 50 components) was performed (tl.pca), followed by graph-based clustering (pp.neighbors, resolution\u0026thinsp;=\u0026thinsp;0.5). The cell populations were visualized in 2D/3D UMAP space (tl.umap). For marker gene identification and functional enrichment, cluster-specific marker genes were identified using Seurat\u0026rsquo;s FindAllMarkers (Wilcoxon rank-sum test; adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, |log2(fold change)| \u0026gt;1) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The enriched pathways were analyzed via g: Profiler2 (v0.2.1) with hypergeometric testing (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) across Gene Ontology (GO), KEGG, and Reactome databases \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For validation and Cell-Cell Communication Analysis, Volcano plots (ggplot2 v3.4.0), heatmaps (EnhancedVolcano v1.14.0), and cell-cell interaction networks (CellChat v1.6.0) were generated in R Studio (v4.2.2).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe sample sizes are detailed in figure legends. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (normal distribution) or median\u0026thinsp;\u0026plusmn;\u0026thinsp;IQR (non-normal distribution). Between-group comparisons used: Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test for two groups; One-way ANOVA with Tukey\u0026rsquo;s post hoc for more than 3 groups; and Wilcoxon rank-sum test for non-parametric data. Analyses were performed in SPSS (v19.0), Prism 9 (v9.0.2), and R Studio. Significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of Microglial Activation and Neurogenic Responses in KA-Induced Epileptogenesis\u003c/h2\u003e \u003cp\u003eTo delineate microglial dynamics during epileptogenesis, we established a kainic acid (KA)-induced seizure model validated through multimodal assessment. Behavioral seizure scoring confirmed progressive ictogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), while FJB histochemistry revealed significant hippocampal neurodegeneration at 3 days post-seizure (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 7 days post-seizure (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) versus controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, E). Electrocorticographic (EEG) analysis demonstrated sustained epileptiform activity post-KA, with mean amplitude increases in frontal (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), parietal (*p\u0026thinsp;=\u0026thinsp;0.01), and occipital (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) cortices (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNeurogenic remodeling was quantified via BrdU pulse-chase labeling. Proliferative BrdU\u003csup\u003e+\u003c/sup\u003e cells localized predominantly to CA3 subfields at 3 and 7 days post-seizure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), paralleled by microgliosis (Iba1\u003csup\u003e+\u003c/sup\u003e cell density: **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Co-staining revealed selective expansion of proliferating microglial subsets in CA3:Iba1\u003csup\u003e+\u003c/sup\u003eBrdU\u003csup\u003e+\u003c/sup\u003e (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); Iba1\u003csup\u003e+\u003c/sup\u003eKi67\u003csup\u003e+\u003c/sup\u003e (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). No significant changes occurred in NeuN\u003csup\u003e+\u003c/sup\u003eBrdU\u003csup\u003e+\u003c/sup\u003e (neuronal) or GFAP\u003csup\u003e+\u003c/sup\u003eBrdU\u003csup\u003e+\u003c/sup\u003e (astroglial) populations, indicating microglia-specific proliferative engagement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRNA-seq analysis of whole-brain lysates identified 1,057 differentially expressed genes (DEGs) at 3 days post-seizure, with 89 persistent changes at 7 days post-seizure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Three-dimensional volcano plots and hierarchical clustering highlighted biphasic expression trajectories that peaking at 3 days post-seizure before partial normalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Microglial signature genes (\u003cem\u003eC1qb\u003c/em\u003e, \u003cem\u003eC1qc\u003c/em\u003e, \u003cem\u003eC3\u003c/em\u003e, \u003cem\u003eCx3cr1\u003c/em\u003e, \u003cem\u003eH2-DMb1\u003c/em\u003e) followed this temporal pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). KEGG pathway analysis implicated stage-specific mechanisms: TNF signaling (neuroinflammation; FDR\u0026thinsp;=\u0026thinsp;1.2\u0026times;10⁻⁵) at 3 days post-seizure, Chemokine signaling (microglial migration; FDR\u0026thinsp;=\u0026thinsp;3.8\u0026times;10⁻⁴) at 7 days post-seizure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Unsupervised clustering partitioned microglial DEGs into three functional modules: Cluster 1: Extracellular matrix regulators (\u003cem\u003eAdamts1\u003c/em\u003e, \u003cem\u003eAif1\u003c/em\u003e); Cluster 2: Immunomodulators (\u003cem\u003eCx3cr1\u003c/em\u003e, \u003cem\u003eCD74\u003c/em\u003e); Cluster 3: MHC-II complex (\u003cem\u003eH2-Eb1\u003c/em\u003e) and calcium-binding proteins (\u003cem\u003eS100a9\u003c/em\u003e, \u003cem\u003eS100a8\u003c/em\u003e), and others (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Notably, P2ry12, as a genetic risk factor for seizures \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, showed stable transcript levels at both timepoints that was confirmed by qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), despite upregulation of P2RY12\u003csup\u003e+\u003c/sup\u003e microglia in CA3 at 3 and 7 days post-seizure in immunohistochemical staining (both **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH,I). This discordance seems to suggest post-transcriptional regulation of P2RY12 during epileptogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSingle-Cell Resolution Reveals Seizure-Induced Microglial Reconfiguration despite Stable P2ry12 Transcript Levels\u003c/h2\u003e \u003cp\u003eTo reconcile the paradox between unchanged whole-brain \u003cem\u003eP2ry12\u003c/em\u003e mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) and CA3-specific P2RY12\u003csup\u003e+\u003c/sup\u003e microgliosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I), we performed single-cell RNA sequencing (scRNA-seq) at 7 days post-seizure. Unsupervised clustering of 44,810 cells identified 30 transcriptomically distinct populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), annotated as microglia (Clusters 6, 8, 20), neurons, astrocytes, and oligodendrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Supplement 2). Unique molecular identifier (UMI) distributions confirmed cluster-specific transcriptional identities (Supplement 3, 4). Microglial clusters (6/8/20) exhibited elevated \u003cem\u003eAif1\u003c/em\u003e (Iba1) expression versus other lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), with kernel density estimates validating inter-cluster heterogeneity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Seizures induced significant microglial subset redistribution (decreased in Cluster 6, increased in Cluster 8; both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F) without altering total microglial abundance (proportion unchanged: p\u0026thinsp;=\u0026thinsp;0.45; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H; Supplement 5). Differential expression analysis of microglia revealed the following: upregulated: \u003cem\u003eCsf1r\u003c/em\u003e (M-CSFR), \u003cem\u003eCcl4\u003c/em\u003e (chemokine), \u003cem\u003eCtss\u003c/em\u003e (cathepsin S); downregulated: \u003cem\u003eTrt\u003c/em\u003e (telomerase), \u003cem\u003eEnPP2\u003c/em\u003e (ectonucleotide pyrophosphatase), and \u003cem\u003eLy6c1\u003c/em\u003e (stem cell marker). Notably, \u003cem\u003eAif1\u003c/em\u003e expression spiked transiently at 3 days post-seizure (qRT-PCR: **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; RNA-seq: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but normalized by 7 days post-seizure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI; Supplement 6), underscoring its limitations as a standalone microglial activation marker given pathway-specific dynamics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubpopulation trajectory analysis uncovered divergent responses: decreased Cluster 6 (homeostatic), marked with down-regulated \u003cem\u003eP2ry12\u003c/em\u003e/\u003cem\u003eCsf1r\u003c/em\u003e/\u003cem\u003eCx3cr1\u003c/em\u003e and up-regulated \u003cem\u003eH2-Ab1\u003c/em\u003e/\u003cem\u003eCD74\u003c/em\u003e; increased Cluster 8 (immunomodulatory, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), marked with up-regulated \u003cem\u003eMs4a7\u003c/em\u003e/\u003cem\u003eRab7b\u003c/em\u003e/\u003cem\u003eSlamt7\u003c/em\u003e and down-regulated \u003cem\u003eTmem119\u003c/em\u003e/\u003cem\u003eSiglech\u003c/em\u003e; and stable Cluster 20 (transitional; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F). KEGG pathway enrichment implicated cytokine-cytokine receptor interactions across all subsets, with Cluster 8 showing dominant involvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). These data demonstrate the seizure-driven microglial repolarization via subset-specific transcriptional rewiring.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVolcano plot analysis confirmed the coordinated upregulation of \u003cem\u003eP2ry12\u003c/em\u003e, \u003cem\u003eTrem2\u003c/em\u003e, \u003cem\u003eCx3cr1\u003c/em\u003e, and \u003cem\u003eCD74\u003c/em\u003e in microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). UMAP visualization revealed selective \u003cem\u003eP2ry12\u003c/em\u003e downregulation within Cluster 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), corroborated by scatter/violin plots showing global microglial \u003cem\u003eP2ry12\u003c/em\u003e reduction post-seizure \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, E). Co-expression analysis identified a diminished \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e subpopulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), indicating that P2Y12R loss accompanies immunomodulatory polarization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicroglia-Specific P2ry12 Ablation Perturbs Neural Cell Homeostasis While Preserving CD74highH2-Ab1high Subpopulation Stability\u003c/h2\u003e \u003cp\u003ePrior studies established that conventional \u003cem\u003eP2ry12\u003c/em\u003e knockout (KO) exacerbates kainic acid (KA)-induced neurotoxicity \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To isolate microglial-specific mechanisms, we employed tamoxifen-inducible \u003cem\u003eCX3CR1CreER\u003c/em\u003e\u003csup\u003e\u003cem\u003e/+\u003c/em\u003e\u003c/sup\u003e:\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (hereafter \u003cem\u003eP2ry12\u003c/em\u003e cKO), and administering four tamoxifen doses (75 mg/kg, IP) at 48-hour intervals post-seizure (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Pre-KO validation confirmed comparable baseline EEG profiles (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.70\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, E) and equivalent KA-induced outcomes between genotypes: hippocampal neurodegeneration (FJB\u003csup\u003e+\u003c/sup\u003e cells: \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.95\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC,F); and seizure severity (Racine scores: NS; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). This protocol ensured equivalent epileptogenic priming prior to microglial \u003cem\u003eP2ry12\u003c/em\u003e deletion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUMAP analysis confirmed efficient \u003cem\u003eP2ry12\u003c/em\u003e knockdown with pan-cellular repercussions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA): increased populations contained microglia (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.03\u003c/em\u003e), endothelial cells (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.01\u003c/em\u003e), and oligodendrocytes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); and decreased populations contained choroid plexus epithelium (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and neurons (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.01\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, E, F). Microglial subpopulations exhibited divergent responses: expanded Cluster 6 (\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003e\u003cem\u003ehigh\u003c/em\u003e\u003c/sup\u003e homeostatic; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.01\u003c/em\u003e), increased Cluster 20 (\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003e\u003cem\u003eint\u003c/em\u003e\u003c/sup\u003e transitional; \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.02\u003c/em\u003e), and stable Cluster 8 (\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.80\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC,F). Violin plots revealed global \u003cem\u003eP2ry12\u003c/em\u003e suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), predominantly driven by Cluster 6 attenuation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Transcriptional profiling confirmed that \u003cem\u003eP2ry12\u003c/em\u003e cKO promoted Cluster 6-specific downregulation of homeostatic markers (\u003cem\u003eHexb\u003c/em\u003e, \u003cem\u003eTmem119\u003c/em\u003e, \u003cem\u003eP2ry12\u003c/em\u003e), with minimal impact on Clusters 8/20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eP2Y12 Receptor Dysfunction Amplifies \u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e Microglial Subsets in Postictal Remodeling\u003c/p\u003e \u003cp\u003eTo interrogate P2Y12R\u0026rsquo;s role in seizure-associated microglial plasticity, we employed a KA-induced epilepsy model in \u003cem\u003eP2ry12\u003c/em\u003e cKO mice. scRNA-seq at 7 days post-seizure revealed profound cellular reorganization in \u003cem\u003eP2ry12\u003c/em\u003e cKO brains, with UMAP visualization showing distinct global transcriptomic shifts versus wildtype (WT) controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Microglial expansion (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) dominated the \u003cem\u003eP2ry12\u003c/em\u003e cKO response, accompanied by neuronal \u003cb\u003e(\u003c/b\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01), endothelial (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and oligodendrocytic (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.02\u003c/em\u003e) depletion, while choroid plexus epithelium remained stable (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.24\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, C, E). Differential gene expression analysis revealed the following: microglia with upregulated \u003cem\u003eLyz2\u003c/em\u003e (lysozyme), \u003cem\u003eCtss\u003c/em\u003e (cathepsin S), \u003cem\u003eC1qb\u003c/em\u003e (complement), and downregulated \u003cem\u003eCD74\u003c/em\u003e, \u003cem\u003eTtr\u003c/em\u003e (transthyretin); neurons with Upregulated \u003cem\u003eSox11\u003c/em\u003e, \u003cem\u003eTubb2b\u003c/em\u003e, \u003cem\u003eSox4\u003c/em\u003e, and downregulated \u003cem\u003eItm2a\u003c/em\u003e, \u003cem\u003eFlt1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). Immunofluorescence staining confirmed sustained P2Y12R suppression in \u003cem\u003eP2ry12\u003c/em\u003e cKO microglia post-seizure (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, D), concomitant with expanded proliferative microglial pools (p\u0026thinsp;=\u0026thinsp;0.02; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB, C). Strikingly, all microglial clusters (6/8/20) exhibited aberrant expansion in \u003cem\u003eP2ry12\u003c/em\u003e cKO mice versus WT (enlarged cluster 8, decreased cluster 6, stable cluster 20; all p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA-D), establishing Cluster 8 with \u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e as the dominant pathological subset.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter P2Y12R ablation, the marker genes of homeostatic Cluster 6, including \u003cem\u003eTmem119\u003c/em\u003e and \u003cem\u003eSiglech\u003c/em\u003e, had partial recoveries and were dramatically down-regulated in WT after seizures, and \u003cem\u003eMs4a7\u003c/em\u003e, \u003cem\u003eRab7b\u003c/em\u003e, \u003cem\u003eSlamt7\u003c/em\u003e in Cluster 8 were hyperactivated (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA). Special transcriptional reprogramming included up-regulated \u003cem\u003eSparc\u003c/em\u003e/\u003cem\u003eCD81\u003c/em\u003e/\u003cem\u003eHexb\u003c/em\u003e, and down-regulated \u003cem\u003eGpnmb/Lyz2\u003c/em\u003e in Cluster 6; up-regulated \u003cem\u003eThbs1/Gpnmb/CD74\u003c/em\u003e, and down-regulated \u003cem\u003eHexb/Sparc\u003c/em\u003e in Cluster 8; down-regualted \u003cem\u003eGpnmb/Thbs1/Lgals3\u003c/em\u003e in Cluster 20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eB-D). UMAP spatial mapping localized \u003cem\u003eCD74\u003c/em\u003e/\u003cem\u003eH2-Eb1\u003c/em\u003e co-expression to Cluster 8, which inversely correlated with P2Y12R\u003csup\u003e+\u003c/sup\u003e microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSub-clustering of 4,463 microglia identified 13 transcriptomic states (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eA), with three principal subsets: Subcluster 1 expressing \u003cem\u003eH2-Eb1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eThbs1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eSaa3\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, and \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e; Subcluster 2 expressing \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eGpr34\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eTmem119\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e, and \u003cem\u003eH2-Eb1\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e; and Others in transitional states (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eB, C, F). Postictal \u003cem\u003eP2ry12\u003c/em\u003e cKO selectively amplified Subcluster 1 after seizure (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eH), validated by \u003cem\u003eH2-Eb1\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and \u003cem\u003eCD74\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eI). UMAP spatial mapping localized \u003cem\u003eCD74\u003c/em\u003e/\u003cem\u003eH2-Eb1\u003c/em\u003e co-expression to Subcluster 1, which inversely correlated with P2Y12R\u003csup\u003e+\u003c/sup\u003e microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMicroglial P2Y12 Receptor Deficiency Suppresses Postictal Neurogenesis via Neuronal-Glial Crosstalk\u003c/h2\u003e \u003cp\u003eAs demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE, microglial P2Y12 receptor deficiency (P2ry12 cKO) abolished seizure-induced neurogenic augmentation. To mechanistically dissect this phenotype, we performed immunofluorescence analysis of the hippocampal dentate gyrus (DG) using DCX as an immature neuronal marker, BrdU as a proliferative cell tracer, and Ki67 as a cell cycle progression indicator. Both DCX\u003csup\u003e+\u003c/sup\u003eKi67\u003csup\u003e+\u003c/sup\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and DCX\u003csup\u003e+\u003c/sup\u003eBrdU\u003csup\u003e+\u003c/sup\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) double-positive cells, as hallmarks of active neurogenesis, were elevated post-seizure in WT but suppressed in P2ry12 cKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eA-C). scRNA-seq volcano plots revealed concomitant downregulation of neurogenic regulators (\u003cem\u003eSox11\u003c/em\u003e, \u003cem\u003eSox4\u003c/em\u003e, and \u003cem\u003eRgs5\u003c/em\u003e) and upregulation of microglial activation markers (\u003cem\u003eCD74\u003c/em\u003e, \u003cem\u003eThbs2\u003c/em\u003e, and \u003cem\u003eClec4e\u003c/em\u003e) in P2ry12 cKO (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eD), suggesting glial-mediated neurogenic inhibition. UMAP-based cell cycle analysis identified neurons as the predominant proliferative population post-seizure (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eA). The key cell cycle drivers, Top2a (DNA topoisomerase IIα) and Mki67 (proliferation marker), presented neuronal-specific expression, with DCX\u003csup\u003e+\u003c/sup\u003e cells peaking in G1 phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eB). P2ry12 cKO attenuated the seizure-induced upregulation of Top2a (S phase; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), Mki67 (M phase; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and DCX (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eC, D; Supplement 7). While global cell cycle distribution remained stable, P2ry12 cKO reduced the G1 neuronal proportion from 64% in WT to 58% in P2ry12 cKO (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eE), indicating subtle cell cycle dysregulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCD74-TNF-α Axis Mediates P2Y12-Regulated Neurogenic Suppression in Postictal Hippocampus\u003c/h2\u003e \u003cp\u003eThe mechanistic link between \u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e microglia and impaired neurogenesis post-seizures remains undefined. Given TNF-α\u0026rsquo;s role as a downstream effector of CD74 signaling in neuroinflammation \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, we performed hippocampal immunofluorescence mapping. Quantitative analysis revealed seizure-induced elongation of both supra- and infra-pyramidal blades\u0026mdash;structural correlates of neuronal maturation\u0026mdash;in WT mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e16\u003c/span\u003eA, B; Supplement 8). This neurogenic plasticity was abolished in \u003cem\u003eP2ry12\u003c/em\u003e cKO mice (supra-blade: \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.01\u003c/em\u003e; infra-blade: \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.03\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e16\u003c/span\u003eA, B). Concomitantly, \u003cem\u003eCD74\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e and TNF-α\u003csup\u003e+\u003c/sup\u003e cell densities surged post-seizure in WT (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with further amplification in \u003cem\u003eP2ry12\u003c/em\u003e cKO hippocampi (\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e: \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.01\u003c/em\u003e; TNF-α\u003csup\u003e+\u003c/sup\u003e: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and CA3 subfields (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e16\u003c/span\u003eC, D). CellChat interrogation of scRNA-seq data identified \u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e Cluster 8 microglia as central interactors, forming putative signaling networks with endothelial cells (angiocrine factors), astrocytes (C1q/C3 complement), erythrocytes (iron homeostasis), and neurons (semaphorin/plexin axes).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this investigation, we delineated the dual regulatory role of microglia in postictal neuronal impairment and compensatory neurogenesis following epileptogenesis. Quantitative analysis revealed a transient microglial expansion peaking at 3 days post-epilepsy, followed by population normalization by 7 days post-epilepsy, as a timeframe coinciding with sustained hippocampal neurogenic activity. Through integrated single-cell transcriptomics and flow cytometric validation, we identified two reciprocally regulated microglial subsets: a diminished \u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e subpopulation and an expanded \u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e subpopulation, collectively designated as epilepsy-associated microglia (EPAM). Conditional ablation of microglial P2Y12 receptors precipitated paradoxical expansion of both subsets post-epileptogenesis, concomitant with significant suppression of neurogenic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e17\u003c/span\u003e). Mechanistically, P2Y12R deficiency upregulated CD74 expression, a MHC-II chaperone, and potentiated hippocampal TNF-α release, establishing a pro-inflammatory milieu inhibitory to neurogenesis. Our proposed model implicates neuronal ADP release as the primary activator of microglial P2Y12R signaling, which normally constrains EPAM expansion while promoting subpopulation equilibrium. Disruption of this pathway destabilizes microglial homeostasis, driving CD74-mediated neuroinflammatory cascades that impair neurogenesis in hippocampus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTraditional neuroimmunology classifies microglia into three broad subtypes (M0 resting, M1 pro-inflammatory, and M2 anti-inflammatory states) through analogy with macrophage polarization \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, emerging evidence underscores fundamental distinctions between microglia and peripheral macrophages in their ontogenetic origins, niche-specific adaptations, ultrastructural organization, and disease-modulatory functions \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This M1/M2 dichotomy, while heuristically useful, fails to capture the context-dependent plasticity of microglia in neurological pathologies or delineate disease-etiological subsets \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The resolution revolution brought by scRNA-seq has enabled high-dimensional deconstruction of microglial heterogeneity \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In AD, temporally dynamic disease- DAM subsets were identified: DAM-1 (early-stage, phagocytic; \u003cem\u003eTrem2\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eApoE\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eTyrobp\u003c/em\u003e \u003csup\u003ehigh\u003c/sup\u003e) and DAM-2 (late-stage, inflammatory; \u003cem\u003eCxcr4\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Similarly, aging-associated subsets marked by \u003cem\u003eP2ry12\u003c/em\u003e, \u003cem\u003eCx3cr1\u003c/em\u003e (chemokine receptor), and \u003cem\u003eCd11b\u003c/em\u003e (integrin) expression demonstrate chronological functional specialization \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In epilepsy research, while lipid-laden reactive astrocytes and pan-glial upregulation of \u003cem\u003eSpp1\u003c/em\u003e (osteopontin), \u003cem\u003eTrem2\u003c/em\u003e, and \u003cem\u003eCd68\u003c/em\u003e (lysosomal marker) have been documented \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, the transcriptomic identity and functional hierarchy of EPAM remained unresolved. Our scRNA-seq analysis revealed two candidate EPAM subsets: Cluster 6: \u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e and Cluster 8: \u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e. These subsets exhibited reciprocal abundance shifts post-epileptogenesis (increased Cluster 6/decreased Cluster 8), potentially explaining the net microglial population stability despite subset reorganization. The decline in P2RY12 expression, as a homeostatic marker, aligns with this phenotypic transition, suggesting functional repolarization toward neuroinflammatory states. While inter-subset plasticity is hypothesized (e.g., Cluster 6 transforms to Cluster 8), alternative mechanisms like proliferative asymmetry require exclusion. This bimodal EPAM model provides a mechanistic scaffold to reconcile microglia\u0026rsquo;s dual roles in postictal neurodegeneration (via \u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e/\u003cem\u003eTNF-α\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e subsets) and neurogenesis (via \u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e surveillance subsets). By mapping these state transitions, we establish a framework for targeting pathology-driving subpopulations while preserving homeostatic functions.\u003c/p\u003e \u003cp\u003eMicroglia exhibit CNS-selective overexpression of the purinergic receptor P2RY12, a Gi-coupled sensor for extracellular ADP that governs surveillance motility and homeostatic maintenance \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In AD, coordinated signaling through Trem2, P2RY12, and TAM receptors (Tyro3/Axl/Mer) enables microglial detection of neurodegeneration-associated molecular patterns, including apoptotic neurons, lipid droplets, and Aβ aggregates, triggering their transition into DAM \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Parallel mechanisms operate in epilepsy, where hippocampal neuronal apoptosis releases ADP, oxidized lipids, and myelin debris that activate microglial P2Y12R\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. As a triad biomarker of homeostatic microglia alongside CX3CR1 and CSF-1R, P2Y12R expression inversely correlates with activation states \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Its functional antagonism against the pro-inflammatory P2X7 receptor mirrors CX3CR1\u0026rsquo;s role in suppressing microglial hyperactivation \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Our seizure model revealed P2Y12R downregulation concurrent with EPAM emergence, suggesting receptor loss destabilizes microglial quiescence\u0026mdash;a hypothesis supported by exacerbated KA-induced neurotoxicity in \u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e models \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Notably, human genetic evidence links \u003cem\u003eP2RY12\u003c/em\u003e enhancer variants (e.g., rs11707416) to Parkinson\u0026rsquo;s disease risk \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, implying conserved roles in neurological disorder susceptibility.\u003c/p\u003e \u003cp\u003eMicroglia exhibits specific overexpression of P2RY12 in the brain \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. As a G protein-coupled receptor, P2RY12 recognizes ADP and plays a critical role in regulating microglial functions \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In AD, microglia detect apoptotic neurons, lipid metabolites, myelin debris, and extracellular protein aggregates through receptors such as Trem2, P2RY12, and TAM (Tyro3/Axl/Mer), leading to their transformation into DAM \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Similarly, EP induces neuronal apoptosis in the hippocampus, accompanied by the release of ADP, lipid metabolites, and myelin debris, which activate microglial P2RY12 receptors \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. P2RY12, along with CX3CR1 and CSF-1R, is classified as a biomarker of homeostatic microglia, with its expression downregulated in activated microglial subpopulations \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Upon ligand binding, CX3CR1 facilitates the transition of microglia from an activated state back to a homeostatic state \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. P2RY12 is hypothesized to counteract the function of another purinergic receptor, P2X7R (Purinergic receptor 2X7), thereby inhibiting microglial activation and the formation of activated subpopulations, similar to the role of CX3CR1 \u003csup\u003e39\u003c/sup\u003e. In this study, we observed alterations in the composition of microglial subpopulations following seizures, accompanied by a reduction in the average expression of P2RY12, and identified candidate EPAM. The decreased expression of P2RY12 may contribute to the instability of the resting state of microglia post-seizure \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In our previous work, we demonstrated that complete knockout of P2RY12 exacerbates KA-induced neuronal damage \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. To avoid the confounding effects of KA, a conditional P2RY12 KO approach was employed in this study. A quantitative trait loci (QTLs) study revealed that mutations at the rs11707416 enhancer of P2ry12 lead to its downregulation, increasing the risk of Parkinson\u0026rsquo;s disease \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Based on these findings, we hypothesize that low expression of P2RY12 in the brain may also influence the risk of epileptic seizures. These insights underscore the potential role of P2RY12 in maintaining microglial homeostasis and its implications in neurological disorders.\u003c/p\u003e \u003cp\u003eThe P2Y12R-CSF1R-CX3CL1 axis orchestrates dichotomous regulation of microglial polarization \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In AD, DAM evolution occurs via two phases: (1) the first stage, TREM2-Independent Priming: \u003cem\u003eP2RY12\u003c/em\u003e downregulation initiates phenotypic shift \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e; (2) The second stage, TREM2-Dependent Maturation: Coordinated \u003cem\u003eTREM2\u003c/em\u003e upregulation stabilizes DAM \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. P2RY12 is hypothesized to regulate the initial transition to DAM in the first stage and collaborate with TREM2 in the second stage to facilitate DAM formation. Our epilepsy models recapitulate this biphasic logic. Conditional \u003cem\u003eP2ry12\u003c/em\u003e ablation precipitated disproportionate EPAM subset expansion\u0026mdash;particularly Cluster 8 (\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e)\u0026mdash;while suppressing homeostatic Cluster 6 (\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e). This skewed amplification suggests P2Y12R normally constrains neuroinflammatory EPAM differentiation, with receptor deficiency permitting uncontrolled Cluster 8 dominance. Such subset reconfiguration likely drives postictal microglial dysfunction, mirroring DAM-mediated neurodegeneration in AD.\u003c/p\u003e \u003cp\u003eThe dualistic influence of microglia on neurogenesis, as context-dependent facilitation versus inhibition, reflects functional specialization across subpopulations \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Emerging evidence implicates microglia in neurogenic suppression through pro-inflammatory activation \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. For instance, intracerebroventricular (I.C.V.) lipopolysaccharide (LPS) administration induces microglial hyperactivation, abolishing basal dentate gyrus (DG) neurogenesis, shown an effect reversible via microglial depletion \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Conversely, genetic disruption of specific receptors (e.g., \u003cem\u003eP2ry13\u003c/em\u003e) enhances DG neurogenesis, suggesting tonic inhibitory regulation \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In epilepsy, microglial TLR9 signaling drives aberrant hippocampal neurogenesis, potentially exacerbating circuit hyperexcitability \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Paradoxically, microglia also exhibit neurogenic support: CX3CR1 deficiency impairs basal neurogenesis \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, while global \u003cem\u003eP2ry12\u003c/em\u003e knockout suppresses both basal and epilepsy-related neurogenesis\u0026mdash;a phenotype recapitulated by microglial ablation \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Notably, we identified seizure-induced proliferation of microglial \"process pouches\" at injured dendrites, structures attenuated by \u003cem\u003eP2ry12\u003c/em\u003e deletion, hinting at unresolved neuroprotective interactions \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. This functional paradox is resolvable through a subpopulation lens. Our single-cell analysis revealed polarized expression of neurogenesis-modulating receptors (P2ry13, TLR9, Cx3cr1, P2ry12) across microglial subsets. P2ry12 ablation skewed EPAM composition toward Cluster 8 dominance (\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e), concomitant with hippocampal CD74 upregulation. As an MHC-II chaperone and macrophage migration inhibitory factor (MIF) receptor, CD74 propagates neuroinflammatory cascades via TNF-α release\u0026mdash;a mechanism implicated in neurogenic suppression \u003csup\u003e\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The mechanistic hierarchy was summarized as: P2Y12R loss induced EPAM subset imbalance, such increased Cluster 8; CD74 overexpression in Cluster promoted TNF-α-driven neuroinflammation; and inflammatory milieu inhibited the neurogenesis in hippocampus. This axis positions CD74\u003csup\u003e+\u003c/sup\u003e EPAM as neurogenic gatekeepers, contrasting with \u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e subsets that may sustain permissive microenvironments. Such bimodal regulation underscores the therapeutic potential of subset-specific modulation over global microglial targeting.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study establishes EPAM as a dynamic bimodal entity regulated by P2Y12R-CD74 signaling. We demonstrated that the transcriptomic profiling of EPAM subpopulations, mechanistic link between P2Y12R signaling and CD74-mediated neuroinflammation, and subpopulation-specific explanation for microglia\u0026rsquo;s dual neurogenic roles. These findings advocate for precision therapeutics targeting EPAM subsets rather than global microglial modulation. For instance, CD74 inhibition could mitigate neuroinflammatory EPAM while preserving homeostatic subsets, as a strategy superior to broad-spectrum anti-microglial agents.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data for each of the graphs presented are provided in this paper and stored in files created using Microsoft® Excel for Mac (Version 16.78.3; Microsoft Corporation) and GraphPad Prism 10 (GraphPad Software Company). The single cell sequencing data generated in this study have been deposited in the SAR database under accession code 9PL6pbW21yR64480, and annotated data provided in attachment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Prof. Pingyi Xu, and Dr. Longjun Wu for their assistance in the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (81701254), General Project of Basic and Applied Basic Research of Guangzhou Bureau of Science and Technology (2060206), Yang-cheng Scholar Project of Guangzhou Municipal Bureau of Education (202032790), General Project of Natural Science Foundation of Guangdong Province (2021A1515011043) and Guangzhou key medical discipline grant\u0026nbsp;(2021-2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMingshu Mo and Juan Ling contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Neurology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China\u003c/p\u003e\n\u003cp\u003eMingshu Mo, Lan Wang, Huishan Deng, Yilin Su, Lijian Wei, Yuting Tang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGuangzhou Key Laboratory for Research and Development of Crop Germplasm Resources, Zhongkai\u0026nbsp;University of Agriculture and Engineering, Guangzhou 510225, China\u003c/p\u003e\n\u003cp\u003eJuan Ling\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.S.M, J.L., W.L., Y.L.S, L.J.W, and Y.T.T. conceived of the experiments and developed the methods. M.S.M, J.L., and W.L. performed the experiments. J.L., W.L., Y.L.S, and M.S.M. analyzed the data. M.S.M and J.L. acquired financial support for this study. M.S.M and J.L. wrote the first draft of the manuscript. J S, L.J.W, and Y.T.T. reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Mingshu Mo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Guangzhou Medical University (Approval No. 2020341)\u0026nbsp;and reported in accordance with the ARRIVE 2.0 guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThijs RD, Surges R, O'Brien TJ, Sander JW. 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Inflamm Res. 2017;66:209\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"P2ry12, Neurogenesis, Epilepsy, Microglia subpopulation","lastPublishedDoi":"10.21203/rs.3.rs-6558062/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6558062/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFollowing epileptogenesis, a pronounced escalation in microglial activation, neuronal dysfunction, and hippocampal neurogenesis is consistently observed. As principal immune sentinels of the central nervous system, microglia perform multifaceted functions including inflammatory mediator secretion, neurotrophic factor synthesis, synaptic material phagocytosis, and homeostatic regulation. Within the epileptic milieu, microglia exhibit dichotomous regulatory effects, paradoxically influencing both neurodegenerative processes and neurogenesis processes. Despite this critical dual functionality, the mechanistic underpinnings of microglial polarization in epileptogenesis remain incompletely characterized. To address this knowledge gap, we implemented an integrative multi-omics approach combining single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) to delineate distinct epilepsy-associated microglial (EPAM) subpopulations. Complementary conditional knockout murine models were employed to elucidate the molecular determinants of EPAM differentiation. Our analytical pipeline identified two microglial subsets demonstrating reciprocal abundance patterns at 7 days post-epilepsy induction: a diminished \u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e population and an expanded \u003cem\u003eP2ry12\u003c/em\u003e\u003csup\u003elow\u003c/sup\u003e\u003cem\u003eCD74\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e\u003cem\u003eH2-Ab1\u003c/em\u003e\u003csup\u003ehigh\u003c/sup\u003e subpopulation, temporally correlated with hippocampal neurogenesis onset. Genetic ablation of P2ry12 precipitated a paradoxical expansion of both subpopulations following epileptogenic challenge, concomitant with significant suppression of neurogenesis. Mechanistic investigations revealed that \u003cem\u003eP2ry12\u003c/em\u003e deficiency upregulated \u003cem\u003eCD74\u003c/em\u003e and \u003cem\u003eH2-Ab1\u003c/em\u003e expression within microglia, enhanced hippocampal TNF-α release, and disrupted neurogenesis processes. These findings collectively demonstrate that P2Y12 receptor-mediated signaling governs the dynamic equilibrium of EPAM subpopulations, with perturbation of this regulatory axis impairing compensatory neurogenesis during epileptogenesis.\u003c/p\u003e","manuscriptTitle":"Microglial P2Y12 Receptor Signaling Governs Epilepsy-Associated Neurogenesis via Bidirectional Regulation of Distinct Microglial Subpopulations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 18:48:39","doi":"10.21203/rs.3.rs-6558062/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"64098ede-8e90-4d18-89f7-ee7c5e3c52d1","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-04T20:08:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 18:48:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6558062","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6558062","identity":"rs-6558062","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}