Biology subjects

Peng, J.

Publications and source records attributed to Peng, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Spatial transcriptomics reveals organizational properties of mouse spinal cord and alterations in neuropathic pain

The spinal cord integrates diverse somatosensory inputs and executes motor outputs through anatomically and functionally distinct circuits. Here, we employed spatially resolved single-cell transcriptomic profiling of the adult mouse spinal cord to gain insights into the organizational logic of the spinal cord. Our results reveal distinct spatial and laminar distributions of neuronal subtypes, including axial level and sex-specific differences. Many neuronal subtypes exhibit close spatial proximity, implicating regionally specific patterns of connectivity and circuit functions. Additionally, neuronal subtypes within the dorsal horn exhibit a wide range of predicted cell-cell communication motifs, as assessed by the spatial distribution of neuropeptide-and other ligand-receptor pairs. Finally, we identified several neuronal subtypes with altered transcriptomic and predicted cell-cell communications in a model of neuropathic pain. This spatially resolved cellular and molecular map of the spinal cord will facilitate the decoding of circuit mechanisms underlying somatosensory and motor functions in health and disease.

neuroscience↗

bioRxiv2026

Designing AAV Capsid Protein with viability-guided Diffusion Model

Adeno-associated virus (AAV) capsids have shown great promise as delivery vectors for gene therapy. However, the natural properties of AAV capsids impose significant limitations on their efficacy. While capsid library screening and directed evolution have facilitated the development of improved vectors, current methods for generating AAV libraries frequently yield nonviable variants that fail to assemble or package DNA. Here, we propose a viability-guided diffusion model AAVDiffusion for de novo viable AAV capsid design. AAVDiffusion iteratively denoises Gaussian vectors into vectors of AAV capsid protein sequences, yielding intermediate latent variables. By leveraging the continuous nature of these latent variables, AAVDiffusion integrates an additional viability classifier that applies gradient updates to enhance the generation of viable AAV sequences. Through extensive computational tests, AAVDiffusion exhibits superior performance in generating viable AAV sequences. Furthermore, 196 AAV candidates were identified through a selection workflow, with the potential to cross the blood-brain barrier, thereby offering safer and more effective gene therapies for brain diseases. AAVDiffusion offers a powerful and efficient computational method for designing viable AAV capsids, advancing the development of AAV vectors for gene therapy.

bioinformatics↗

bioRxiv2025

Cross-tissue transcriptome-wide association study prioritizes druggable targets for hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is known as the sixth most common cancer and the third leading cause of death related to cancer. While genome-wide association study (GWAS) has uncovered risk loci, mapping variants to causal genes and tissues is challenging. The aim of this study is to translate HCC GWAS signals from variants to genes and tissues to identify genetically regulated causal candidates and potential therapeutic targets. MethodsWe collected and integrated 12 benchmark GWAS summary statistics from different research including various areas and causes of HCC. We applied S-PrediXcan using GTEx 49 tissues to infer genetically regulated expression. Multiple testing was controlled using group-wise Benjamini-Hochberg FDR within tissues (primary, q[≤]0.05). For visualization, suggestive signals with p[≤]0.01 were highlighted; heatmaps display FDR-thresholder results, whereas other figures show the full distribution. We then performed cross-tissue prioritization, gene-trait network construction, gene set enrichment analysis (GSEA), and pathway enrichment. ResultsWe identified 422 significant gene-tissue associations (BH-FDR within tissue, q[≤] 0.05) regarding HCC, consolidating to 34 unique genes. Top tissues included breast mammary, left ventricle, and esophagus muscularis. Excluding established HCC loci (e.g., PNPLA3, TM6SF2, TERT, HSD17B13), we prioritized 32 druggable "novel" candidates, with top-ranked examples including DHCR24 and HLA-DPB1. ConclusionsCross-tissue TWAS integrating GWAS and GTEx models identifies genetically regulated genes associated with HCC, refines tissue specificity, and prioritizes plausible drug targets. This gene-centric framework complements variant-level GWAS and supports mechanism-guided prevention and therapy development.

bioinformatics↗

bioRxiv2025

Ectopic engraftment of nociceptive neurons derived from hPSCs for pain relief and joint homeostasis

Chronic pain arises from the interplay of inflammatory signals that activate and sensitize nociceptors within injured tissues. Most analgesics fail clinically due to their mono-targeted mechanisms. Here, we apply human pluripotent stem cell-derived nociceptive neurons (hPSC-NNs) as therapeutic agents for osteoarthritis, targeting both pain and joint degeneration. We generated sensory neurons from hPSCs and identified CD200 as a nociceptor marker. Transcriptomic and functional profiling revealed that CD200highhPSC-NNs closely resemble human nociceptors, expressing pain-relevant receptors and ion channels. Strikingly, ectopic transplantation of CD200highhPSC-NNs into the knee joint of osteoarthritic mice reduced pain and promoted bone and cartilage repair, whereas CD200low cells exhibited no benefit. Mechanistically, human and mouse proteomics suggest that CD200highhPSC-NNs act as decoys by sequestering inflammatory ligands while secreting reparative factors in joint tissues. These findings uncover a fundamental role of nociceptors in tissue repair, providing a multi-targeted, disease-modifying strategy for OA and chronic pain. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/694733v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@15f0948org.highwire.dtl.DTLVardef@5a59bforg.highwire.dtl.DTLVardef@1ba4439org.highwire.dtl.DTLVardef@1d518d1_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIhPSC-derived nociceptors (hPSC-NNs) as Decoy Engraftment for Cellular Interception and Repair (DECIR) when transplanted into the knee joint, extending beyond conventional regenerative strategies C_LIO_LICD200 serves as a clinically actionable surface marker for the purification of hPSC-NNs C_LIO_LIEctopic grafting of CD200high hPSC-NNs delivers dual benefits, alleviating pain and modulating the neuro-immune environment within joint tissues C_LIO_LIProteomic analyses reveal that CD200highhPSC-NNs sequester inflammatory mediators and secrete reparative factors to support joint homeostasis C_LI

neuroscience↗

bioRxiv2025

A haplotype-resolved T2T genome assembly of Indigofera pseudotinctoria reveals the genetic basis of flavonoid biosynthesis in Chinese Indigo

Indigofera pseudotinctoria, commonly known as Chinese Indigo, is a multifunctional leguminous shrub widely distributed in East Asia, valued for its medicinal, ecological, and forage importance, and naturally adapted to drought and nutrient-poor soils. However, the absence of genomic resources has hindered insights into the genetic basis of its bioactive metabolites and adaptive traits. Here, we present a haplotype-resolved, telomere-to-telomere (T2T) reference genome of I. pseudotinctoria, which represents the first complete genome within the genus Indigofera. By integrating PacBio HiFi sequencing, Oxford Nanopore ultra-long reads, and Hi-C chromatin conformation sequencing technologies, we successfully constructed two gap-free haplotypes assemblies (647.38 Mb and 657.52 Mb), characterized by high BUSCO completeness and QV scores, and fully captured all telomeric sequences. Comparative analysis between haplotypes revealed extensive synteny but notable structural heterozygosity but notable structural heterozygosity. Approximately 46% of structural variations overlap with genic or regulatory regions, leading to allele-specific expression divergence. Between the two haplotypes, we identified 2,607 and 2,331 haplotype-specific genes, reflecting a complementary functional specialization in defense and repair versus metabolism and growth. Integrative transcriptomic and metabolomic profiling across six tissues reconstructed the flavonoid biosynthetic network and identified MYB, bHLH, NAC, WRKY, and ERF transcription factors regulating five key pharmacologically active flavonoids (Calycosin, Butein, Sulfuretin, Chrysoeriol, and Genistin). These results collectively uncover the haplotype-specific regulatory and structural basis of Indigofera pseudotinctoria and establish a high-quality genomic framework for evolutionary, medicinal, and metabolic engineering studies in leguminous plants, and provide a framework for molecular breeding and bioactive compound discovery in medicinal legumes.

genomics↗

bioRxiv2025

Astrocyte cell-surface proteomics identified CD44 as an OPN/SPP1 receptor regulating lipid metabolism and glial crosstalk in Alzheimer s disease

Abstract/SummaryThe development of {beta}-amyloid (A{beta}) pathology in Alzheimers disease (AD) is accompanied by profound changes in astrocytes and microglia. How these responses are orchestrated by cell surface proteins, key mediators of cell-cell communication, remain unclear. Using in situ astrocyte cell-surface proteome profiling in 5xFAD mice, we identified a set of dysregulated surface proteins induced by A{beta} pathology, including CD44. CD44 was selectively upregulated in plaque-adjacent astrocytes and interacted with osteopontin (OPN), encoded by the disease-associated microglia gene Spp1, to promote lipid accumulation, and this effect is {gamma}-secretase dependent. Astrocytic CD44 in turn regulated Spp1 expression and microglial activity. Conditional deletion of Cd44 in adult astrocytes of 5xFAD mice attenuated glial reactivity, reduced A{beta} pathology, and improved cognition. These findings define a plaque-proximal OPN-CD44 axis that controls astrocyte lipid metabolism and glial activity, positioning CD44 as a surface-accessible therapeutic target in AD.

neuroscience↗

bioRxiv2025

The Human-In-the-Loop Drug Design Framework with Equivariant Rectified Flow

Advancements in AI-based drug design often face obstacles due to incomplete datasets, hindering progress to clinical trials. Human experts bring invaluable expertise and nuanced contextual understanding to drug design. There are two main difficulties in integrating human knowledge into the drug development process. First, human annotations are costly, and traditional machine learning algorithms require a large number of samples to be effective. Second, human experts are unable to accurately describe their expertise using natural language, nor can they precisely specify what kind of molecule is needed before seeing the generated molecules. To address these problems, we propose a new platform, called HIL-DD, for experts to infuse their experience by selecting molecules generated by AI that meet their criteria, or discarding those that do not. The core generative technology utilizes an Equivariant Rectified Flow Model (ERFM), which offers faster generation speeds than conventional diffusion models, enabling efficient human-AI collaboration. More importantly, we provide a user-friendly interface to ensure smooth and effective collaboration between human experts and AI systems. Rigorous experiments demonstrate that our system can produce 3D molecules that align with expert expectations in minimal interactive sessions. These generated molecules maintain drug-like qualities comparable to those created by current state-of-the-art models.

bioinformatics↗

bioRxiv2025

Induction of menstruation in mice reveals the regulation of menstrual shedding

During menstruation, an inner layer of the endometrium is selectively shed, while an outer, progenitor-containing layer is preserved to support repeated regeneration. Progress in understanding this compartmentalization has been hindered by the lack of suitable animal models, as mice and rats do not menstruate. Here, we present transgenic mouse models that recapitulate the key anatomical, functional, and transcriptional features of human menstruation through targeted chemogenetic activation of premenstrual differentiation. Using single-cell spatial transcriptomics, we define a new paradigm for spatially regulated fibroblast differentiation that drives pre-menstrual endometrial layering and ultimately determines the extent of tissue shedding. Our results revise a century-old view of endometrial shedding and regeneration and establish new transgenic mice as powerful tools to advance menstruation research.

developmental biology↗

bioRxiv2025

Gravity-Dependent Metabolomic Responses in Starbor Kale Brassica oleracea: Comparisons With Simulated Microgravity Versus Gravity Grown

As science continues to push the frontiers on the length of space flight, we begin to experience an enhanced need for food that is nutritionally dense. Nasa-grants have worked to explore the ability of growing superfoods in space on aircrafts. However, it is paramount to ensure the physiological health of the plants being grown in space flight, perform relatively similar to the plants that grow on earth. An aspect which needs to be further probed is the comparison of microgravity and gravity on the plants seeking to be grown in space flight. Within this NASA-funded research, we grew a superfood vegetable, Kale. The kale was grown in simulated microgravity environments to mimic life in outer space. This simulation was induced by means of a 2-D clinostat. Additionally, kale was grown in gravity conditions as well. We used multivariate statistical tools such as Principal Component Analysis (PCA), together with differential-expression visualizations like volcano plots, to summarize complex data rendered from LC-MS. In this study, we used these approaches to examine how horizontal and vertical orientations both in static and rotating configurations simulate aspects of simulated-microgravity and influence metabolic responses; this combined analysis provided a broad perspective on gravity-related metabolic adaptations and points to potential molecular markers that could guide future research on spaceflight health and countermeasure development.

plant biology↗

bioRxiv2025

Establishing a Canine Urothelial Organoid Repository: A Platform for Comparative and Translational Carcinoma Studies

Despite the increasing number of treatment options for patients with muscle-invasive bladder Cancer (MIBC), many of these patients ultimately have a poor prognosis. Drug responses vary considerably among patients and in many who respond initially, drug resistance may ultimately develop leading to tumor progression. Scope for improvement has been limited by the phenotypic and molecular diversity of MIBC, which impacts the selection pressures of therapy. To address this translational gap, we describe the largest known canine urothelial carcinoma organoid bioarchive, with in-depth phenotypical and molecular characterization. Immunohistochemistry confirmed the expression of key biomarkers including UPKIII, E-cadherin, Vimentin, and Ki-67 in organoids. Single-nucleus RNA sequencing revealed cellular heterogeneity, while bulk RNA sequencing showed canine patient-to-patient variability in transcriptomic profiling. Bulk RNA sequencing also displayed highly similar expression profiles between tissue- and urine-derived organoids, as well as similarity to previously published human MIBC transcriptome data. Whole genome sequencing in a subset of patients further supported the overall genomic fidelity of the organoids with their tissue of origin. Finally, for proof-of-concept of usefulness for drug testing studies, organoid cytotoxicity to vinblastine was investigated in several organoid lines. These canine bladder cancer organoid lines can be thawed, expanded, and screened for response to potential novel therapeutics to expand personalized medicine approaches. In addition, the organoid lines can serve as a valuable resource for comparative bladder cancer research, and as a pre-clinical screening tool to identify efficacious drugs before taking them into canine clinical trials to support future human clinical trials.

cancer biology↗

bioRxiv2025

Integrative Systems Biology Identifies the AHR Signaling Axis as a Mechanistic Basis for Xiaobi Plaster in Lumbar Disc Degeneration

BackgroundLumbar disc degeneration (LDD) is characterized by chronic inflammation, oxidative stress, and extracellular matrix (ECM) breakdown, yet current therapies provide only symptomatic relief. Xiaobi Plaster (XBG), a traditional Chinese transdermal herbal preparation, has shown clinical benefit, but its molecular mechanisms remain unclear. MethodsWe applied an integrative systems biology framework combining network pharmacology, machine learning, and transcriptomic validation to identify candidate targets of Xiaobi Plaster in LDD. Molecular docking, dynamics simulations, and immune infiltration analysis further elucidated the underlying mechanisms, which were experimentally validated in a rat puncture-induced degeneration model. ResultsThree key targets--AHR, PTPN2, and SOAT1--were identified, with AHR emerging as the central regulator. Differential expression and enrichment analyses highlighted inflammatory and ECM-remodeling pathways, which overlapped with AHR-specific GSEA, underscoring its hub role in integrating immune and redox signaling with matrix turnover. Molecular docking and 100-ns molecular dynamics confirmed rutaecarpine (MOL002662) as the most stable AHR ligand. Transcriptomic data further showed AHR expression correlates with degeneration severity and mast cell infiltration. In vivo qPCR and Western blotting validated upregulation of AHR and supporting roles of PTPN2 and SOAT1 in degenerative discs. ConclusionThis study elucidates the multi-target and multi-pathway mechanisms of XBG in LDD, highlighting AHR as a pivotal therapeutic target modulated by rutaecarpine, with PTPN2 and SOAT1 serving as auxiliary regulators. These findings provide mechanistic insights into the transdermal application of XBG and support the therapeutic relevance of targeting the AHR signaling axis in LDD.

bioinformatics↗

bioRxiv2025

NeuronID: An automatic toolkit for identifying neurons in two-photon calcium imaging data

Two-photon calcium imaging has emerged as a powerful technique for monitoring neuronal activity in neuroscience; however, its data processing remains challenging. Here, we introduce NeuronID, an automatic toolkit designed to process two-photon calcium imaging data. The NeuronID toolkit features a modular architecture that includes motion correction, noise reduction, segmentation of neuronal components, and extraction of neuronal signals. Notably, the NeuronID toolkit offers an optimized strategy for segmenting neuronal components, which systematically integrates morphological boundary identification, cross-correlation analysis between pixels, and evaluation of neuronal signal quality. Compared to existing tools or manual annotation by experts, the NeuronID toolkit reduces the likelihood of over-segmentation while achieving near-human accuracy. Overall, this study provides a standardized analytical tool for processing two-photon calcium imaging data.

neuroscience↗

bioRxiv2025

Extracellular Vesicles of Salivary Mesenchymal Stem Cells Mitigate Acute Irradiation Injury: Use of an ex-vivo organotypic human slice tissue culture as a disease model

AbstractIonizing radiation (IR) therapy for cancer patients can damage surrounding healthy tissues, particularly the salivary glands (SGs), leading to oral and systemic health issues reducing the quality of life of the patients. The mechanisms behind IR damage in SGs are not fully understood, and current therapies often fail to meet patient needs adequately. Therefore, identifying targeted pathways and alternative treatments is essential. To address this, we developed an ex vivo model of SG damage using human salivary glands obtained from patients. Healthy submandibular glands were harvested, cultured, and exposed to IR. RNA sequencing revealed elevated markers for DNA damage, inflammation, and ferroptosis, with four specific genes--FDXR, MDM2, H2AX, and p21--showing increases in expression that correlated with the IR dose. Using them, we developed a high-throughput genetic screening method to evaluate stem cell therapies aimed at mitigating IR injury. Conditioned media from mesenchymal stem/stromal cells (MSC-CM) were found to reduce the expression of all four markers, maintain tissue viability, promote cell proliferation, and decrease oxidative stress. Further analysis involved separating MSC-CM into two fractions: Extracellular Vesicles (EV)-rich and EV-depleted. The EV-depleted fractions retained elevated levels of DNA damage response markers, indicating that EVs play a crucial role in mediating tissue repair. In contrast, the EV-rich fractions reduced the markers of DNA damage response and were readily absorbed by the tissue slices. In conclusion, we have developed a genetic screening method to evaluate treatments for acute IR injury, emphasizing the significant role that EVs play in the repair process.

molecular biology↗

bioRxiv2025

A predicted structural interactome reveals binding interference from intrinsically disordered regions

Proteins function through dynamic interactions with other proteins in cells, forming complex networks fundamental to cellular processes. While high-resolution and high-throughput methods have significantly advanced our understanding of how proteins interact with each other, the molecular details of many important protein-protein interactions are still poorly characterized, especially in non-mammalian species, including Drosophila. Recent advancements in deep learning techniques have enabled the prediction of molecular details in various cellular pathways at the network level. In this study, we used AlphaFold2 multimer to examine and predict protein-protein interactions from both physical and functional datasets in Drosophila. We found that functional associations contribute significantly to high-confidence predictions. Through detailed structural analysis, we also found the importance of intrinsically disordered regions in the predicted high-confidence interactions. Our study highlights the importance of disordered regions in protein-protein interactions and demonstrates the importance of incorporating functional interactions in predicting physical interactions between proteins. We further compiled an interactive web interface to present the predictions, facilitating functional exploration, comparative analysis, and the generation of mechanistic hypotheses for future studies. Author SummaryUnderstanding which proteins interact with each other and how they interact is essential for uncovering fundamental biology and for identifying new pathways involved in health and disease. However, identifying protein-protein interactions experimentally is often challenging and error-prone, and many organisms still lack comprehensive interaction maps. In this study, we use AlphaFold2-multimer, a powerful artificial intelligence tool, to generate high-confidence predictions of PPIs in Drosophila melanogaster, a widely used model organism. We highlight the importance of incorporating often neglected functional associations when predicting protein-protein interactions at a genomic scale. The predictions enable us to examine how intrinsically disordered regions can mediate binding across large interaction networks, revealing widespread, structurally plausible interactions in vivo. Overall, our work demonstrates how AlphaFold predictions can greatly expand our understanding of the structural forces that shape protein interaction networks and help reveal hidden layers of cellular complexes and pathways.

genomics↗

bioRxiv2025

Integrated spatial proteomics of human PDAC uncovers an expanded tumour-immune-stroma spectrum with genomic associations

Distinctively, pancreatic ductal adenocarcinoma (PDAC) consists of sparse tumour lesions intertwined with extensive desmoplastic stroma. The complexity of tumour-microenvironment interactions within this desmoplasia poses a challenge for accurate tumour profiling and patient stratification, and characterizes a profoundly chemoresistant tumour. Here we mapped the spatial relationships between tumour, stroma, and immune cell compartments delineating tumour and microenvironment types that expand the classical to basal spectrum of human PDAC. We used imaging mass cytometry to profile the in situ multi-cellular organization of 81 cell types in resected cases with paired whole genome sequencing. Cell types, functions, and pathway activation were distributed as highly reproducible environments in discrete locations throughout these tumours, which we deep-profiled using laser-capture mass spectrometry. We show that the connections between tumour phenotypes, vascularization, immune response, and stromal biophysical state are reinforced by genomic aberrations, altered by treatment, and associated with patient outcome. Predictive machine-learning models showed that spatial single cell data outperformed genomic or clinical features but integrated multi-omics models provide the best prediction of patient survival with compressed models requiring only 10 non-redundant robust molecular measures associated with the phenotypic spectrum of PDAC. Together, these findings define a phenotypic and molecular framework of PDAC that captures tumour-microenvironment co-dependencies and offers a refined basis for patient stratification and therapeutic targeting.

cancer biology↗

bioRxiv2025

mTORC1-Dependent Regulation of the CCL24-CCR3 Axis Controls Granuloma Formation and Maintenance in Sarcoidosis

Sarcoidosis is a chronic granulomatous disease marked by persistent inflammation and immune cell aggregation, yet its molecular underpinnings remain incompletely understood, hindering the development of effective targeted therapies. Here, we report that deletion of TSC1 or TSC2 in mice using a Fsp1-Cre leads to spontaneous formation of sarcoid-like granulomas, driven by hyperactivation of the mTORC1 pathway in fibroblasts and interstitial macrophages. Through inflammatory cytokine/chemokine array, we identified CCL24, a chemokine ligand for CCR3, as a key immunoregulatory molecule downregulated in both our murine model and sarcoid cohort plasma. Mechanistically, mTORC1 suppresses CCL24 expression via aberrant STAT3 signaling in fibroblasts and promotes CCR3 expression in interstitial macrophages, uncovering a novel regulatory axis in granuloma formation and maintenance. Pharmacological inhibition using rapamycin and azithromycin markedly attenuated granuloma burden and normalized CCL24-CCR3 signaling, underscoring the therapeutic relevance of this axis. Together, our study establishes a mechanistic link between mTORC1 activation, CCL24-CCR3 dysregulation, and granuloma persistence, offering not only a new insight into molecular mechanisms in sarcoidosis but also identifying promising targets for clinical intervention.

immunology↗

bioRxiv2025

PLK1-mediated phosphorylation of PHGDH reprograms serine metabolism in advanced prostate cancer

Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to meet their increased biosynthetic and energetic demands. While cells possess the capacity for de novo serine biosynthesis, most transformed cancer cells heavily depend on exogenous serine uptake to sustain their growth, yet the regulatory mechanisms driving this metabolic dependency remain poorly understood. Here, we uncover a novel mechanism by which Polo-like kinase 1 (PLK1), often overexpressed in prostate cancer, orchestrates a metabolic shift in serine and lipid metabolism through the phosphorylation of phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme of the serine synthesis pathway (SSP). We demonstrate that PLK1 phosphorylates PHGDH at three specific sites (S512, S513, S517), leading to a marked reduction in its protein level and enzymatic activity. This downregulation of SSP forces cancer cells to increase their reliance on exogenous serine uptake via the ASCT2 transporter, which, in turn, fuels the biosynthesis of lipids, including sphingolipids essential for tumor growth and survival. Targeting the SSP, serine uptake, or downstream lipid biosynthetic pathways may offer promising therapeutic avenues in PLK1-high advanced cancers.

cancer biology↗

bioRxiv2025

Spatial profiling of chromatin accessibility reveals alteration of glial cells in Alzheimer's disease mouse brain

Abnormal epigenetic modifications, including altered chromatin accessibility, have been implicated in the development and progression of Alzheimers disease (AD). In this study, we applied spatially resolved chromatin accessibility profiling by performing spatial assay for transposase-accessible chromatin using sequencing (ATAC-seq) to analyze brain tissues from 5xFAD AD model and C57BL/6J control mice. Our analysis identified seven major cell types across 11 brain regions and further characterized glial subtypes--microglia and astrocytes-- revealing subtype-specific chromatin accessibility changes in 5xFAD mice relative to controls. These alterations were associated with AD-related pathways, including neuroinflammation and immune dysregulation, and synaptic dysfunction and neuronal signaling in microglia, as well as lysosomal and proteasomal activity, lipoprotein metabolism, and mitochondrial dysfunction in astrocytes. We also characterized cell-type-specific enrichments of motifs and transcription factors, including enrichment of Bcl11a in microglia. Furthermore, we linked chromatin accessibility changes in 5xFAD mice to human AD risk genes, highlighting altered epigenetic signatures in genes such as Trem2. Supporting the spatial ATAC-seq findings, flow cytometry validated a selective increase in VISTA (encoded by Vsir) protein expression in 5xFAD microglia, supporting the spatial ATAC-seq findings and indicating a shift toward a pro-inflammatory, disease-associated microglial (DAM-like) phenotype. These findings underscore the utility of spatial chromatin accessibility profiling for uncovering brain-region specific cell identities and epigenetic mechanisms underlying AD pathogenesis.

bioinformatics↗