Biology subjects

Chen, K.

Publications and source records attributed to Chen, K..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

PPP1R3G Deletion Blocks RIPK1-Mediated Apoptosis and Necroptosis in Doxorubicin-Induced Cardiotoxicity

Cardiotoxicity is a major limitation of cancer chemotherapy, exemplified by doxorubicin (DOX), yet its underlying mechanisms remain incompletely defined. Here, we identify Protein Phosphatase 1 Regulatory Subunit 3G (PPP1R3G) as a critical amplifier of DOX-induced cardiotoxicity. We show that DOX activates both apoptosis and necroptosis in vitro. Mechanistically, DOX first induces p38-dependent inhibitory phosphorylation of receptor-interacting protein kinase 1 (RIPK1), providing a transient brake on cell death. PPP1R3G facilitates the removal of inhibitory phosphorylation, thereby permitting RIPK1 activation, oligomerization, and downstream apoptotic signaling. Activated RIPK1 further promotes mitochondrial DNA (mtDNA) release, which induces IFN-{beta}-mediated ZBP1 expression and establishes a positive feedback loop that amplifies late-stage necroptosis. Genetic ablation of Ppp1r3g suppresses both apoptosis and necroptosis in cardiomyocytes, attenuates inflammatory cytokine production, and protects mice from DOX-induced cardiac injury and mortality. These findings delineate a PPP1R3G-RIPK1 axis that converts an early protective phosphorylation checkpoint into sustained death signaling and identify PPP1R3G as a potential therapeutic target for cardioprotection.

molecular biology↗

bioRxiv2025

A New Aneurysm Rupture-Prone Marfan Mouse Model with FBN1Q2467X Nonsense Mutation Reveals Adventitial Inflammation

Marfan syndrome (MFS) is a genetic disorder caused by mutations in fibrillin-1(FBN1), which encodes FBN1, a key structural component of the extracellular matrix. Mutations in FBN1 influence the severity of aortic disease and therapeutic responses, with aortic aneurysm being the leading cause of mortality in patients with MFS. To investigate the mechanisms driving aneurysm progression, we generated a mouse model (Fbn1Q2469X/+) carrying the FBN1Q2467X nonsense mutation identified in MFS patients. This mutation results in FBN1 deficiency. Although Fbn1Q2469X/+ mice appear normal, showing only mild, nonprogressive dilation of the aortic root and ascending aorta with minor reductions in blood pressure, homozygous Fbn1Q2469X/Q2469X mice develop spontaneous thoracic aortic aneurysms (TAA) that progress to rupture between 10 and 25 days of age, with 100% penetrance. Histopathology shows progressive vessel wall degeneration characterized by disorganized vascular smooth muscle cells, collagen loss, and elastic fiber fragmentation from early to late stages. RNA-seq analysis identifies inflammation as the dominant process in late-stage aneurysms. Immunofluorescence assay reveals inflammatory cells prominently localized to the adventitia near rupture sites, linking adventitial inflammation to aneurysm progression. This genetically modified Fbn1Q2469X/Q2469X mouse model consistently develops progressive aortic aneurysms and provides a reliable, cost-effective platform to investigate the molecular mechanisms of aneurysm progression and to evaluate therapeutic strategies in aneurysm diseases, including Marfan syndrome and related disorders.

genetics↗

bioRxiv2025

Bodily Self in Macaque Monkeys and Human Infants

Whether monkeys possess the concept of bodily self remains controversial. We found that macaque monkeys trained for mirror self-recognition (MSR) could spontaneously recognize themselves when viewing video images of their back from a third-person perspective (3PP), but this generalization required a few minutes of "exploration". In contrast, through visual-tactile synchronization training similar to that used in human out-of-body studies, naive monkeys could recognize themselves from 3PP back images and demonstrated MSR within seconds. Interestingly, in human infants, we found that 3PP back self-recognition developed spontaneously several months after MSR. These results demonstrate the existence of hierarchical bodily self in macaques and similar stepwise development of bodily self in human infants, offering insights into the emergence and manifestation of bodily self in natural and artificial intelligence.

neuroscience↗

bioRxiv2025

Biodegradation of the endocrine-disrupting compound bisphenol F by Sphingobium yanoikuyae DN12

Bisphenol F (BPF) is an emerging environmental pollutant widely present in surface water and wastewater systems. Microbial activity is crucial in driving its degradation, offering a potential avenue for mitigating its environmental impact. Although the degradation pathway for BPF has been identified in various bacteria, the biodegradation mechanisms remain unclear. In this study, we isolated a highly efficient BPF-degrading strain of Sphingobium yanoikuyae DN12, which could utilize BPF as the sole carbon source and energy source for growth, from a river sediment in Anhui Province China. Through Ultra performance liquid chromatography high-resolution mass spectrometry (UPLC-HRMS) analysis, we found that oxidation and hydrolysis are key steps for BPF biodegradation. Utilizing whole-genome sequencing, comparative transcriptomics analysis and biochemical identification, a gene cluster bpf was identified to be involved in BPF degradation. BpfAB is a two-component oxidoreductase responsible for converting BPF to 4,4-dihydroxybenzophenone (DHBP). BpfC is a Baeyer-Villiger monooxygenase (BVMO) responsible for converting DHBP to 4-hydroxyphenyl-4-hydroxybenzoate (HPHB). Isotope tracing demonstrated that the oxygen atom incorporated by BpfAB originates from water, whereas that incorporated by BpfC derives from molecular oxygen (O2). BpfD is an /{beta} hydrolase responsible for converting HPHB to 4-hydroxybenzoate (4HB) and 1,4-hydroquinone (HQ). Analysis of the taxonomic and habitat of 325 prokaryotic genomes revealed that BpfA-like homologs are predominantly found in the phylum Pseudomonadota, primarily inhabiting soil and aquatic environments. This study enhances our understanding of the biodegradation mechanism of BPF, and provides guidance for the effective remediation of BPF-contaminated environments. IMPORTANCEBPF is a widely used alternative to bisphenol A and poses a growing threat to ecosystems and human health due to its environmental persistence and endocrine-disrupting effects. Although microbial degradation pathways for BPF have been reported, the key enzymes involved and their catalytic mechanisms remain unclear. This work reports the isolation of a Sphingobium strain capable of mineralizing BPF and the genetic basis for the catabolic pathway. Three enzymes--a two-component oxidoreductase, a Baeyer-Villiger monooxygenase, and an /{beta} hydrolase--were biochemically characterized and shown to catalyze the three critical steps in BPF degradation. These findings provide insights into the biochemical processes involved in the microbial degradation of BPF.

microbiology↗

bioRxiv2025

A comprehensive RNA virome from molluscan transcriptomes reveals extensive diversity and modular genome evolution

Mollusca is the second-largest animal phylum and an important marine food resource for humans. While DNA viruses that threaten molluscan aquaculture have received much attention, molluscan RNA viromes remain poorly explored. Here, based on 223 molluscan metatranscriptomes covering eight animal classes, we identified 80 species-level RNA viruses spanning three viral phyla and nine viral families. Phylogenetic result combined structural modeling found a major increase in the number of Pisuviricota-related lineages. Extensive modular evolution in viral genomes was observed including gene rearrangements and co-evolution of capsid and RdRP genes. Host prediction linked 78% of the RNA viruses to a diverse range of eukaryotes, including vertebrates, invertebrates, and algae. These findings expand the known diversity of RNA viruses in molluscs and shed light on their phylogenetic relationships, highlighting molluscs as valuable models for RNA virus evolution. Graphical abstractRNA virus diversity within 223 molluscan transcriptome samples were analyzed based on RdRP identification. Viruses belonging to the phylum Pisoviricota were the most abundant; co-evolution between RdRP and capsid proteins of molluscan Picornavirales was revealed, and a complex virus-host interaction network was constructed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/673676v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1ce8fc0org.highwire.dtl.DTLVardef@3f9068org.highwire.dtl.DTLVardef@121cc2forg.highwire.dtl.DTLVardef@1e431a0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIComprehensive metatranscriptomic analysis found extensive RNA viral diversity in molluscs. C_LIO_LIPisuviricota dominate the molluscan RNA virome, substantially broadening known viral lineages. C_LIO_LIModular genome evolution involving gene rearrangements and conserved structural features characterize molluscan RNA viruses. C_LIO_LIMolluscs potentially serve as major ecological reservoirs for diverse RNA viruses infecting various eukaryotic hosts. C_LI

bioinformatics↗

bioRxiv2025

Cryptic splicing in synaptic and membrane excitability genes links TDP-43 loss to neuronal dysfunction

TDP-43 pathology is a defining pathological hallmark of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major feature of TDP-43 pathology is its nuclear depletion, leading to the aberrant inclusion of cryptic exons during RNA splicing. STMN2 and UNC13A have emerged as prominent TDP-43 splicing targets, but the broader impact of TDP-43-dependent cryptic splicing on neuronal function remains unclear. Here, we report new TDP-43 splicing targets critical for membrane excitability and synaptic function, including KALRN, RAP1GAP, SYT7 and KCNQ2. Using human stem cell-derived neurons, we show that TDP-43 reduction induces cryptic splicing and downregulation of these genes, resulting in impaired excitability and synaptic transmission. In postmortem brains from patients with FTD, these cryptic splicing events occur selectively in neurons with TDP-43 pathology. Importantly, suppressing individual cryptic splicing events using antisense oligonucleotides partially restores neuronal function, and combined targeting almost fully rescues the synaptic deficit caused by TDP-43 loss. Together, our findings provide evidence that cryptic splicing in these synaptic and membrane excitability genes is not only a downstream marker but instead a direct driver of neuronal dysfunction, establishing a mechanistic link between TDP-43 pathology and neurodegeneration in ALS and FTD.

neuroscience↗

bioRxiv2025

SARS-CoV-2 NSP14 inhibitor exhibits potent antiviral activity and reverses NSP14-driven host modulation

The emergence of SARS-CoV-2 variants and drug-resistant mutants highlights the urgent need for novel antiviral therapeutics. SARS-CoV-2 NSP14, an N7-guanosine methyltransferase, plays a critical role in viral RNA capping, enabling viral replication and immune evasion. While NSP14 has emerged as a promising drug target, its role in host-virus crosstalk and the cellular consequences of NSP14 inhibition remain poorly understood. Here, we present the identification and characterization of C10, a highly potent and selective first-in-class non-nucleoside inhibitor of the NSP14 S-adenosylmethionine (SAM)-binding pocket. C10 demonstrates robust antiviral activity against SARS-CoV-2, including its variants, with EC50 values ranging from 64.03 to 301.9 nM, comparable to the FDA-approved drug remdesivir in our cell-based assays. C10 also exhibits broad-spectrum activity against other betacoronaviruses and inhibits SARS-CoV-2 at the replication stage. C10 suppresses viral translation and exhibits immunostimulatory effect. Additionally, C10 specifically reversed NSP14-mediated alterations in host transcriptome. The antiviral efficacy of C10 was further validated in a transgenic mouse model of SARS-CoV-2 infection. Our findings highlight C10 as a promising candidate for the development of effective treatments against SARS-CoV-2 and its emerging variants. This study also uncovers a novel mechanism of NSP14 in SARS-CoV-2 pathogenesis and its therapeutic potential, providing insights that may extend to other viral capping methyltransferases.

microbiology↗

bioRxiv2025

Human single-cell atlas analysis reveals heterogeneous endothelial signaling

Endothelial cells (ECs) play complex roles across tissues and vessel types. Yet, systematic investigations of EC heterogeneity in the combined context of vessel type and tissue microenvironment are still largely lacking. We integrated over three million single cells of scRNA-seq datasets in 15 human tissues and found that ECs in some tissues (e.g., heart and kidney) exhibited greater tissue specificity, while others displayed more substantial vessel specificity. We developed a computational pipeline to analyze cell-cell communications (CCC) mediated by metabolites or proteins to explore microenvironmental regulation. Interestingly, our results showed that CCC events involving ECs varied vastly across tissues, highlighting tissue-specific EC interactions. Using topic modeling, we identified CCC patterns, termed CCC topics, representing metabolite- and protein-mediated interactions between ECs and other tissue-resident cells. Most CCC topics exhibited high tissue specificity, potentially explaining microenvironmental regulations for EC heterogeneity. The work systematically investigates EC heterogeneity and provides insights into how EC heterogeneity was regulated across diverse tissue microenvironments.

bioinformatics↗

bioRxiv2025

A genetically engineered vertebrate animal model of NAA15-related heart disease uncovers myocardial growth, contractility, and mitochondrial defects

Delineating the genetic and environmental instigators of congenital heart disease (CHD), affecting up to 1% of newborns, will improve preventative, diagnostic, and therapeutic efforts to mitigate disease outcomes. Although mutations in NAA15, which encodes the auxiliary subunit of N-alpha-acetyltransferase A (NatA), are associated with CHD in humans, vertebrate animal models of NAA15-related heart disease have yet to be described. Therefore, we isolated zebrafish strains carrying null mutations in paralogs naa15a and naa15b, which we co-localized to the early larval myocardium. Double knockout (DKO) naa15-deficient larvae exhibited diminutive, lowly contractile, and bradycardic ventricles composed of fewer and smaller cardiomyocytes (CMs) incapable of proliferation. On a subcellular level, mutant CMs exhibited moderately disorganized myofibrils. CM-specific re-expression of naa15a partially rescued the contractility and growth deficits, revealing an indispensable myocardial function. Ubiquitous mis-expression of human NAA15 achieved complete rescue, enabling functional testing of human NAA15 variants. Animals with a reduced dosage (RD) of naa15 survived to adulthood and shared phenotypes with DKO larvae, including smaller ventricular chambers and CMs, which exhibited mosaic myofibril disarray. Deep quantitative proteomic profiling of WT and naa15RD adult hearts revealed differential expression of multiple protein classes, including several subunits of mitochondrial respiratory complex I, a component of the electron transport chain. Accordingly, we documented reduced mitochondrial content and function in the myocardium of naa15-deficient larvae. Taken together, our data reveal myocardial functional and structural abnormalities associated with mitochondrial dysfunction in a vertebrate animal model of NAA15-related heart disease.

developmental biology↗

bioRxiv2025

Biphasic Mechanical Loading Disrupts Cytoskeletal Symmetry in 3D Architected Scaffolds

Cells in load-bearing tissues experience both solid deformation and interstitial fluid flow during physiological loading, but the mechanisms by which they integrate these biphasic mechanical signals remain poorly understood. Here, we develop a porous, nanoarchitected 3D scaffold that allows simultaneous delivery and control of matrix strain and fluid shear stress. We validated the platform through fatigue loading experiments and simulations of fluid-structure interactions. In static culture, osteoblast-like cells adopted shapes, cytoskeletal architectures, and focal adhesion patterns templated by scaffold geometry. Under cyclic compression, the combined influence of matrix deformation and induced fluid flow disrupted this alignment, producing disordered actin structures and reduced focal adhesion eccentricity. These changes emerged even under low-frequency loading, within the drained poroelastic regime, indicating a high sensitivity of cytoskeletal organization to fluid-solid coupling. Our findings establish a tractable and tunable platform to investigate how cells sense and respond to dynamic biphasic mechanical environments in 3D. Significance StatementCells in tissues such as bone experience mechanical inputs from both matrix deformation and interstitial fluid flow. However, existing in vitro systems often isolate one type of input or lack the ability to control both independently. We engineered a nanoarchitected 3D scaffold that delivers tunable biphasic mechanical inputs by combining structural compression and fluid flow. Without external loads, cells align their cytoskeleton and focal adhesions to the scaffold geometry. When subjected to dynamic loading, they transition to disordered morphologies and less mature focal adhesions, suggesting a transition to migratory states. These results highlight the sensitivity of cells to even subtle biphasic cues and provide a new platform to study how cells integrate multiple mechanical signals in 3D environments.

bioengineering↗

bioRxiv2025

Derivation of Genetically-Defined Murine Hepatoblastoma Cell Lines with Angiogenic Potential

Background/ObjectivesHepatoblastoma (HB) is the most common form of pediatric liver cancer, with the vast majority of these tumors evidence of mutation and/or deregulation of the oncogenic transcription factors {beta}-catenin (B), YAP (Y) and NRF2 (N). HB research has been hampered by a paucity of established cell lines, particularly those bearing these molecular drivers. All combinations of B, Y and N (i.e. BY, BN, YN and BYN) are tumorigenic when over-expressed in murine livers but it has not been possible to establish cell lines from primary tumors. Recently, we found that concurrent Crispr-mediate targeting of the Cdkn2a tumor suppressor locus allows for such immor-talized cell lines to be generated with high fidelity. MethodsWe generated 5 immortalized cell lines from primary Cdkn2a-targeted BN and YN HBs and characterized their properties. Notably, 4 of the 5 retain their ability to grow as subcutaneous or pulmonary tumors in the immune-competent mice from which they originated. Most notably, when maintained under hypoxia conditions for as little as 2 days, BN cells reversibly up-regulated the expression of numerous endothelial cell (EC)-specific genes and ac-quired EC-like properties that benefited tumor growth. ConclusionsThe above approach is currently the only means by which HB cell lines with pre-selected, clinically relevant oncogenic drivers can be generated and the only ones that can be studied in immune-competent mice. Its generic nature should allow HB cell lines with other oncogenic drivers to be derived. A collection of such cell lines will be useful for studying tumor cell-EC trans-differentiation, interactions with the immune environment and drug sensitivities. Simple SummaryMost hepatoblastomas (HB) are associated with aberrant expression of {beta}-catenin (B), YAP (Y) and/or NRF2 (N) transcription factors and can be modeled in mice by over-expressing pairwise of triple combination of these. Virtually no human or murine HB cell lines exist that bear these mutations. We describe here an efficient way to generate cell lines from primary BN and YN tumors. Moreover, one of the BN lines shows a remarkable ability to trans-differentiate into endothelial cells under hypoxic conditions that may facilitate angiogenesis. These cell lines along with previousl-derived BN and BYN lines showed similar sensitivities to drugs commonly used to treat HB. Because the approach for cell line derivation we describe is quite general, it should allow for the generation of additional lines driven by less common factors. A collection of such permanent and well-characterized cell lines will facilitate studies that are difficult or impractical to perform in vivo.

cancer biology↗

bioRxiv2025

BioinAI: a general bioinformatic framework for multi-level transcriptomic data analysis using multiple semi-agents

Clinical and biological insights from large-scale transcriptomic data are often limited by technical variability and analytical complexity. Here, we introduce BioinAI, a general bioinformatic framework designed for multi-level transcriptomic data analysis. As part of the BioinAI framework, two algorithms named DeepAdvancer and stNiche were developed to improve data integration and analytical efficiency. DeepAdvancer leverages a class-aware adversarial autoencoder to reconstruct gene-expression profiles from 49,738 samples across 131 skin conditions. These profiles revealed a conserved inflammatory axis and a transcriptomic continuum which link diverse diseases through shared immune responses and structural programs. stNiche leverages spatial graph networks and symmetry-aware matching to identify functional cellular niches and reveals microstructural alterations across development, homeostasis, and disease. For instance, it identified a fibroblast-immune niche surrounding hair follicles in healthy skin, which disappears in pathological states. In addition, BioinAI provides a user-friendly online analysis platform powered by multiple semi-agents (www.bioinai.com), significantly facilitating the extraction of biological insights to advance scientific research.

bioinformatics↗

bioRxiv2025

Genome-Wide Investigation of Transcription Factor Occupancy and Dynamics Using cFOOT-seq

Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences. Notably, cFOOT-seq, combined with FootTrack analysis, predicts TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.

molecular biology↗

bioRxiv2025

Decoding Lymphangioleiomyomatosis (LAM) Niche Environment via Integrative Analysis of Single Cell Multiomics and Spatial Transcriptomics

Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease characterized by cystic destruction and metastatic growth of smooth muscle-like cells. Despite advances in understanding its genetic basis, the cellular heterogeneity, regulatory mechanisms, and microenvironmental interactions driving LAM progression remain poorly defined. In this study, we employed an integrative multi-omics approach combining single-cell RNA sequencing (scRNA-seq), single-nucleus ATAC sequencing (snATAC-seq), and high-resolution spatial transcriptomics (Visium, Visium HD, and Xenium) to decode the LAM niche in its native environment. We identified two spatially and functionally distinct LAM subtypes: LAMCORE1 and LAMCORE2. LAMCORE1 cells exhibited a uterine smooth muscle-like phenotype, expressing associated markers (ACTA2, MYH11) and were enriched in MTORC1 signaling and myogenic pathways, supporting a uterine origin. In contrast, LAMCORE2 cells displayed fibroblast-like features, with upregulated extracellular matrix (ECM) remodeling genes (COL1A1, MMP11) and epithelial-to-mesenchymal transition (EMT) pathways, suggesting a role in niche formation. Pseudotime and regulon analyses revealed dynamic transitions between these subtypes, driven by distinct transcriptional networks (e.g., HOX/PBX in LAMCORE1, TWIST/ZEB in LAMCORE2). The presence of the two distinct LAM subtypes was further validated by RNAscope and immunofluorescence microscopy. We identified LAM-associated fibroblasts (LAFs) as activated stromal cells expressing canonical markers (FAP, S100A4, VIM, IGFBP7, SPARC) and localized within LAM lesions. Subpopulations of LAFs, LAF-seed (proximal to LAMCORE1) and LAF-niche (surrounding LAM niches), exhibited unique functional profiles, including ECM deposition, TGF-{beta} signaling, and myofibroblast activation. Regulatory network analysis pinpointed EGR1 as a central hub governing LAF phenotype. Our comprehensive spatial profiling revealed niche structures dominated by LAMCORE1 cells and surrounded by lymphatic endothelial cells (LECs), LAFs, scattered LAMCORE2 cells, macrophages, and reprogrammed alveolar epithelial cells (AT2). ECM remodeling and aberrant organization of cable-like structures (-smooth muscle actin+) of the LAM niches were further validated by second harmonic generation microscopy. These findings provide a high-resolution blueprint of LAM pathogenesis, highlighting the interplay between uterine-derived LAMCORE cells, activated fibroblasts, and the remodeled lung microenvironment. They significantly enhance our understanding of the LAM niche microenvironment and offer insights into potential therapeutic targets and strategies for managing this complex disease.

genomics↗

bioRxiv2025

GeneInsight: Condensing Gene Set Knowledge via Language Models

Interpreting gene sets is often complicated by the overwhelming number of annotations associated with individual genes, making it difficult to extract meaningful biological insights. To address this issue, we developed GeneInsight, an AI-powered tool that combines advanced topic modelling with large language models to automatically synthesise diverse biological annotations from literature, gene ontologies, and databases such as STRING. GeneInsight consolidates extensive annotations into coherent thematic summaries that render such data readily interpretable, thereby enabling the rapid extraction of biologically significant insights that conventional enrichment analyses often overlook.

bioinformatics↗

bioRxiv2025

Hypermyelination Improves Strength and Detection of Neuronal Activity in the CA1 Hippocampus and Facilitates Neuroprotection in FusOLcKO Mice

1.0.Loss of oligodendrocytes (OLs) and myelin impairs cortical neuronal firing and network stability, whereas enhancement of oligodendrogenesis improves electrophysiological stability in cortex and, to a lesser extent, hippocampus. OLs exhibit regional heterogeneity, especially in their ability to synthesize cholesterol, a critical driver of myelin wrapping and ensheathment of axons. Conditional depletion of the Fused in sarcoma (Fus) gene in OLs, referred to as FusOLcKO, increases cholesterol biosynthesis, myelin thickness, and tissue cholesterol content. We examine whether this hypermyelination alters extracellular recordings across the layers of visual cortex and the underlying hippocampal CA1 over 16 weeks. In FusOLcKO mice, visually-evoked single-unit detectability and firing rate in CA1 increased relative to wild-type littermates, whereas cortical recordings showed no improvement. At the population level, FusOLcKO cortex exhibited reduced firing rates and lower functional connectivity, indicating altered network dynamics. Post-mortem histology revealed higher neuron density in recorded cortex and greater excitatory synapse density in CA1 of FusOLcKO mice suggesting region-specific neuroprotection and synaptic strengthening. These results demonstrate that cholesterol-driven hypermyelination enhances chronic hippocampal recordings while disrupting cortical network communication. Our study highlights myelins region-dependent roles in supporting single-cell reliability, tuning population dynamics, and maintaining circuit integrity under chronic perturbation. 2.0. SIGNIFICANCE STATEMENTMyelin critically regulates neural circuit function via conduction and metabolic support. Here, we show that cholesterol-driven hypermyelination in FusOLcKO mice augments single-unit detection and firing in hippocampal CA1 but reduces population firing and interlaminar connectivity within the cortex. These findings reveal a dual role for myelin: it can both safeguard specific circuit activity and perturb large-scale cortical communication. Understanding these dynamics is essential for designing myelin-targeted therapies in neurodegenerative disorders.

neuroscience↗

bioRxiv2025

Development of a method for large-scale single-molecule analysis of tau proteoforms

Proteins exist as diverse proteoforms resulting from a combination of genetic variation, alternative splicing, and post-translational modifications. Current methods struggle to capture this complexity at the single-molecule level. Here we introduce Iterative Mapping of Proteoforms (IMaP), a method that enables the massively-parallel interrogation of millions to billions of single-protein molecules through iterative probing with fluorescently labeled antibodies. Using 12 site-specific antibodies, the method is capable of measuring 212 (4,096) potential proteoform groups. We used IMaP to measure proteoform group profiles of the tau protein, a key player in neurodegenerative diseases, using two pan anti-tau antibodies (Tau-13, Tau-216), three isoform-specific antibodies (Anti-0N, Anti-2N, Anti-4R), and seven phosphosite-specific antibodies (Anti-pT181, Anti-pS202+pT205, Anti-pT205, Anti-pS214, Anti-pT217, Anti-pT231, and Anti-pS396). The method demonstrates high sensitivity (detecting proteoforms at 0.1% abundance), high reproducibility (median CV <5.5%), and broad dynamic range (>3 orders of magnitude), outperforming conventional techniques in resolving closely related proteoform groups. We demonstrated that the method can be used on relevant biological samples by examining various neuronal models (iNeuron cells, organoids, MiBrains, and mouse brains) and human samples. This examination revealed 130 distinct tau proteoform groups with as many as six phosphorylation events. The non-random distribution of these phosphorylation events suggests ordered and site-specific modification processes rather than random, stochastic accumulation. Certain combinations of phosphorylation events were more abundant than others; for example, pT217 preferentially co-occurred with pT181. In validating the applicability of the assay to human disease samples, we noted a specific pattern of multiple phosphorylation events in an advanced Alzheimers disease patient that suggests a sequential pathway of pathological tau modification. Iterative Mapping of Proteoforms provides insights into proteoform complexity at the single-molecule level, with significant implications for understanding protein regulation in neurodegenerative diseases and beyond.

biochemistry↗

bioRxiv2025

Microglia surveillance is directed toward neuron activation during sustained intracortical microstimulation

Intracortical microstimulation (ICMS) is a widely used tool for neuroprostheses, but its long-term efficacy is often limited by biofouling and neuroinflammatory responses at the electrode-tissue interface. Microglia orchestrate neuroinflammation and regulate synaptic plasticity, and low-frequency stimulation has been shown to promote anti-inflammatory microglial phenotypes. We therefore examined, over the first three days post-implantation, how 10-Hz ICMS influences microglial-neuronal interactions in vivo using two-photon imaging in Cx3cr1-GFP/jRGECO1a mice. A one-hour session of 10-Hz ICMS did not induce overt morphological activation of microglia but increased their process motility, directing extensions toward both the electrode and neurons exhibiting elevated calcium activity. By post-implantation Day 2, microglial extensions were significantly biased toward neurons whose {Delta}F/F surpassed a 3 standard deviation threshold after stimulation onset (74.26{degrees} {+/-} 11.83{degrees}) but shifted away from those same neurons after 40 min of continuous stimulation (116.99{degrees} {+/-} 9.19{degrees}) (p = 0.001732), suggesting a dynamic, homeostatic response to sustained neuronal calcium elevations. Although multi-day electrode insertion accelerated microglial motility and polarization toward the device, 10-Hz ICMS alone did not alter microglial branching or soma shape. Microglial contact frequency scaled with neuronal adaptation profiles: depressed neurons received the most contacts immediately post-implant (1.15 {+/-} 0.3 contacts; p = .0460). These findings reveal stimulus-associated, neuron-dependent surveillance behaviors of microglia during early post-implantation ICMS and implicate them as active participants in short-term modulation of cortical circuits.

bioengineering↗