Aug 2026· Frontiers in Cell and Developmental Biology· Vol 14· 0 citations· 41 references
TL;DR
TIM-4 is established as a critical driver of neuroinflammation-associated neuronal death in TBI, representing a promising therapeutic target for TBI-related cognitive dysfunction.
Abstract
Traumatic brain injury (TBI) is a leading cause of severe disability, frequently resulting in persistent cognitive dysfunction. Microglial M1/M2 polarization is critically involved in TBI pathogenesis, yet its molecular regulatory mechanisms remain poorly understood. TIM-4, a TIM family member implicated in cerebral ischemia-reperfusion injury, has an unknown function in TBI—particularly regarding its regulation of neuronal death.
We employed a comprehensive multi-omics strategy integrating bulk RNA-seq, publicly available single-cell RNA sequencing (scRNA-seq; GEO: GSE101901), weighted gene co-expression network analysis (WGCNA), quantitative proteomics, phosphoproteomics, and epigenomic profiling (ATAC-seq and H3K27ac ChIP-seq) to systematically investigate TIM-4 in TBI. Functional validation included TIM-4 knockdown experiments, TUNEL apoptosis detection, Golgi staining for dendritic spine analysis, and behavioral assessments.
TIM-4 was the most significantly upregulated gene and protein across all omics layers, with expression positively correlated with pro-inflammatory factors and negatively correlated with anti-inflammatory markers. ScRNA-seq revealed TIM-4 upregulation was restricted to activated M1-like microglia, and pseudotime trajectory analysis demonstrated TIM-4-driven M1 polarization. WGCNA identified a TIM-4-associated co-expression module strongly correlated with TBI severity and behavioral outcomes (r = 0.92, p < 0.001). Phosphoproteomics identified TIM-4 Y
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hyper-phosphorylation as a key post-translational regulatory event, and ATAC-seq/ChIP-seq revealed NF-κB-driven chromatin remodeling at the TIM-4 locus. Multi-omics integration ranked TIM-4 as the master regulatory hub (composite evidence score = 0.89). Functionally, TIM-4 knockdown promoted microglial M2 polarization, reduced neuronal apoptosis and dendritic spine loss, and significantly improved spatial memory deficits and motor coordination following TBI.
TIM-4 is established as a critical driver of neuroinflammation-associated neuronal death in TBI, representing a promising therapeutic target for TBI-related cognitive dysfunction.
Apoptosis contributes substantially to neurological deficits after traumatic brain injury (TBI), yet the molecular drivers and cellular regulation of astrocyte-associated apoptosis remain poorly defined. We integrated bulk RNA-seq, single-cell RNA-seq, and spatial transcriptomics to identify apoptosis-related hub genes in TBI. Differentially expressed genes were intersected with apoptosis-related genes and ranked by protein-protein interaction network topology. Astrocyte heterogeneity was examined through sub-clustering, pseudotime, regulon, and in silico transcription-factor perturbation analyses. Findings were validated in vivo using a Feeney weight-drop model and in vitro using an astrocyte scratch model, with Western blot, immunofluorescence co-staining, siRNA knockdown, wound-healing, CCK-8, and flow-cytometric apoptosis assays. VIM and LGALS3 were identified as the apoptosis-related hub genes and showed reproducible injury-associated upregulation across multiple independent datasets. Single-cell analysis showed their expression in a reactive astrocyte subpopulation, with levels rising along the homeostatic-to-reactive trajectory. CEBPB, FOSL1/FOSL2, and MAFF showed higher regulon activity in TBI-reactive astrocytes, and there in silico knockout shifted reactive astrocytes toward the homeostatic state. Spatially, both transcripts accumulated in the lesion core and peri-lesional zones, coinciding with elevated apoptotic activity. Western blot confirmed their upregulation in vivo and in vitro, and immunofluorescence co-staining of injured cortical tissue showed their expression in GFAP-positive astrocytes; silencing either genes reduced astrocyte migration, and flow cytometry together with an increased cleaved-caspase-3/caspase-3 ratio indicated enhanced apoptotic signaling. In astrocytes after TBI, VIM and LGALS3 modulate migration and apoptotic signaling, representing candidate molecular markers and potential therapeutic targets for secondary brain injury that warrant further validation.
Binyang Wang, Yuxue Wang, Wen Cao et al.· Neuroscience· 0 citations
BACKGROUND
Microglia-mediated neuroinflammation and oxidative stress are pivotal drivers of secondary injury following traumatic brain injury (TBI). While neddylation governs essential cellular functions, its specific contribution to microglial activation and TBI pathology remains poorly understood.
METHODS
We integrated bulk microglial RNA sequencing profiles with single-cell RNA sequencing (scRNA-seq) datasets from TBI mouse brains. To assess therapeutic potential, we employed a controlled cortical impact mouse model and treated animals with the neddylation inhibitor MLN4924. The role of microglia was validated using microglia-depleted mice. Mechanistically, a combinatorial approach utilizing AlphaFold 3 molecular docking predictions, quantitative proteomics, and immunoprecipitation-mass spectrometry was performed to identify molecular targets.
RESULTS
We revealed a specific and robust up-regulation of neddylation exclusively within microglial clusters. Pharmacological inhibition of neddylation using MLN4924 significantly ameliorated neurological deficits, attenuated brain edema, and preserved blood-brain barrier integrity. Crucially, these neuroprotective benefits were abrogated in microglia-depleted mice, pinpointing microglia as the primary cellular target. We identified the glutamate-cysteine ligase modifier subunit (GCLM) as a novel substrate of the CUL3-KLHL12 E3 ligase complex. MLN4924 inhibits CUL3 neddylation, thereby impeding the CUL3-KLHL12-mediated ubiquitination and degradation of GCLM. Consequently, GCLM stabilization restores intracellular glutathione synthesis, effectively scavenging reactive oxygen species and mitigating neuroinflammation.
CONCLUSIONS
Our findings characterize the Neddylation-CUL3-KLHL12-GCLM axis as a critical regulator of microglial redox homeostasis and highlight this pathway as a promising therapeutic target for TBI intervention.
Yu Ni, Yuan-feng Liu, Xincheng Zhang et al.· Journal of Translational Med...· 0 citations
The secondary injury cascade following spinal cord injury (SCI) involves substantial metabolic reprogramming and immune responses. However, whether transcriptional signatures related to lactylation, ferroptosis, and neuroinflammation show coordinated changes after SCI, and which cellular states are associated with these signatures, remain unclear.
Public bulk transcriptomic datasets (GSE47681 and GSE5296) and a single-cell RNA-seq dataset (GSE162610) were integrated. Bioinformatic analyses included single-sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network analysis (WGCNA), protein-protein interaction analysis, pseudotime trajectory inference, and
in silico
perturbation analysis. A C5 unilateral contusion SCI mouse model was used for transcriptomic validation, behavioral assessments, Western blotting, and immunofluorescence. Human peripheral blood RNA-seq data (GSE151371) were used as an exploratory external reference for systemic SCI-associated expression patterns.
Bulk transcriptomic analysis revealed coordinated elevation of lactylation-related, ferroptosis-related, and inflammation-related signature scores after SCI, with the highest scores observed at 3 days post-injury. Single-cell RNA-seq localized these signatures predominantly to expanded disease-associated microglia (DAM)-like states. Network screening prioritized vimentin (Vim) as a candidate hub gene associated with the three signatures, and Vim-high pathological microglia exhibited an activated inflammatory-metabolic transcriptional state. In the SCI mouse model, VIM, pan-lysine lactylation (pan-Kla), acyl-CoA synthetase long-chain family member 4 (ACSL4), and interleukin-1β (IL-1β) were upregulated in whole-lesion spinal cord tissue. Immunofluorescence further showed increased VIM, pan-Kla, and ACSL4 signals with spatial overlap within IBA1-positive cells after SCI.
Pathological DAM-like microglia represent major microglial states exhibiting concurrent lactylation-related, ferroptosis-related, and inflammatory signatures after SCI. VIM was prioritized as a candidate hub associated with this pathological microglial program. These findings provide a cellular and molecular framework for future mechanistic studies of secondary SCI.
Jia-yu Chen, Kai Chen, Zu-cheng Huang et al.· Frontiers in Genetics· 0 citations
Spinal cord injury (SCI) leads to a complex cascade of cellular events, among which necroptosis plays a critical role in exacerbating neuronal injury and inflammation. In this study, we aimed to identify and validate key genes associated with necroptosis in SCI using bulk RNA‐seq data, followed by differential analysis and weighted gene coexpression network analysis (WGCNA). We identified several candidate necroptosis‐related genes, and further least absolute shrinkage and selection operator (LASSO) regression highlighted five SCI‐necroptosis differentially expressed genes (DEGs): toll‐like receptor 4 (Tlr4), Nlrp3, Il1b, Tnfaip3, and Stat4. These genes were validated using RT‐qPCR and western blot experiments. Our analysis revealed that necroptosis scores were significantly elevated following SCI. Single‐cell RNA sequencing (scRNA‐seq) and spatial transcriptomics (ST) analysis revealed that Tlr4 was upregulated in myeloid cells (microglia and macrophages) and played a pivotal role in triggering downstream necroptosis, which was confirmed by protein levels. In vitro and in vivo experiments confirmed that Tlr4 inhibition attenuated necroptosis and inflammation. This study is the first to establish Tlr4 as a direct upstream regulator of the pRIPK1/pRIPK3/pMLKL necroptotic axis in SCI, distinct from its role as a general inflammatory mediator, suggesting Tlr4 as a promising therapeutic target for functional recovery.
Wanzhou Wang, Lu Sun, Wei Xie et al.· Mediators of Inflammation· 0 citations
: Ischemic stroke (IS) is the most common form of stroke. It triggers complex neuroinflammatory responses involving diverse cell populations within the central nervous system. Among these, microglia exhibit significant functional heterogeneity in IS. MicroRNAs (miRNAs), which regulate gene expression at the post-transcriptional level, are increasingly recognized as key modulators of neuroinflammation and potential biomarkers for IS; however, their specific relationships with microglial subtypes remain poorly defined. This research obtained 29 miRNAs through comparing correlation analysis between mRNA and miRNA. Through manual annotations and microglial populations, microglial functions are grouped into 5 groups. The process obtained increasing amounts of M1-like pro-inflammatory cells and decreasing amounts of M2-like anti-inflammatory populations. Several miRNAs, including miR-21-5p, let-7e-5p, miR-149-5p, miR-615-3p, and miR-218-5p, were shared across both M1 and M2, suggesting central roles in regulating polarization balance. Biological processes linked M1-associated targets to immunity, whereas M2-associated genes were enriched for cellular division and differentiation. These findings highlight miRNA-mediated networks that may shape microglial responses and influence IS progression. These findings provide insight into the mechanisms underlying microglial heterogeneity and offer potential directions for developing diagnostic biomarkers and therapeutic targets for ischemic stroke.
Jianyang Su· Frontiers in Medical Science...· 0 citations
A high-resolution map of transcriptional and cellular dynamics during the EIP of AMI is delineates a coordinated network of inflammatory mediators linked to early myeloid cell recruitment and activation, revealing a coordinated network of inflammatory mediators linked to early myeloid cell activation.
Zeyang Wang, Jinhu Shi, Yinchuan Lai et al.· Frontiers in Cardiovascular...· 0 citations