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ALKBH5 Demethylates the m6A Modification of SOCS3 in Microglia/Macrophages and Alleviates Neuroinflammation after Brain Injury [scRNA-seq]

ABSTRACT: Microglia/macrophage-induced neuroinflammation plays a crucial role in the progression of traumatic brain injury (TBI). However, the involvement of N6-methyladenosine (m6A) RNA modifications in this process remains elusive. Single-cell RNA sequencing (scRNA-seq) and m6A RNA immunoprecipitation sequencing (MeRIP-seq) across multiple time points post-injury revealed a strong correlation between m6A modifications and genes enriched in microglia/macrophage. Furthermore, the m6A demethylase ALKBH5 was identified as a key regulator of dynamic m6A patterns at the injury site. ALKBH5 suppression in microglia/macrophage exacerbated neuroinflammation in vitro and worsened neurological deficits in controlled cortical impact (CCI) models. MeRIP-qPCR and RNA pull-down assays revealed SOCS3 was a downstream target of ALKBH5-mediated m6A demethylation. This demethylation stabilized Socs3 mRNA and enhanced its protein expression, which in turn suppressed neuroinflammation via inhibiting the JAK2-STAT3 pathway. Conversely, SOCS3 depletion impaired functional recovery after injury. These findings unveiled a critical ALKBH5-m6A-SOCS3 regulatory axis that mitigated microglia/macrophage-driven neuroinflammation after TBI, underscoring its potential as a therapeutic intervention target for TBI progression.

ORGANISM(S): Mus musculus

PROVIDER: GSE302863 | GEO | 2025/09/05

REPOSITORIES: GEO

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Project description:Senescent foamy macrophages are the key pathogenic drivers of atherosclerosis (AS) progression and plaque stability. The RNA N6-methyladenosine (m6A) modification plays an important role in the development of various diseases. However, its role in foamy macrophage aging during AS is still not clarified. To assess m6A methylation levels and m6A modifying enzyme profiles, the infiltrated macrophages were sorted using CD68+ magnetic beads from plaque tissues of patients with AS and high-fat diet feeding Apoe-/- mice. The ALKBH5f/f, Lyz2Cre, Apoe-/- mice were constructed to observe senescent macrophage infiltrated in plaques and AS progression. The RNA-seq, MeRIP, RIP and dual luciferase assays were performed to explore the mechanisms of ALKBH5-mediated formation of senescent macrophage. Downregulated m6A methylation levels and upregulated ALKBH5 expression were observed in aortic plaques and infiltrated macrophages from AS patients and mice. Compared with control mice, ALKBH5f/f, Lyz2Cre, Apoe-/- mice decreased AS plaques and enhanced plaque stability by suppressing senescence of foamy macrophages and senescence-associated secretory phenotypes. In addition, the data of the RNA-seq, MeRIP, RIP and dual luciferase assays showed that ALKBH5 deletion reduced the mRNA level of CC chemokine ligand 5 (CCL5) by increased m6A methylation in macrophages. Actinomycin D assay indicated ALBH5 knockout destroyed stability of Ccl5 mRNA. Mechanismly, ALKBH5 promotes senescent macrophage formation by CCL5/CC chemokine receptor 5 (CCR5)/mTORC1/autophagy signaling. And ALKBH5 induced macrophage derived CCL5 recruits increasingly CD8+IFNγ+ T cells by CCL5-CCR5 axis. Furthermore, the application of ALKBH5 inhibitor IOX-1 and CCR5 monoclonal antibody Adaptavir are potential clinical interventions for the inhibition of senescent macrophage formation and AS progress. Myeloid ALKBH5 deletion mitigates AS progression by suppressing the formation of senescent macrophages and the recruitment of CD8+IFNγ+ T cells. These findings favour the ALKBH5/CCL5/CCR5 signaling as novel targets for atherosclerosis therapy.

Project description:Traumatic brain injury (TBI) induces neuroinflammatory innate immune responses that plays roles in both worsening brain damage and facilitating functional recovery. A major goal is to understand the heterogeneity of the immune responses to TBI, and to precisely identify key components that impact functional outcomes. We previously demonstrated that genetically targeting Ccr2 in a mouse model of controlled cortical impact led to neuroprotection in TBI. Our current studies of TBI use single cell RNA sequencing of over 10,000 TBI ipsilateral brain leukocytes to examine the mechanisms associated with the observed benefit in Ccr2-/- mice by comparing gene expression in leukocyte subsets from Ccr2-/- mice to gene expression in C57BL/6 wild type mice. Unbiased clustering identified two monocyte subsets, Chil3hi Ly6Chi classical monocytes and Gpnmbhi Ly6Clo nonclassical monocytes, and nine microglia states in the ipsilateral TBI brain. Comparative analysis between the genotypes revealed that Ccr2-/- TBI mice contained reduced numbers of inflammatory macrophages. In TBI, we observed a subset of microglia highly expressing several type I interferon-stimulated genes (ISGs) and is designated as Irf7hi microglia. Notably, unbiased differential expression analysis detected a two-fold reduction in the type I interferon response in multiple Ccr2-/- TBI microglia subsets compared to wild type TBI microglia. Treatment post-injury with a human CCR2 (hCCR2) inhibitor, CCX872, in hCcr2 knock-in mice improved cognitive function post-TBI, and also correlated with reduced expression of a key ISG, Irf7. We identified and characterized macrophage and microglia subsets during acute TBI. Our data showed that a reduction in macrophage expansion in TBI by both genetic and pharmacological methods, improved TBI and correlated with a reduction in the type I IFN response. These data indicate that macrophage expansion co-directs a type IFN response in microglia, and that targeting macrophage expansion in the brain can alter the profile of microglia subsets and lead towards improved functional outcomes.

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ALKBH5 Demethylates the m6A Modification of SOCS3 in Microglia/Macrophages and Alleviates Neuroinflammation after Brain Injury [scRNA-seq]

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