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Chronic stress promotes intestinal stem cell aging [RNA-seq]

ABSTRACT: To elucidate alterations in intestinal cell types under chronic stress, we conducted scRNA-seq analysis of intestinal crypts from NT and ES models. After that, we found that significant changes in ISCs in the ES group compared to the NT group. To validate the functional roles of ISCs, we performed RNA-seq of ISCs under different treatment conditions, we identified Chrm3-dependent differential genes between NT and ES groups, particularly noting downregulated genes associated with stemness and proliferation (e.g., Olfm4, Lgr5, and Mcm4), and upregulated genes linked to aging and calcium signaling pathways (e.g., Cdkn1a, Orai1, and Chp2), which contribute to ISC aging. These findings provided mechanistic insights into targeting these pathways to enhance intestinal function and integrity. Furthermore, to assess the impact of stress-induced changes in microbiota composition on ISC stemness, we synchronized microbiota between NT and ES groups through co-housing conditions and employed 16S rDNA sequencing. This analysis aimed to ascertain the possibility that changes in the microbiota composition whether contribute to the decline in ISC stemness under stress conditions. scRNA-seq of crypts were used to to characterize the diversity of cell lines under chronic stress. RNA-seq of ISC in Chrm3Lgr5+/+ and Chrm3Lgr5-/- mice from NT and ES mice were taken to delineate altered pathways and the mechanisms underlying ISC changes in ES model. 16S rDNA-seq were employed to confirm microbiota synchronization between NT and ES groups under co-housing conditions.

ORGANISM(S): Mus musculus

PROVIDER: GSE262228 | GEO | 2025/02/08

REPOSITORIES: GEO

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Project description:Background & Aims: Hierarchical organization of intestine relies on their stem cells by self-renew and producing committed progenitors. Although signals like Wnt are known to animate the continued renewal by maintaining intestinal stem cells (ISCs) activity, molecular mechanisms especially E3 ubiquitin ligases that modulate ISCs ‘stemness’ and supportive niche have not been well understood. Here, we investigated the role of Cullin 4B (Cul4b) in regulating ISC functions. Methods: We generated mice with intestinal epithelial-specific disruption of Cul4b (pVillin-cre; Cul4bfn/Y), inducible disruption of Cul4b (Lgr5-creERT2; Cul4bfn/Y, CAG-creERT2; Cul4bfn/Y) and their control (Cul4bfn/Y). Intestinal tissues were analyzed by histology, immunofluorescence, RNA sequencing and mass spectrum. Intestinal organoids deprived from mice with pVillin-Cre; Cul4bfn/Y, Lgr5-Cre; Cul4bfn/Y, Tg-Cul4b and their controls were used in assays to measure intestinal self-renewal, proliferation and differentiation. Wnt signaling and intestinal markers were analyzed by immunofluorescence and immunoblot assays. Differential proteins upon Cul4b ablation or Cul4b-interacting proteins were identified by mass spectrometry. Results: Cul4b specifically located at ISCs zone. Block of Cul4b impaired intestinal homeostasis maintenance by reduced self-renewal and proliferation. Transcriptome analysis revealed that Cul4b-null intestine lose ISC characterization and showed disturbed ISC niche. Mechanistically, reactivated Wnt pathway could recover intestinal dysfunction of Cul4b knockout mice. Analysis of differential total and ubiquitylated proteins uncovered the novel targeting substrate of Cullin-Ring ubiquitin ligase 4b (CRL4b), immunity-related GTPase family M member 1 (Irgm1) in intestine. Decreased Irgm1 rescued abnormally interferon signaling, overemphasized autophagy and downstream phosphate proteins in Cul4b knockout mice. Conclusion: We conclude that Cul4b is essential for ISC self-renewal and Paneth cell function by targeting Irgm1 and modulating Wnt signaling. Our results demonstrate that Cul4b is a novel ISC stemness and niche regulator.

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Chronic stress promotes intestinal stem cell aging [RNA-seq]

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