Targeting RhoA nuclear mechanoactivity rejuvenates aged hematopoietic stem cells [scRNA-seq]

Biomechanical alterations contribute to the decreased regenerative capacity of hematopoietic stem cells (HSCs) upon aging. RhoA is a key regulator o­f m­ec­ha­no­si­gn­aling, but its role i­n m­ec­ha­no­tr­an­sd­uction in stem cell aging remains unclear. Here we show that murine HSCs respond to increased nuclear envelope (NE) tension by inducing NE translocation of P-cPLA2, which cell-intrinsically activates RhoA. Aged HSCs experience physiologically higher intrinsic NE tension, but reducing RhoA activity lowers NE tension in aged HSCs. Feature image analysis of HSC nuclei reveals that chromatin remodeling is associated with RhoA inhibition, including restoration of youthful levels of t­he h­et­er­oc­hr­omatin m­ar­ker H3K9me2 and a decrease in chromatin accessibility and transcription at retrotransposons. Finally, we demonstrate that RhoA inhibition upregulates Klf4 expression and transcriptional activity, improving aged HSC regenerative capacity and lympho/myeloid skewing in vivo. Together, our data outline an intrinsic RhoA-dependent mechanosignaling axis, which can be pharmacologically targeted to restore aged stem cell function. Overall design: Comparative gene expression profiling analysis of scRNA-seq data from young, aged and aged treated with RhoA-inhibitor (Ri) LSKs. For each condition, 3 biological replicates were performed. LSKs were sorted from bone marrow cells as Lin- Sca1+ cKit+.生物力学改变可导致衰老状态下造血干细胞（hematopoietic stem cells, HSCs）的再生能力下降。RhoA是机械信号转导的关键调控因子，但目前尚不明确其在干细胞衰老过程中机械转导环节的作用。本研究发现，小鼠造血干细胞可通过诱导磷酸化胞质磷脂酶A2（P-cPLA2）的核膜易位，响应升高的核膜张力，进而在细胞内激活RhoA。衰老造血干细胞本身存在更高的生理性固有核膜张力，而抑制RhoA活性可降低衰老造血干细胞的核膜张力。对造血干细胞细胞核的特征图像分析显示，染色质重塑与RhoA抑制存在关联，具体包括恢复年轻态的异染色质标记物H3K9me2水平，以及降低反转录转座子（retrotransposons）的染色质开放性与转录活性。最后，本研究证实，抑制RhoA可上调Kruppel样因子4（Klf4）的表达与转录活性，在体内改善衰老造血干细胞的再生能力以及淋巴/髓系分化偏向性。综上，本研究数据揭示了一条依赖于RhoA的内在机械信号转导轴，可通过药理学靶向该轴以恢复衰老干细胞的功能。总体实验设计：对年轻、衰老以及经RhoA抑制剂（Ri）处理的衰老LSK细胞的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据开展比较基因表达谱分析。每组实验设置3次生物学重复。LSK细胞通过分选骨髓来源的Lin⁻ Sca1⁺ cKit⁺群体获得。