Chemically induced senescence prompts functional changes in human microglia-like cells

In response to various stressors, cells can enter a state called cellular senescence which is characterized by irreversible cell cycle arrest and a senescence-associated secretory phenotype (SASP). The progressive accumulation of senescent glial cells in the central nervous system (CNS) with aging suggests a potential role for senescence as driver of aging and inflammation in the brain. As the main immune cell population residing in the CNS, microglia are thought to play a pivotal role in the progression of age-associated neuroinflammation. Furthermore, due to their slow turnover, microglia are highly susceptible to undergoing cellular senescence. However, current understanding of age-related changes in microglia and their impact on brain aging is limited. Due to the challenge in accessing human primary microglia and the lack of models to adequately recapitulate aging, this knowledge is predominantly limited to rodent studies. Here, we chemically induced senescence in a human immortalized microglia cell line with a cocktail of senescence inducing molecules. We demonstrate that chemically induced senescent microglia adopt a pro-inflammatory phenotype, have reduced phagocytic activity and impaired calcium activity. Our results show that chemically induced senescence can mimic features of cellular aging and can provide insight on the impact of aging and cellular senescence on human microglia. To validate that a cocktail of the three small molecules SBI-026965, Lopinavir and O-151 (SLO) could induce features of aging in human microglia, we treated human immortalized microglia cells HMC3 for 6 consecutive days. We then performed bulk RNA seq of 3 different experimental groups (vehicle control, SLO full dose, as published in Fathi et al., 2021 (DOI: 10.1111/acel.13541) and SLO 1/2 dose) and validated our findings with an array of experiments evaluating aging features, including nuclear structure, proliferation, p21 expression, beta-galactosidase activity, secretion of proinflammatory molecules, phagocytosis and calcium imaging Comparative gene expression profiling of our RNA-seq data were conducted for VEH controls and each SLO doses (SLO 1, SLO1/2).

面对各类应激源时，细胞可进入一种被称为细胞衰老（cellular senescence）的状态，其特征为不可逆的细胞周期阻滞与衰老相关分泌表型（senescence-associated secretory phenotype, SASP）。随着衰老进程，中枢神经系统（central nervous system, CNS）内衰老胶质细胞的进行性积累，提示衰老可能是大脑衰老与炎症的驱动因素之一。作为中枢神经系统内的主要免疫细胞群，小胶质细胞（microglia）被认为在年龄相关性神经炎症的进展中发挥关键作用。此外，由于其更新速率缓慢，小胶质细胞极易发生细胞衰老。然而，目前学界对衰老相关的小胶质细胞变化及其对大脑衰老的影响认知仍十分有限。由于获取人类原代小胶质细胞存在难度，且缺乏能充分复现衰老过程的模型，相关认知主要局限于啮齿类动物研究。本研究通过使用衰老诱导分子组合，对人类永生化小胶质细胞系诱导化学性衰老。研究证实，化学诱导的衰老小胶质细胞会呈现促炎表型，吞噬活性降低，且钙活动受损。研究结果表明，化学诱导的衰老可模拟细胞衰老的特征，有助于揭示衰老与细胞衰老对人类小胶质细胞的影响。为验证由SBI-026965、洛匹那韦（Lopinavir）与O-151组成的三小分子组合（SLO）可诱导人类小胶质细胞出现衰老特征，我们对人类永生化小胶质细胞系HMC3连续6天进行处理。随后我们对3组不同的实验样本（溶剂对照组、按Fathi等人2021年发表的研究[DOI: 10.1111/acel.13541]使用的SLO全剂量组与SLO半剂量组）进行批量RNA测序（bulk RNA seq），并通过一系列评估衰老特征的实验验证了研究结果，包括核结构、细胞增殖、p21蛋白表达、β-半乳糖苷酶活性、促炎分子分泌、吞噬作用与钙成像。我们对溶剂对照组与各SLO剂量组（SLO全剂量组、SLO半剂量组）的RNA测序数据进行了对比基因表达谱分析。