Investigation of mechanisms underlying cellular senescence and the Senescence-associated secretory phenotype (SASP) during brain ageing

大脑老化与神经炎症、神经变性和认知能力下降有关。我们最近已经表明，神经元可以获得衰老样表型，显示出各种衰老标志，如持续DNA损伤反应的激活，衰老相关的β-半乳糖苷酶活性以及促炎细胞因子和活性氧（ROS）的产生增加。由衰老细胞产生的ROS和促炎因子都可以分泌并有助于诱导周围年轻细胞的衰老，这种现象被称为“旁观者效应”。然而，在神经元衰老的背景下，衰老旁观者效应还没有研究。在这个项目中，我们假设，从衰老的神经元和/或小胶质细胞分泌的因子可以促进旁观者效应，这可能会加速神经退行性变和功能下降。为此，学生将：1）比较衰老小胶质细胞和从小鼠分离的神经元中衰老相关分泌表型（SASP）的组成。2）研究衰老神经元和小胶质细胞中的SASP因子是否可以诱导旁分泌衰老，以及是否需要细胞与细胞的接触。3）测试参与SASP调节的信号通路如p38 MAPK和mTORC 1的下调是否可以抑制神经元/小胶质细胞的衰老并改善体内神经元的功能。4）最后，学生将研究衰老过程中神经元核膜稳定性和SASP之间的联系。该项目将使用团队中已建立的方法（包括固定和活细胞的共聚焦显微镜、蛋白质印迹和免疫组织化学）。SASP和分子机制将使用神经元培养物，分离的成年神经元和小鼠大脑进行检查。将通过测量mRNA水平（PCR、PCR阵列或RNAseq）和蛋白质分泌（抗体阵列）来研究SASP的变化。SASP对神经元衰老的影响将使用一组成熟的标记物如端粒相关DNA损伤灶（TAF）、Sen-beta-Gal、p21和核膜维持标记物来研究。

Brain ageing is associated with neuroinflammation, neurodegeneration and cognitive decline. We have recently shown that neurons can acquire a senescent-like phenotype displaying various senescent hallmarks such as activation of a persistent DNA damage response, Senescence-associated-beta-Galactosidase activity and increased production of both pro-inflammatory cytokines and Reactive Oxygen Species (ROS). Both ROS and pro-inflammatory factors which are produced by senescent cells can be secreted and contribute to induction of senescence in surrounding young cells, a phenomenon termed as the "bystander-effect". However, the senescence bystander effect has not been investigated in the context of neuronal senescence.In this project, we hypothesise that secreted factors from senescent neurons and/or microglia can promote a bystander effect which may accelerate neurodegeneration and functional decline. For that, the student will:1) Compare the composition of the senescent associated secretory phenotype (SASP) in aged microglia and neurons isolated from mice.2) Investigate whether SASP factors from senescent neurons and microglia can induce paracrine senescence and if cell-to-cell contact is required.3) Test whether downregulation of signalling pathways involved in SASP regulation such as p38MAPK and mTORC1 can suppress neuronal/microglia senescence and improve neuronal function in vivo.4) Finally, the student will investigate links between nuclear envelop stability and the SASP in neurons during ageing.The project will use established methods in the team (including confocal microscopy of fixed and live cells, western blotting and immunohistochemistry). SASP and molecular mechanisms will be examined using neuronal cultures, isolated adult neurons and mouse brains. Changes in SASP will be investigated by measuring mRNA levels (PCR, PCR array or RNAseq) and secretion of proteins (antibody arrays). Effect of SASP on neuronal senescence will be studied using a set of well-established markers like Telomere Associated DNA damage Foci (TAF), Sen-beta-Gal, p21 and markers of nuclear envelop maintenance.