5×FAD转基因痴呆鼠小胶质细胞异常老化的端粒调控机制

Both senescence and β-amyloid-induced immunological abnormity in brain are the key mechanisms in the progress of Alzheimer's disease (AD). Of note, one of pivotal driving senescence factors is telomere damage in the process of aging in mammalian brain. Our preliminary data showed that a progressive cognitive impairment accompanied with intracellular Aβ or Aβ plaque accumulating in the brain of 5×FAD transgenic mice occurred with aging, as well as abnomal microglial activation and cellular senescence. Moreover, we also found that the early Aβ form- - Aβ oligomers, not fibrils, could induced a potent level of intracellular reactive oxygen species (ROS) in microglia, which is similar to tert-Butyl hydroperoxide (t-BHP) that can typically induce "replicative senescence" in the WI-38 cellular senescence model. However, it is unclear that how endogenous Aβ triggers telomere-damage signaling, whereby leading to microglial aging and dysfunction in AD. Hence, we propose an important and significant hypothesis that whether or not endogenous Aβ, with aging advance, step-by-step induces the telomere-damage of microglia, which triggers the cellular senescence and subsequently leads to microglial dysfunction and immonological abnormity, then eventually exacerbates the development of AD pathology. Therefore, this project will study the changes of microglial function and senescence indexes, and the relationship between them in 5×FAD transgenic mice wih aging.This study will reveal the essence that how endogenous Aβ in different stages of AD induce microglial "replicative senescence", thereby leading to the development of AD pathology and cognitive impairments. Furthermore, using a cultured microglia derived from the aged 5×FAD transgenic mice or Aβ-induced microglial senescence model, this study will explore the mechanisms of telomere-damage signaling on endogenous Aβ affecting microglial dysfunction with aging in AD.

衰老及Aβ诱导的胶质细胞免疫异常是触发Alzheimer病(AD)发生发展的重要机制。端粒损伤是脑老化的重要驱动因素。我们前期研究显示5×FAD转基因鼠随增龄过程出现明显的认知障碍、脑内细胞内外Aβ沉积加剧、小胶质细胞免疫异常活化和老化现象；而且早期的Aβ寡聚体比晚期的凝聚体诱导了小胶质细胞产生更强烈的触发端粒损伤的ROS。然而，内源性的Aβ随增龄进程如何触发端粒损伤、引起小胶质细胞衰老和免疫功能紊乱、进而加剧AD进展的机制还不明确。本项目通过研究增龄过程中5×FAD转基因痴呆鼠小胶质细胞免疫功能与衰老指标的变化及其关系，揭示AD不同疾病期内源性Aβ诱发小胶质细胞衰老、功能缺陷、继而引起AD病理级联和认知障碍的本质；在此基础上通过体外分离老化的AD鼠来源的小胶质细胞和Aβ诱导小胶质细胞的"复制衰老"模型，应用端粒酶激活剂和抑制剂，探讨老化过程Aβ诱导小胶质细胞端粒损伤及调控衰老的分子机制。

衰老及Aβ 诱导的胶质细胞免疫异常是触发Alzheimer 病(AD)发生发展的重要机制。端粒损伤是脑老化的重要驱动因素。我们前期研究显示 5×FAD 转基因鼠随增龄过程出现认知障碍、脑内Aβ沉积加剧、小胶质细胞免疫异常活化和老化现象及早期的 Aβ寡聚体诱导小胶质细胞产生更强的触发端粒损伤的ROS。然而， Aβ随增龄进程如何触发端粒损伤、引起细胞衰老和免疫紊乱、加剧AD 进展的机制还不明确。本项目研究增龄过程5×FAD 转基因痴呆鼠小胶质细胞免疫功能与衰老指标的变化及其关系，揭示AD 不同疾病期内源性Aβ诱发脑细胞衰老、功能改变、引起AD 病理级联、导致认知障碍的本质。研究结果发现：AD转基因痴呆鼠Aβ高负荷环境加速海马神经组织衰老、认知损害以及神经免疫功能紊乱并与衰老相关的细胞周期蛋白P16、P21以及端粒酶相关因子TRF1和TRET等密切相关；同时AD痴呆鼠小胶质细胞相关受体TGFβ、FPR2、TREM2、CX3CR1、DAP12、炎症细胞因子IL-1β、TNFα、IL-6等mRNA水平随增龄过程变化。在高龄5XFAD鼠脑内，TNFα、IL-6、FPR2等炎症与吞噬相关基因比年轻鼠表达明显减少，强烈提示：年老的5XFAD鼠在Aβ内环境诱导下加剧了小胶质细胞功能退行性改变、免疫防御下降、细胞反应能力（分泌、趋化、吞噬功能）减退。有趣的是，小胶质细胞关键的吞噬、趋化功能相关基因TREM2、CX3CR1、DAP12表达在5XFAD痴呆鼠老化过程明显上调，经分子筛选发现小胶质细胞增殖、老化的关键小分子PU.1，能够精细调控AD模型脑小胶质细胞表达的TREM2膜蛋白（AD的风险因子），从而揭示了老化过程PU.1分子调控小胶质细胞免疫吞噬功能的分子机制。在上述基础上，本项目创新性地发现：植物单体环黄芪醇CAG具有端粒酶活性，CAG通过调节衰老相关的细胞周期蛋白P16、P21和P53等基因表达、改善中年痴呆模型鼠认知行为异常，同时对痴呆鼠的非认知功能（运动功能）也具有明显改善作用；小动物PET-CT显像显示，CAG明显改善老化的5XFAD小鼠和野生鼠脑18F-FDG 葡萄糖代谢。这些发现揭示了老化过程Aβ诱导小胶质细胞端粒损伤及端粒酶调控免疫衰老的分子机制，本项目的创新性发现为AD的防治提供了一个新的策略和思路，具有非常重要的临床转化意义。

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