Investigating the role of autophagy-mitochondria-NAD+ axis in ageing

背景：自噬是降解功能障碍的蛋白质、蛋白质聚集体和细胞器(如受损的线粒体)的一种重要的细胞质量控制途径。通过自噬选择性地降解线粒体称为有丝分裂。来自我们和其他机构的数据强烈表明，一般自噬功能受损，特别是有丝分裂吞噬功能受损，是细胞和有机体老化的原因。该项目将确定自噬/有丝分裂功能障碍对细胞的影响，并制定干预措施以延缓与年龄相关的功能衰退。目的：1.研究自噬/有丝分裂吞噬功能障碍对细胞功能和存活的影响。这些模型的表型特征表明存在细胞衰老的关键特征-线粒体功能障碍、氧化应激和不可逆转的细胞周期停滞。此外，我们观察到应激反应通路，sirtuins和parps的过度激活，以及一种特殊的代谢特征-烟酰胺腺嘌呤二核苷酸(NAD+)的耗竭。该项目的第一个目标是使用哺乳动物和酵母(Supervisor 2实验室放置)细胞模型来建立表型之间的关系，并解释导致衰老的事件序列。这些机制研究将允许测试药物干预(监督员3的专业知识)，这将挽救自噬/有丝分裂功能障碍细胞的衰老表型。利用干细胞来源的人类神经元建立生理相关性。初级主管实验室还建立了两个具有自噬和有丝分裂功能障碍的人类可诱导多能干细胞(IPSC)来源神经元模型。第二个目标将测试在人类神经元的自噬/有丝分裂基因敲除模型中观察到的关键表型和机制的相关性。正在研究中的小分子靶向通路将测试它们潜在的治疗相关性。新奇：尽管线粒体功能障碍、自噬/有丝分裂缺陷和代谢缺陷已被确定为细胞和生物衰老的标志，但我们缺乏对它们如何相互联系并导致年龄相关下降的了解。该项目将有助于我们建立一个更全面的细胞衰老模型。时效性：通过建立将自噬/有丝分裂功能障碍与细胞和生物衰老联系起来的机制，该项目将确定新的抗衰老干预措施点，并使用与生理相关的人类神经元模型对其进行测试。实验方法：该项目将使用我们实验室建立的广泛的模型和技术。生化(免疫印迹、酶分析)、成像(包括超分辨率显微镜)、功能(线粒体呼吸)和代谢分析将被用来测量自噬/有丝分裂吞噬、线粒体功能、应激反应通路和细胞存活/衰老的变化。关键词：衰老/衰老/自噬/蛋白沉积/NAD

Background: Autophagy is an essential cellular quality control pathway degrading dysfunctional proteins, protein aggregates and organelles, such as damaged mitochondria. Selective degradation of mitochondria by autophagy is called mitophagy. Data from us and others strongly implicate impairment of general autophagy, and specifically mitophagy, as a cause of cellular and organismal ageing. This project will determine consequences of autophagy/mitophagy dysfunction for the cell and establish interventions to delay age-related functional decline. Objectives:1. Investigate the impact of autophagy/mitophagy dysfunction on cell function and survival.Mouse cell lines with the loss of autophagy and mitophagy have been generated in Primary supervisor's lab using CRISPR/Cas9. Phenotypic characterisation of these models indicated the presence of the key features of cellular senescence - mitochondrial dysfunction, oxidative stress, and irreversible cell cycle arrest. Additionally, we observed hyperactivation of stress response pathways, Sirtuins and PARPs, and a specific metabolic signature - depletion of nicotinamide adenine dinucleotide (NAD+). The first objective of this project is to use mammalian and yeast (Supervisor 2 laboratory placement) cell models to establish the relationship between the phenotypes and explain the sequence of events leading to senescence. These mechanistic studies will allow to test drug interventions (Supervisor 3 expertise) that would rescue senescence phenotypes in cells with autophagy/mitophagy dysfunction.2. Establish physiological relevance of the mechanisms under study using stem cell-derived human neurons.The laboratory of Primary supervisor also established 2 human inducible pluripotent stem cell (iPSC)-derived neuronal models with autophagy and mitophagy dysfunction. The second objective will test the relevance of the key phenotypes and mechanisms observed in the autophagy/mitophagy knockout models in human neurons. Targeting pathways under study with small molecules will test their potential therapeutic relevance.Novelty: Although mitochondrial dysfunction, autophagy/mitophagy defect and metabolic deficit have been established as hallmarks of cellular and organismal ageing, we lack in our understanding of how they interconnect and contribute to age-related decline. The project will contribute to our attempts to build a more comprehensive model of cellular ageing.Timeliness: By establishing mechanisms linking autophagy/mitophagy dysfunction to cellular and organismal ageing the project will identify new points for anti-ageing interventions and test them using physiologically relevant human neuronal model.Experimental approach: The project will use a wide range of models and techniques established in our laboratories. Biochemical (immunoblotting, enzymatic assays), imaging (including super-resolution microscopy), functional (mitochondrial respiration) and metabolic assays will be used to measure changes in autophagy/mitophagy, mitochondrial function, stress response pathways, and cell viability/senescence.Keywords: ageing/senescence/autophagy/proteostasis/NAD