Is the epigenetic clock accelerated by mitochondrial reprogramming via mtDNA heteroplasmy and deemed quality control?

目的本项目旨在评估年龄相关应激源中线粒体DNA异质性：同质性比率和线粒体质量控制（线粒体自噬）过程之间的相互作用，以揭示它们对表观遗传时钟的影响以及这些影响如何被调控。线粒体自噬（针对线粒体的自噬）已被提出通过去除线粒体网络中表现出功能障碍的部分来纠正线粒体DNA异质性3。因此，它的衰变将增加mtDNA异质性，推动年龄表型。一种与年龄相关的细胞应激源是病原体感染的累积效应;甲型流感病毒通过每年的流行病对老年人造成重大疾病负担，并破坏线粒体自噬8。因此，我们将采用实验条件，包括流感感染的细胞，营养修饰和药物/配体治疗，以模拟衰老相关的细胞应激源。在这些细胞中，我们将分析线粒体DNA异质性、线粒体稳态和核DNA的表观遗传重组，以了解这些是如何导致细胞脆弱和死亡的。即：目的I-评估年龄相关应激因素引起的线粒体DNA异质性和甲基化程度;目的a.建立要用流感病毒感染的衰老细胞系;B.提取线粒体和基因组DNA、RNA和蛋白质;测序DNA库并评估其甲基化;AIM II-线粒体健康、质量控制机制和与细胞核的反向相互作用的特征参数;目的。使用在AIM I中建立的细胞，我们将：测量细胞氧化还原应激、线粒体OXPHOS和脂质过氧化;B.细胞自噬、线粒体自噬和病毒自噬（异种自噬）的图像事件;c.这两种质量控制途径的决定因素的概况表达和功能;d.绘制线粒体与细胞核逆行通讯的真实读数（例如NfkB）;AIM III-测试逆转mtDNA异质性的化学物质和诱导线粒体自噬的化学物质对线粒体稳态、DNA甲基化和基因组重编程的影响;目的。在用ShifBioscience化学品和线粒体自噬药物治疗后，我们将：重新评估呼吸和氧化的线粒体参数;B.验证自噬、线粒体自噬和异噬;c.监测线粒体和基因组DNA的甲基化;d.分析通过激活线粒体与细胞核的逆行反应诱导的基因;方法学将人成纤维细胞和肺细胞系A549在早期和晚期传代数用流感A/PR 8/34（H1N1）感染，其是显示破坏线粒体自噬的菌株8。PD患者来源的细胞系中含有60%的mtDNA异质性，将作为阳性对照纳入分析。Shift Bioscience设计的mtDNA异质性和甲基化程度测序方案将沿着他们的化学文库，能够降低mtDNA突变程度。营养品（如尿石素）和药物（如p62/SQSTM 1介导的线粒体自噬诱导剂PMI）来源的线粒体自噬诱导剂6也将与TSPO的配体组合入组，TSPO是一种保守的线粒体自噬抑制剂，随年龄增长而积累7。将测试线粒体自噬和线粒体健康，测量活性氧（ROS）、OXPOS参数和Ca 2+信号传导。转录组分析将用于基因表达谱分析，并对应激诱导的线粒体状态影响中涉及的细胞通路进行全局无偏分析。该项目将为表观遗传重编程的线粒体机制的隐藏方面提供信息，突出显示在年龄模拟条件下异质性和细胞器功能障碍之间的层次结构。将获得通过绘制线粒体与细胞核的逆向通讯来延缓细胞衰老的新方案以及潜在地改变这一点的方法。

AimsThis project aims to assess the interplay between mtDNA heteroplasmy:homoplasmy ratio and autophagic processes of mitochondrial quality control (mitophagy) in age associated stressors to unveil their impact on the epigenetic clock and how these could be pharmacologically controlled.Mitophagy (autophagy targeted to mitochondria) has been proposed to rectify mitochondrial DNA heteroplasmy by removing that portion of the mitochondrial network which manifests dysfunctions3. Its decay will therefore increase mtDNA heteroplasmy, propelling the age phenotype. One age associated cellular stressor is the cumulative effects of pathogen infections; influenza A virus is a major disease burden on the elderly via annual epidemics and it subverts mitophagy8. Therefore we will employ experimental conditions including influenza-infected cells, nutrient modification and treatment with drugs/ligands to model ageing associated cellular stressors. In these cells we will profile mtDNA heteroplasmy, mitochondrial homeostasis, and the driven epigenetic repogramming of nuclear DNA in order to learn how these underlie cellular frailty and demise.Namely we shall:AIM I- Assess the degree of mtDNA heteroplasmy and methylation caused by age-associated stressors;Objectivesa. Establish senescent cell lines to be infected with influenza virus;b. Extract mitochondrial and genomic DNA, RNA and proteins;c. Sequence DNA pools and assess their methylation;AIM II- Profile parameters of mitochondrial health, quality control mechanisms and retro-interplay with the nucleus;Objectives. Using cells established in AIM I we will:a. Measure cellular redox-stress, mitochondrial OXPHOS and lipid peroxidation;b. Image events of cellular Autophagy, Mitophagy and Viral Autophagy (Xenophagy);c. Profile expression and function of determinants of both these pathways of quality control;d. Map bonafide read-outs of mitochondrial retrograde communication with the nucleus (e.g. NfkB);AIM III- Test the effects of chemicals reversing mtDNA heteroplasmy and those inducing mitophagy on mitochondrial homeostasis, DNA methylation and genome reprogramming;Objectives. Following treatment with ShifBioscience chemicals and mitophagy drugs we will:a. Re-assess mitochondrial parameters of respiration and oxidation;b. Verify Autophagy, Mitophagy and Xeniphagy;c. Monitor methylation of mitochondrial and genomic DNA;d.Profile genes induced by activation of the mitochondrial retrograde response with the nucleus;MethodologyHuman fibroblasts and lung cell line A549, at early and late passage number will be infected with influenza A/PR8/34 (H1N1), which is the strain shown to subvert mitophagy8. PD patient-derived lines containing 60% mtDNA heteroplasmy will be enrolled in the analysis as positive control.Shift Bioscience devised protocols to sequence mtDNA heteroplasmy as well as degree of methylation will be adopted along with their chemical library able to reduce the degree of mtDNA mutation. Mitophagy inducers of both nutraceutical (e.g. Urolithin) and pharmaceutical (e.g. the p62/SQSTM1-Mediated Mitophagy Inducer PMI) sources6 will be also enrolled in combination with ligands of TSPO, the conserved mitophagy inhibitor which accumulates with ageing7. Mitophagy and mito-health will be tested measuring Reactive Oxygen Species (ROS), OXPOS parameters and Ca2+ signalling. Transcriptome analysis will be run to profile genes expression and enable global unbiased analysis of the cellular pathways involved in stressor-induced effects on mitochondrial status.This project will inform on hidden aspects of mitochondrial mechanisms of epigenetic reprogramming highlighting the hierarchy between heterplasmy and organelle dysfunction in age- mimicking conditions. Novel protocols of biomarking cellular ageing by mapping the mitochondrial retro-comunication with the nucleus will be obtained as well as approaches to potentially modify this.