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

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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.