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

Research in gerontology is largely focused on the complex relationship between mitochondrial dysfunction and biological decay. Even though the mitochondrial free radical theory of ageing (MFRTA) is accepted and extensively investigated1, recent evidence has expanded the focus from free radical accumulation to heterogeneity and mutations of the mitochondrial DNA (mtDNA) as factors which is estimated by the epigenetic clock: degree of DNA methylation2.

Our working hypothesis for this research project is that the degree of mtDNA heteroplasmy impairs mitochondrial quality driving cellular ageing via epigenetic reprogramming.

Rationale
Mitochondria differ from all other organelles in having their own DNA (mtDNA)3. Given its multi-copy nature, spontaneous mtDNA mutations often affect only a small proportion of cellular mtDNA, a state termed heteroplasmy, whereas if all mtDNA molecules harbor a specific mutation, it is said to be in a state of homoplasmy. A shift towards increased heteroplasmy is implicated in several age associated conditions as it greatly affects mitochondrial homeostasis thus compromising cellular integrity overall. Malfunctioning mitochondria retro-communicate with the nucleus to prime global cellular reprogramming which occurs via DNA methylation and so impacting gene transcription. DNA methylation is a novel biomarker of ageing that enables accurate age estimates for tissues across their lifespan4. This ‘epigenetic clock theory’ is applicable as an ageing biomarker in viral infection5 and holds great promise for unifying cellular homoestatic processes with biological ageing.

Aims
This 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. We will therefore 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 reprogramming of nuclear DNA in order to learn how these underlie cellular frailty and demise.

Methodology
Human fibroblasts and lung cell line A549, at early and late passage number will be infected with influenza A/PR8/34, specifically with the H1N1 which is the strain shown to subvert mitophagy8. PD patients derived lines containing 60% of mtDNA heteroplasmy will be enrolled in the analysis as positive control.

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.

Outcome and Remit
This project will inform on hidden aspects of mitochondrial mechanisms of epigenetic reprogramming highlighting the hierarchy between heteroplasmy and organelle dysfunction in age-mimicking conditions.

Novel protocols of biomarking cellular ageing by mapping the mitochondrial retro-communication with the nucleus will be obtained as well as approaches to potentially modify this. This proposition of research naturally falls in the BBSRC highlight areas of “New approaches to Ageing Research” besides meeting the Council priorities in “Data Driven Biology” “Science underpinning Health” which will be integrated together with “Collaborative Research with the Users”.

The work will therefore provide a solid training opportunity for the PostGraduate Student to be enrolled in the project.