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

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