An attempt to localise Atf5 at the subcellular level in the ageing brain of Nothobranchius furzeri

Details

Supervisors

Page, Melissa

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

Life expectancy has increased significantly, resulting in a globally ageing population and a corresponding rise in age-related diseases. Mitochondrial dysfunction, a hallmark of ageing, plays a key role in the onset of several such diseases. The mitochondrial unfolded protein response (UPRmt) aims to mitigate mitochondrial stress, including issues related to protein import. This response involves signalling from the mitochondria to the nucleus, leading to the upregulation of chaperone and protease proteins. Activating transcription factor 5 (Atf5), one of the four known UPRmt activators, monitors mitochondrial import capacity and, in the event of an import defect, relocates to the nucleus to induce the expression of stress-relieving genes. This Master’s thesis investigates the localisation and function of Atf5 within the subcellular compartments of the brain of the animal model of ageing, Nothobranchius furzeri, focusing on mitochondria and nuclei. The research employed differential centrifugation, immunofluorescence microscopy, oxygen consumption assays, and transcriptomic analysis. Whilst subcellular fractionation protocols encountered challenges with contamination affecting the purity of mitochondrial and nuclear fractions, immunofluorescence provided valuable insights into Atf5 colocalisation, albeit highlighting the technical limitations of the equipment. Efforts to measure mitochondrial oxygen consumption rates faced unexpected technical difficulties, resulting in inconclusive data. Furthermore, transcriptomic analysis did not reveal significant age-related changes in gene expression associated with Atf5 and the UPRmt, suggesting a potential post-transcriptional regulatory mechanism that warrants further proteomic studies. The findings of this research underscore the need for methodological refinements and highlight the complexities of studying mitochondrial function and its implications in the ageing process in N. furzeri.