Investigating the expression of Topoisomerase II Beta in aged neurons: development of a Murine Cell Line and Drosophila model

Bainbridge, Callum S. (2020) Investigating the expression of Topoisomerase II Beta in aged neurons: development of a Murine Cell Line and Drosophila model. Doctoral thesis, Northumbria University.

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Abstract

The enzyme Topoisomerase II Beta (Top2B) has previously been shown to be a crucial component of neuronal differentiation and development in mammals. It is also expressed in adult neuronal tissue where it plays important roles in facilitating transcription of long genes and early response genes and may also be involved in DNA repair. To date, studies investigating age-related changes in Top2B expression in neuronal tissue are limited and the importance of Top2B in the maintenance of neuronal function and integrity during ageing has not yet been fully elucidated, thus this study aimed to further investigate Top2B during the ageing process.

The development of an in vitro murine model of neuronal ageing was successfully achieved using the Cath.a-differentiated (CAD) cell line. A neuronal-like phenotype in CAD cells was achieved through serum starvation and cells were then chronologically aged. Levels of Top2B mRNA and protein were seen to decline significantly during ageing of the cells in RT-qPCR and western blotting experiments, respectively. Concomitant increases in protein levels of the tumour suppressor gene p21 were also observed as well as a significant accumulation of double strand breaks as shown by γH2AX assays. In addition, preliminary in vivo experiments also revealed age-related declines of Top2B in mouse hippocampus. The development of an equivalent human in vitro model using the human neuroblastoma cell line SH-SY5Y was unsuccessful.

Further in vivo experiments using Drosophila brain tissue also revealed significant age-related declines in Topoisomerase II (Top2) protein levels with ageing in both males and females, which was accompanied by a decline in locomotor function and increases in advanced glycation end-products (AGEs) in females. Interestingly, in Drosophila this was not accompanied by a reduction in Top2 mRNA levels.

Reduction in the levels of mouse Top2B and Drosophila Top2 with age may have profound effects on transcription and the ability of cells to repair DNA damage and may result in increased vulnerability to oxidative stress, ultimately having detrimental effects on longevity and normal ageing. Thus, these models offer an opportunity to further elucidate the functional effect of this loss, its causes and potential pharmaceutical interventions to reverse these effects. Importantly, they also illustrate the need for such research to be carried out in human neuronal cells and brain tissues.

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