37 kDa LRP::FLAG enhances telomerase activity and reduces ageing markers in vitro and in vivo

Otgaar, Tyrone C
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Ageing is a degenerative process characterised by detrimental changes which accumulate to cause a decline in physiological functioning of the organism. It is characterised by stem cell exhaustion, mitochondrial dysfunction as well as genomic instability. One of the core regulators of cellular ageing are telomeres, repetitive DNA sequences of TTAGGG that cap the ends of chromosomes and are maintained by the ribonucleoprotein DNA polymerase complex, telomerase. Age-dependent progressive loss of telomere functions due to the “end replication” problem and insufficient telomerase activity eventually induces cell cycle exit for the induction of either replicative senescence or apoptosis. To bypass senescence, most epithelial and tumorigenic cells re-activate telomerase allowing for telomere extension thereby, extending their proliferative potential and promoting overall cell viability. It was recently established that knockdown of the 37kDa/ 67kDa laminin receptor (LRP/LR), a proliferative-related protein which maintains cell viability in cancerous and normal cells, reduces telomerase activity. Therefore, it was hypothesized that elevating LRP/LR may increase telomerase activity and hinder the ageing process. The core aims of this research included: To prove in vitro that LRP::FLAG overexpression influences telomere dynamics and senescent related proteins in cells. Thereafter, to prove in vivo that LRP::FLAG overexpression (i) has an effect on the ageing process, (ii) positively impacts on telomere dynamics and (iii) significantly reduces the levels of tissue senescence in aged C57BL/6J mice. To this end, cell lines were successfully developed that stably overexpressed LRP::FLAG and have further overexpressed LRP::FLAG in aged C57BL/6J mouse models. Western blotting, confocal microscopy and qPCR were used to assess the effects of overexpression of LRP::FLAG on ageing markers as well as telomere dynamics in both cell lines and mice. In addition, various physiological tests (balance beam, puzzle box, nesting, wirehang, social interaction, open field and hair greying tests) and histological analyses were performed to assess overall mouse fitness as well as to discern the treatments ability at reducing tissue degeneration and atrophy. In terms of the in vitro research, it was found that the overexpression of LRP::FLAG induced a significant elevation hTERT levels, telomerase activity and telomere length. Concomitantly, it was shown that LRP::FLAG overexpression also reduced the levels of the senescence/ageing markers β-galactosidase and λH2AX in both HEK293 and MRC 5 cells. Moreover, for the in vivo component it was found that mice overexpressing LRP::FLAG displayed improved physiological characteristics and markedly less tissue degeneration and atrophy when compared to control and non-treated mice. Alongside these improvements, certain organs displayed increased telomerase activity with a corresponding elongation in average telomere length, further substantiating that a functional relationship exists between LRP and telomerase. In addition to the improved aspects of telomere biology it was found that the overexpression of LRP::FLAG significantly improved various proliferative and anti-ageing associated proteins (Klotho, MDM2, SIRT1, Akt and c-Myc) while causing a concomitant decrease in senescent associated proteins (p16, NFkB and γH2AX). These findings are indicative of a novel function of LRP/LR impeding the onset of senescence, while also promoting healthier ageing through elevating TERT and telomerase activity. Therefore, LRP::FLAG could act as a novel drug for healthier ageing through the impediment of the cellular ageing process
A thesis submitted in fulfilment of the degree of Doctor of Philosophy in Biochemistry and Cell Biology in the Faculty of Science, University of the Witwatersrand, Johannesburg, 2020