37 kDa LRP::FLAG enhances telomerase activity and reduces ageing markers in vitro and in vivo
Date
2020
Authors
Otgaar, Tyrone C
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Abstract
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
Description
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