Date of Award

12-4-2024

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr.Gunjan Guha

Second Advisor

Dr.Dipita Guha

Keywords

Cell Biology, Mitochondria and mTOR, Cellular Pathways, Rapamycin, Ridaforolimus, Oxidative Stress

Abstract

Cellular aging is a gradual process involving structural and functional deterioration of cells. Mitochondria, the biological clock in cells, are known to play a crucial role in the progression of cell aging. According to the mitochondrial free radical theory of aging, oxidative phosphorylation in the mitochondria produces free radicals, such as, reactive oxygen species (ROS), which lead to oxidative stress in cells. This, in turn, results in mitochondrial dysregulation, and eventually causes degeneration of cellular components, thereby propagating cell aging. However, it is to be underscored that aging, at its core, is a multifaceted phenomenon that involves a number of complex cross-talks of diverse aspects within a cell.

While it has been known that improved mitochondrial function is instrumental in mitigating cell aging, some studies have correlated a reduced rate of aging with partial/incomplete inhibition of mTOR activity. Studies conducted in the Intervention Testing Program of the National Institute of Aging (NIA-ITP) showed an increase in longevity in animals on treatment with mild doses of rapamycin (RAP), which is a well-known inhibitor of the multifaceted cellular regulator, mTOR. Intriguingly, RAP is also known to be extensively toxic towards cells. There is, thus, a clear dichotomy in its functions — it is cytotoxic at moderate/high doses, while it can restrain aging and extend lifespan at low doses. Such biphasic effect of drugs is known as hormesis.

Earlier studies have suggested that the hormetic (low) doses of RAP might cause partial/incomplete inhibition of mTOR, which may be attributed for the reported life-prolonging effect. However, understanding is still limited on the actual interplay of mitochondria and mTOR in the precincts of aging. The actual modus operandi of how such partial mTOR inhibition might modulate the mTOR-mitochondria cross-talk remained to be deciphered in the context of cellular aging. It is, therefore, prudent to investigate the mechanisms and impacts on cell aging by modulating facets of mTOR-mitochondria cross-talk via hormetic effects of RAP, and its analogs (rapalogs), such as temsirolimus, everolimus, and ridaforolimus (RFL), a non-prodrug rapalog.

Although prior reports have stated the role of ‘mild’ or incomplete inhibition of mTOR in slowing down of the aging process, till date no study has reported ‘an mTOR-mitochondria cross-talk’ that governs cellular aging. Moreover, it was also unknown how ‘mild’ mTOR inhibition affects the mitochondria-governed aging. We hypothesized that ‘mild’ degrees of mTOR inhibition (incomplete inhibition) by the hormetic function of RAP/RFL can cause mitigation of oxidative stress-governed injury to the mitochondrial paraphernalia, thereby alleviating degenerative conditions in cells and augmenting cellular longevity. We aimed to investigate an ‘mTOR- mitochondria cross-talk’ by administration of very low doses of RAP and RFL. No prior report was available on the molecular roleplay of RAP and RFL in the cross-talk between mTOR and mitochondria in the aging process. Furthermore, no literature showed the specialized hormetic function (at very low doses) of RFL and RAP on the molecular modulation of the mTOR-mitochondria functional axis that we have hypothesized.

In the undertaken study, we investigated the hormetic nature of RAP and RFL, in modulating the mTOR-mitochondria cross-talk to manifest an anti-aging outcome in WRL-68 cells. Cells treated with low doses of RAP/RFL were explored to comprehend how the hormetic function of the drugs affected the aging-associated parameters, such as, mitochondrial metabolism and biogenesis, mitophagy, oxidative damage to mtDNA/mtRNA and mitochondrial proteins, viability, cell death and senescence, mitochondrial density and Δψm. In conclusion, we report that low doses of RAP and RFL can hormetically amend the mTOR-mitochondria cross-talk, and can consequently promote anti-aging outcome in cells.

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