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Hormetic effect of low doses of rapamycin triggers anti-aging cascades in WRL-68 cells by modulating an mTOR-mitochondria cross-talk

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Abstract

Background

Rapamycin is hormetic in nature—it demonstrates contrasting effects at high and low doses. It is toxic at moderate/high doses, while it can restrain aging and extend lifespan at low doses. However, it is not fully understood how rapamycin governs cellular aging. On the other hand, aging is putatively correlated to mitochondrial dysregulation. Although previous studies have suggested that hormetic (low) doses of rapamycin can cause partial/incomplete inhibition of mTOR, 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. The present study was designed to understand the hormetic effects of rapamycin on cellular factors that govern aging-associated changes in mitochondrial facets, such as functional and metabolic homeostases, sustenance of membrane potential, biogenesis, mitophagy, and oxidative injury to mitochondrial macromolecules.

Methods and results

WRL-68 cells treated (24 h) with variable doses of rapamycin were studied for estimating their viability, apoptosis, senescence, mitochondrial density and Δψm. Expression levels of key functional proteins were estimated by immunofluorescence/immunoblots. Oxidative damage to mtDNA/mtRNA/proteins was measured in mitochondrial lysates. We demonstrated that hormetic doses (0.1 and 1 nM) of rapamycin can alleviate aging-associated mitochondrial dyshomeostasis in WRL-68 cells, such as oxidative injury to mitochondrial nucleic acids and proteins, as well as disequilibrium of mitochondrial density, membrane potential, biogenesis, mitophagy and overall metabolism.

Conclusions

We established that low doses of rapamycin can hormetically amend the mTOR-mitochondria cross-talk, and can consequently promote anti-aging outcome in cells.

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Abbreviations

4E-BP1:

Eukaryotic translation initiation factor 4E-binding protein 1

8-OH-dG:

8-Hydroxy-2’-deoxyguanosine

8-OH-g:

8-Hydroxyguanine

8-OH-G:

8-Hydroxyguanosine

BECN1:

Beclin 1

COXII:

Cytochrome c oxidase subunit II

DAPI:

4′,6-Diamidino-2-phenylindole

DHA:

Dinitrophenyl hydrazone adduct

DHE:

Hydroethidine/dihydroethidium

DNPH:

2,4-Dinitrophenylhydrazine

Dox:

Doxorubicin

eIF4E:

Eukaryotic translation initiation factor 4E

FOXO1:

Forkhead box protein O1

ITP:

Interventions Testing Program

mtDNA:

Mitochondrial DNA

mtOGR:

Mitochondrial OGR

mTOR:

Mechanistic target of RAP

mtRNA:

Mitochondrial RNA

MTT:

3-(4,5- Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

OGR:

Oxidized guanine residues

p70S6K:

70 kDa ribosomal protein S6 kinase

PGC-1α:

Peroxisome proliferator-activated receptor gamma coactivator 1α

PINK1:

PTEN-induced kinase 1

RAP:

Rapamycin

rTdT:

Recombinant terminal deoxynucleotidyl transferase

SA-β-gal:

Senescence-associated β-galactosidase

SDH:

Succinate dehydrogenase

SDS:

Sodium dodecyl sulfate

SIRT1:

Sirtuin 1

TFAM:

Mitochondrial transcription factor A,

TUNEL:

Terminal deoxynucleotidyl transferase dUTP nick-end labeling

X-gal:

5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside

YY1:

Yin yang 1

Δψm:

Mitochondrial membrane potential

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Acknowledgements

The authors acknowledge Dr. Sudarshan Singh Rathore, Dr. M.R. Charan Raja, Dr. Sandeep Miryala and Ms. Nirekshana Krishnasagar for assistance with experiments and blinded confirmatory estimations. The authors also thank Dr. N. Saisubramanian, SCBT, SASTRA University, for his support. This study was funded by the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India (Grant Nos. YSS/2014/000139 & YSS/2015/000025), Department of Science and Technology “Fund for Improvement of S&T Infrastructure in Universities and Higher Educational institutions” (DST-FIST), Government of India [Grant No. SR/FST/ETI-331/2013 (SASTRA)], and Department of Biotechnology (DBT), Government of India Grant No. BT/PR22434/MED/30/1901/2017.

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Authors and Affiliations

  1. Department of Biotechnology, School of Chemical and Bio Technology, Cellular Dyshomeostasis Laboratory, SASTRA University, Tamil Nadu, Thanjavur, 613401, India

    R. Mahalakshmi, J. Priyanga, Dipita Bhakta-Guha & Gunjan Guha

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  1. R. Mahalakshmi
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Contributions

GG and DBG: Conception and design of the study; Acquisition of data: RM and JP; Analysis and interpretation of data: RM, JP, GG and DBG; Drafting of the article: GG and DBG; Critical revisions for intellectual content: GG and DBG; Final approval of the submitted version: GG, DBG, RM and JP.

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Correspondence to Dipita Bhakta-Guha or Gunjan Guha.

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R. Mahalakshmi, J. Priyanga, Dipita Bhakta-Guha and Gunjan Guha declare that there is no conflict of interest.

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Mahalakshmi, R., Priyanga, J., Bhakta-Guha, D. et al. Hormetic effect of low doses of rapamycin triggers anti-aging cascades in WRL-68 cells by modulating an mTOR-mitochondria cross-talk. Mol Biol Rep 49, 463–476 (2022). https://doi.org/10.1007/s11033-021-06898-6

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