Rejuvenation of Senescent Cells, in vitro and in vivo, by Low-frequency Ultrasound without Senolysis

The presence of an appreciable number of senescent cells causes age-related pathologies as their removal by genetic or pharmacological means, as well as possibly by exercise, improves outcome in animal models. An alternative to depleting such cells would be to rejuvenate them to promote their return to a replicative state. Means to do so have not been explored, but here we report that treatment of non-growing senescent cells with low-frequency ultrasound (LFU) rejuvenates the cells. Notably, we find 15 characteristics of senescent cells that are reversed by LFU, including decreased cell and organelle motility. Mechanistically, LFU causes Ca2+ entry that precedes dramatic increases in autophagy and an inhibition of mTORC1 signaling and the movement of Sirtuin1 from the nucleus to cytoplasm. Also, there is inhibition of SASP secretion, as well as β-galactosidase expression, telomere length is increased, while nuclear 5mc, H3K9me3, γH2AX, nuclear p53, ROS and mitoROS levels are all restored to normal levels. Repeated LFU treatments enables expansion of primary cells and stem cells beyond normal replicative limits without altering their phenotype. The rejuvenation process is enhanced by co-treatment with rapamycin or Rho kinase inhibition but is inhibited by blocking Sirtuin1 or Piezo1 activity. We further optimized the LFU treatment parameters to increase mouse lifespan and healthspan. These results suggest that mechanically-induced pressure waves alone can reverse senescence and aging effects at the cellular and organismal level, providing a non-pharmacological way to treat the effects of aging. One-Sentence Summary Low frequency ultrasound is sufficient to rejuvenate senescent cells by activating autophagy in aged mice to improve healthspan. ### Competing Interest Statement Authors (MS, SK, FM, and RM) are co-authors of patents related to these studies and MS and FM have financial interests in a company, Mechanobiologics, Inc that is planning to market LFU devices suitable for senescent cell rejuvenation. All data are available in the main text or the supplementary materials.