Cyclophilin D plays a critical role in the survival of senescent cells.

Senescent cells play a causative role in many diseases, and their elimination is a promising therapeutic strategy. Here, through a genome-wide CRISPR/Cas9 screen, we identify the gene PPIF, encoding the mitochondrial protein cyclophilin D (CypD), as a novel senolytic target. Cyclophilin D promotes the transient opening of the mitochondrial permeability transition pore (mPTP), which serves as a failsafe mechanism for calcium efflux. We show that senescent cells exhibit a high frequency of transient CypD/mPTP opening events, known as 'flickering'. Inhibition of CypD using genetic or pharmacologic tools, including cyclosporin A, leads to the toxic accumulation of mitochondrial Ca2+ and the death of senescent cells. Genetic or pharmacological inhibition of NCLX, another mitochondrial calcium efflux channel, also leads to senolysis, while inhibition of the main Ca2+ influx channel, MCU, prevents senolysis induced by CypD inhibition. We conclude that senescent cells are highly vulnerable to elevated mitochondrial Ca2+ ions, and that transient CypD/mPTP opening is a critical adaptation mechanism for the survival of senescent cells.