A critical role of the mitochondrial permeability transition pore (mPTP) in the survival of senescent cells

Senescent cells are present in many pathological contexts and their elimination by pharmacological agents, known as senolytics, is a promising therapeutic strategy. Senescent cells exhibit distinctive mitochondrial adaptations, including an abnormally high influx of calcium from the endoplasmic reticulum. Here, through a genome-wide CRISPR/Cas9 screen, we identify the gene PPIF, encoding the mitochondrial protein cyclophilin D (CypD), as a senolytic target. CypD promotes the opening of the mitochondrial permeability transition pore (mPTP) and we show that senescent cells undergo frequent events of transient mPTP opening, known as mPTP flickering. Inhibition of CypD using genetic or pharmacologic tools, including cyclosporin A, impairs mPTP flickering, leading to toxic accumulation of mitochondrial calcium and cell death specifically in senescent cells. Pharmacological inhibition of NCLX, another mitochondrial Ca2+ efflux channel, also leads to senolysis. Our study links mPTP flickering to the survival of senescent cells and identifies novel targets for aging-associated diseases. Mouse embryonic stem cells (mESC) were induced to differentiate by the removal of leukimia inhibitory factor (LIF) and addition of retinoic acid for 5 days. Differentiated cells (diff) were induced to senesce via irradiation (IR), CDK4/6 inhibition (palbociclib, PD) or doxorubicin (doxo). 7 days after the senescence inducers were adminstered, Cas9 was activated in senescent cells by the addition of doxycycline to the media for 10 days. Doxycyline was also adminstered to non-senescent differentiated cells (doxy), and to the untreated mESCs (ESC). Screen was conducted in biological triplicate (R1, R2, R3)