Periodic Reporting for period 1 - MitoSen (Detection and elimination of senescent cells targeting Cyclophilin D)
Período documentado: 2022-09-01 hasta 2024-08-31
Senescence is a complex cellular response to various stressors characterised by stable cell cycle arrest and secretion of a complex mixture of inflammatory, fibrogenic, and mitogenic soluble factors known as senescence-associated secretory phenotype (SASP). While the development of this phenotype is physiologically important during embryonic development and tissue regeneration, the unrestricted accumulation of senescent cells is a hallmark of ageing-related diseases and cancer. Indeed, in pathological conditions, the reduced clearance of these cells by the immune system can lead to chronic inflammation and impaired tissue regeneration abilities.
The selective elimination of senescent cells has been shown to delay or prevent numerous age-associated pathologies, thus, the generation of novel therapeutics, termed senolytics, able to clear senescent cells while being non-toxic for other cell populations, is a highly attractive strategy in the treatment of these disorders. To develop successful senolytic approaches, a deep understanding of senescent cells’ biology is necessary, to identify which unique aspects can be targeted.
Numerous studies have reported that senescent cells are characterised by highly altered mitochondrial and metabolic adaptations, including changes in morphology and dynamics, altered respiratory capacity, and reactive oxygen species (ROS) and calcium accumulation. These differences between senescent and non-senescent cells’ mitochondrial biology could offer a promising and still largely unexplored research field for senolytic therapies.
Through a CRISPR-based screening, we identified PPIF, the gene encoding for the mitochondrial matrix isomerase cyclophilin D (CypD), as a novel senolytic candidate. Reduced steady-state levels of CypD, indeed, significantly decrease the viability of senescent human fibroblasts and cancer cell lines but not of their proliferative counterparts. CypD is known to be the main modulator of the mitochondrial permeability transition pore (mPTP), triggering its opening by sensitising it to calcium, inorganic phosphate, and reactive oxygen species (ROS). The nature of the pore is still largely debated, since its structural components have not been fully identified, and the knowledge around its biological function is still evolving. The sustained and irreversible opening of the mPTP, which takes place in conditions of elevated and irreparable damage, leads to mitochondrial swelling, uncontrolled diffusion of molecules of <1,500 Da across the inner mitochondrial membrane, and loss of mitochondrial membrane potential, resulting in cell death. On the other side, the transient and intermitting opening of the pore is believed to maintain mitochondrial homeostasis and favour the exchange of molecules between the mitochondrial matrix and the cytoplasm. In our cellular models, we expect the downregulation of CypD to lead to decreased transient opening of the mPTP in senescent cells and a consequential increase in the mitochondrial matrix calcium levels.
Our final aim is to identify and characterise in vitro and in vivo a new senolytic target that can be use to develop therapeutics for the treatment of ageing-associated diseases and cancer.
The selective elimination of senescent cells has been shown to delay or prevent numerous age-associated pathologies, thus, the generation of novel therapeutics, termed senolytics, able to clear senescent cells while being non-toxic for other cell populations, is a highly attractive strategy in the treatment of these disorders. To develop successful senolytic approaches, a deep understanding of senescent cells’ biology is necessary, to identify which unique aspects can be targeted.
Numerous studies have reported that senescent cells are characterised by highly altered mitochondrial and metabolic adaptations, including changes in morphology and dynamics, altered respiratory capacity, and reactive oxygen species (ROS) and calcium accumulation. These differences between senescent and non-senescent cells’ mitochondrial biology could offer a promising and still largely unexplored research field for senolytic therapies.
Through a CRISPR-based screening, we identified PPIF, the gene encoding for the mitochondrial matrix isomerase cyclophilin D (CypD), as a novel senolytic candidate. Reduced steady-state levels of CypD, indeed, significantly decrease the viability of senescent human fibroblasts and cancer cell lines but not of their proliferative counterparts. CypD is known to be the main modulator of the mitochondrial permeability transition pore (mPTP), triggering its opening by sensitising it to calcium, inorganic phosphate, and reactive oxygen species (ROS). The nature of the pore is still largely debated, since its structural components have not been fully identified, and the knowledge around its biological function is still evolving. The sustained and irreversible opening of the mPTP, which takes place in conditions of elevated and irreparable damage, leads to mitochondrial swelling, uncontrolled diffusion of molecules of <1,500 Da across the inner mitochondrial membrane, and loss of mitochondrial membrane potential, resulting in cell death. On the other side, the transient and intermitting opening of the pore is believed to maintain mitochondrial homeostasis and favour the exchange of molecules between the mitochondrial matrix and the cytoplasm. In our cellular models, we expect the downregulation of CypD to lead to decreased transient opening of the mPTP in senescent cells and a consequential increase in the mitochondrial matrix calcium levels.
Our final aim is to identify and characterise in vitro and in vivo a new senolytic target that can be use to develop therapeutics for the treatment of ageing-associated diseases and cancer.
Cellular viability after CypD downregulation is reduced by ~40% in A549, SK-MEL and IMR90 senescent cells, while proliferative cells are unaffected. In vivo, the pharmacological inhibition of CypD by Cyclosporine A treatment in nude mice effectively halted the growth of xenografts when in combination with Palbociclib, a senescence inducer, and significantly reduced the level of P21-positive cells, an important marker of senescence.
Our results show that senescent cells rely more than their proliferative counterparts on transient opening of the mPTP and that CypD downregulation strongly reduced this event. As a consequence, CypD downregulation in senescent cells (A549 and IMR90) increased mitochondrial matrix calcium levels at rest and affected mitochondrial morphology. Indeed, mitochondrial length and volume is reduced, while average cross-section is increased. Our data suggest that this phenotype can induce cell death via pyroptosis. mRNA levels of ER and mitochondrial calcium transporters were not affected by the depletion of CypD in A549 and IMR90, suggesting that an alteration in the mPTP activity is likely to be mainly responsable for the observed phenotype.
The involvement of calcium accumulation in the mitochondrial matrix of senescent cells in senolysis was further validated by additional experiments using pharmacological modulators of matrix calcium influx and efflux. Firstly, cells were treated with CGP37157, an inhibitor of NCLX (main efflux pathway for matrix calcium), which caused an increase in mitochondrial matrix calcium. Senescent cells treated with CGP37157 showed similar phenotype to cells treated with siRNA against CypD for what concerns viability (A549, IMR90, SK-MEL) and mitochondrial morphology (A549, IMR90). Secondly, cells downregulating CypD were treated with Ru360, an inhibitor of MCU, main transporter of calcium inside mitochondria. Ru360 partially rescued the viability phenotype in A549 and SK-MEL cells, and mitochondrial morphology in A549 cells. In senescent IMR90, Ru360 showed a partially toxic effect.
Our results show that senescent cells rely more than their proliferative counterparts on transient opening of the mPTP and that CypD downregulation strongly reduced this event. As a consequence, CypD downregulation in senescent cells (A549 and IMR90) increased mitochondrial matrix calcium levels at rest and affected mitochondrial morphology. Indeed, mitochondrial length and volume is reduced, while average cross-section is increased. Our data suggest that this phenotype can induce cell death via pyroptosis. mRNA levels of ER and mitochondrial calcium transporters were not affected by the depletion of CypD in A549 and IMR90, suggesting that an alteration in the mPTP activity is likely to be mainly responsable for the observed phenotype.
The involvement of calcium accumulation in the mitochondrial matrix of senescent cells in senolysis was further validated by additional experiments using pharmacological modulators of matrix calcium influx and efflux. Firstly, cells were treated with CGP37157, an inhibitor of NCLX (main efflux pathway for matrix calcium), which caused an increase in mitochondrial matrix calcium. Senescent cells treated with CGP37157 showed similar phenotype to cells treated with siRNA against CypD for what concerns viability (A549, IMR90, SK-MEL) and mitochondrial morphology (A549, IMR90). Secondly, cells downregulating CypD were treated with Ru360, an inhibitor of MCU, main transporter of calcium inside mitochondria. Ru360 partially rescued the viability phenotype in A549 and SK-MEL cells, and mitochondrial morphology in A549 cells. In senescent IMR90, Ru360 showed a partially toxic effect.
No previous research had analysed the role of the mPTP opening in cellular senescence, nor tested this pathway as a potential senolytic target. We identified a novel target for therapies and described the biological mechanism behind it.
Future research should be focused on identifying new compounds able to specifically inhibit CypD or the transient mPTP opening without inducing toxicity.
Future research should be focused on identifying new compounds able to specifically inhibit CypD or the transient mPTP opening without inducing toxicity.