ROLE OF CDK4 IN THE REGULATION OF CELLULAR SENESCENCE AND ORGANISMAL AGING

Aging is a complex biological process, characterized by multiple hallmarks and increased vulnerability to disease and death. At cellular level, one key hallmark and driver of aging is cellular senescence, a cell intrinsic response to non-lethal stress, characterized by permanent cell cycle arrest and a pro-inflammatory secretome. This arrest is mediated by inhibitors of cell cycle progression such as p16 and p21, which suppress cyclin-dependent kinases (CDKs). Among them, CDK4 inhibition has been shown early to induce senescence in vitro; however, its relevance in vivo and potential impact on organismal aging have not yet been characterized. Using a viable CDK4-knockout mouse model, preliminary findings from the hosting laboratory reveal a shortened lifespan and premature appearance of macroscopic aging traits. In addition, with local increase in molecular markers, the cardio-renal system appears to be the most affected by senescence. The specific aim of this internship is to validate the role of CDK4 in the induction of cellular senescence, extending previous studies, focusing specifically on the kidney and heart. To do so, I first completed the molecular characterization of this model by analysing the expression of key senescence regulators (p16, p21) in several organs (liver, lungs, kidney and heart) and extended the analysis temporally to investigate how the senescent phenotype evolves over time in cardiac tissue, with a particular focus on fibrosis and transcriptomic changes. Secondly, the in vivo findings were validated in primary kidney- and heart-derived cell cultures assessing senescence features using genetic (conditional or constitutive) knockout or CDK4/6 inhibition, and in the MRC-5 human fibroblast cell line via CDK4 knockdown or pharmacological inhibition to extend the relevance of the findings to human biology. Finally, given CDK4’s influence on mitochondrial biology, I explored specific structural, functional, and regulatory features either in vivo or in vitro to provide potential mechanistic insights linking CDK4, mitochondrial impairment, and senescence. These analyses highlighted that the sole ablation of CDK4 is sufficient to accelerate the accumulation of fibrogenic and inflammation-associated signatures in the heart. Moreover, all in vitro models, including MRC-5 cells, consistently exhibited senescence features, such as reduced proliferation and increased β-galactosidase activity, despite variable SASP profiles. Mechanistically, CDK4 loss ultimately leads to mitochondrial dysfunction, impairing oxidative phosphorylation, reducing fission, and increasing ROS production. In summary, this internship concludes a research project unveiling the protective role of CDK4 against mitochondrial dysfunction, thereby preventing premature senescence induction and decline of the cardio-renal system at the cellular and organismal levels, respectively.