Reverse Engineering of Cell Senescence

摘要

Abstract Cellular senescence is an elusive cell state - it is recognized in embryogenesis, wound healing and aging, where it is not only a symptom, but a major contributor to aging pathology. The striking experiments in the mouse, where eliminating senescent cells bestows broad health benefits and even a reversal of the aging process, drives our interest in understanding and ultimately controlling the transition of normal cells towards senescence. If we understood how cell senescence arises, we would have a chance to find ways to suppress it or reverse it, as well as to develop practical ways of clearing such cells from our bodies. This proposal is to use new tools on the microscopic and on the molecular level to observe the passage of cells from a normal to senescent state and reveal the changes in their molecular circuitry. Among the transitions that cells make is a dramatic increase in cell size. Hypertrophy is known to be driven by mTOR, and inhibited by the drug rapamycin, which is tied to life extension in several phylogenetically diverse species. We focus on hypertrophy to help crack senescence because, as a phenotype, it is in such stark violation of the normal cellular economy, where across cell types size is precisely maintained. Unfortunately, cell size has been one of the hardest phenotypes to study. This situation has changed radically with the development of new forms of microscopy by our group, that directly measure cell dry mass or even directly measure protein and lipid mass separately. Using such instruments, we propose to study with unprecedented temporal and mass resolution how cells, prompted by stress or aging, become hypertrophic and how hypertrophy is connected to other phenotypes of senescence. We will follow these same trajectories with deep quantitative mass spectrometry to correlate protein expression and phosphorylation with size and other senescent markers. We cross-reference some of our findings between in-vitro studies in cell culture and in-vivo studies in young and aged mice. Once we build a baseline description of the process of senescence, the same induction-maturation-death lifecycle of senescent cells will be repeated under perturbation by drugs. We can progress from description to causal analysis using the knowledge that several senolytic drugs are kinase inhibitors. When we perturb senescing cells by a small optimally informative, pre-selected set of poly-specific kinase inhibitors, whose inhibitory activities tile the whole kinome, we can identify key kinases that regulate senescence. Further coupling this to phospho-mass spectrometry will allow us to trace the signaling cascades to specific protein substrates and phosphosites. These observations and pharmacological perturbations can suggest new senolytic strategies and even suggest specific senolytic drugs.