Understanding the heterogeneity of senescence and ageing at the single-cell level
This thesis contains two main research projects, both of which demonstrate the power of singlecell approaches in the interrogation of complex biological systems. The first part of my studies focuses on cellular heterogeneity in oncogene-induced senescence (OIS). Senescence is a cellular response triggered by diverse stresses. It can be beneficial, as a tumour suppressive response to oncogene activation, or detrimental as it drives inflammation and pathology of ageing. Senescence can be transmitted to neighbouring cells through secreted factors of the senescence associated secretory phenotype (SASP), a phenomenon known as secondary senescence. Thus far, primary and secondary senescence have been considered identical phenotypes. Here, I used single-cell transcriptomics in co-culture systems to decipher heterogeneity between primary and secondary Ras-induced senescence and observed two distinct transcriptional trajectories, one marked by Ras and the other by Notch. Furthermore, secondary senescence in vitro and in vivo were found to be driven by Notch, rather than by the SASP alone as previously thought. In conclusion, primary and secondary senescence showed functional diversification and were distinct molecular endpoints. In the second part of this thesis, I explored cellular heterogeneity in Hutchinson-Gilford progeroid syndrome (HGPS), which represents a sporadic, rare, autosomal dominant genetic disease characterised by clinical features of premature ageing and has been extensively studied as a model for the ageing process. Ageing remains indisputably the largest risk factor for the majority of prevalent human pathologies such as cancer, cardiovascular diseases and neurodegenerative disorders. An attractive interpretation of ageing is that cells age as a result of a ‘toxic environment’ created from damaged or defected cells, which then toxically impact on their healthy and normal neighbouring cells and tissues. Studies that lend support to this model reported that removal of senescent cells, namely stably non-proliferating cells induced by insulting stimuli, from mouse tissues can delay the onset of age-associated disorders in adipose tissues, skeletal muscles and eyes, as well as extend their healthy lifespan. Provided that persistent secretion of inflammatory cytokines and other systemic factors during chronic senescence can favour both degenerative and hyperplastic pathologies, it is plausible that accumulation of senescent cells might systematically promote an ageing environment and therefore the ensuing loss of cellular function. A study in mice whose cells were half progeria and half normal demonstrated that these mosaic mice age normally, with no overt abnormalities in the proliferative capacity in cell culture or increased levels of progeria markers, suggesting cell-extrinsic mechanisms in the pathogenesis of progeria. This finding further supports the interpretation of the toxic ageing model. Using confocal microscopy and single-cell technologies, I aimed to understand the heterogeneity in progeria by quantifying the proportion of progeria cells that were compromised phenotypically and transcriptionally. By combining the morphological profiles with the transcriptional profiles, I hope to dissect the disease state of progeria and propose a mechanism by which organismal ageing occurs. The molecular insight into the pathophysiology this premature ageing disease will help pave the way for novel development of therapeutic strategies against age-related disorders with the improvement of both lifespan and healthspan.