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

Induction and transmission of oncogene-induced senescence

Senescence is a cellular stress response triggered by diverse stressors, including oncogene activation, where it serves as a bona-fide tumour suppressor mechanism. Senescence can be transmitted to neighbouring cells, known as paracrine secondary senescence. Secondary senescence was initially described as a paracrine mechanism, but recent evidence suggests a more complex scenario involving juxtacrine communication between cells. In addition, single-cell studies described differences between primary and secondary senescent end-points, which have thus far not been considered functionally distinct. Here we discuss emerging concepts in senescence transmission and heterogeneity in primary and secondary senescence on a cellular and organ level

Functional heterogeneity in senescence

Senescence is a tumour suppressor mechanism which is cell-intrinsically activated in the context of cellular stress. Senescence can further be propagated to neighbouring cells, a process called secondary senescence induction. Secondary senescence was initially shown as a paracrine response to the secretion of cytokines from primary senescent cells. More recently, juxtacrine Notch signalling has been implicated in mediating secondary senescence induction. Primary and secondary senescent induction results in distinct transcriptional outcomes. In addition, cell type and the stimulus in which senescence is induced can lead to variations in the phenotype of the senescence response. It is unclear whether heterogeneous senescent end-points are associated with distinct cellular function in situ, presenting functional heterogeneity. Thus, understanding senescence heterogeneity could prove to be important when devising ways of targeting senescent cells by senolytics, senostatics or senogenics. In this review, we discuss a role for functional heterogeneity in senescence in tissue- and cell-type specific manners, highlighting potential differences in senescence outcomes of primary and secondary senescence

Notch signalling mediates secondary senescence- imaging data

Chandra, Tamir; Rattanavirotkul, Nattaphong; Kirschner, Kristina (2019), “Notch signalling mediates secondary senescence- imaging data”, Mendeley Data, v2 http://dx.doi.org/10.17632/y76pb7s8h3.

Sex difference in pathology of the ageing gut mediates the greater response of female lifespan to dietary restriction

Abstract Women live on average longer than men but have greater levels of late-life morbidity. We have uncovered a substantial sex difference in the pathology of the aging gut in Drosophila. The intestinal epithelium of the aging female undergoes major deterioration, driven by intestinal stem cell (ISC) division, while lower ISC activity in males associates with delay or absence of pathology, and better barrier function, even at old ages. Males succumb to intestinal challenges to which females are resistant, associated with fewer proliferating ISCs, suggesting a trade-off between highly active repair mechanisms and late-life pathology in females. Dietary restriction reduces gut pathology in aging females, and extends female lifespan more than male. By genetic sex reversal of a specific gut region, we induced female-like aging pathologies in males, associated with decreased lifespan, but also with a greater increase in longevity in response to dietary restriction

Polymer Modelling Predicts Chromosome Reorganisation in Senescence

This dataset contains the data related to the figures and supplemental figures in the manuscript "Polymer Modelling Predicts Chromosome Reorganisation in Senescence". Lamina-associated domains (LADs) cover a large part of the human genome and are thought to play a major role in shaping the nuclear architectural landscape. Here, we perform polymer simulations, microscopy and mass spectrometry to dissect the roles played by heterochromatin- and lamina-mediated interactions in nuclear organisation. Our model explains the conventional organisation of heterochromatin and euchromatin in growing cells and the pathological organisation found in oncogene-induced senescence and progeria. We show that the experimentally observed changes in the locality of contacts in senescent and progeroid cells can be explained as arising due to phase transitions in the system. Within our simulations LADs are highly stochastic, as in experiments. Our model suggests that, once established, the senescent phenotype should be metastable even if lamina-mediated interactions were reinstated. Overall, our simulations uncover a generic physical mechanism that can regulate heterochromatin segregation and LAD formation in a wide range of mammalian nuclei.Chiang, Michael; Michieletto, Davide; Brackley, CA; Rattanavirotkul, Nattaphong; Mohammed, Hisham; Marenduzzo, Davide; Chandra, Tamir. (2019). Polymer Modelling Predicts Chromosome Reorganisation in Senescence, [dataset]. University of Edinburgh. https://doi.org/10.7488/ds/2593