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Radiotherapy is a common treatment modality for NSCLC which despite continuing advances in the field still presents very high rates of recurrence, with secondary tumours typically more difficult to treat, due in part to the radioresistance of these tumour's cells. This ultimately leads to poor treatment outcomes. One contributing factor may be the induction of senescence, a form of permanent cell cycle arrest, following radiotherapy treatment. While senescence is initially considered beneficial to the outcome of radiotherapy, an accumulation of senescent cells is known to disrupt tissue homeostasis and even favour the growth and survival of cancer cells. Therefore, the use of senolytics in combination with radiotherapy could greatly benefit NSCLC patients. Thus far use of such drugs has not been possible as there are no targetable markers of senescence specific enough to prevent damage to other cell types. The relative radiosensitivity of a panel of four NSCLC cell lines was assessed following different doses of ionising radiation (IR) and then investigated for the presence of senescence under the same conditions. This showed a correlation between radioresistance and the cells' ability to induce senescence following radiation. The senescence associated secretory phenotype (SASP) of the panel was analysed using shotgun proteomics to search for the presence of novel markers of senescence. Several interesting candidates were found which could present better targets for novel senolytics. The effects of these SASP on other cells were also tested, which showed increases in wound healing activity and invasion, indicating that the secretome of senescent cells had pro-proliferative activity on neighbouring cells. Our results demonstrate that clinically relevant doses of IR induce senescence in NSCLC cells and that their SASP can affect surrounding cells. We propose that the presence of senescent cells post IR treatment may be leading to high rates of radiotherapy failure and recurrence.

Autophagy is an essential quality control function of the cell. It selectively degrades harmful protein aggregates and/or damaged organelles, enabling maintenance of cellular homeostasis. Basal autophagy also mediates proper cardiovascular function. A variety of cardiovascular risk factors cause defective autophagy, consequently, pharmacotherapy with compounds to re-establish physiological levels of autophagy and stimulate pro-survival effects in the vasculature is an emerging strategy for cardiovascular disease. Therefore, it is crucial for the design of new pharmacological therapy the identification of novel key genes involved in the regulation of autophagy in vascular cells. In two recent studies Caporali et al showed how miR-503 might be considered a suppressor of postishemic neovascularization in type I diabetes and suggested some critical genes targeted by miR-503 whom downregulation may be crucial for the detrimental effect on endothelial cells and pericyte. Among the putative targets of miR-503 not prioritized in the previous studies but of potential interest for endothelial cell functions there was TCHP. Trichoplein (TCHP) was first identified as a keratin binding protein in 2005 and was described having a role in cancer progression, cellular apoptosis, cell cycle, primary cilium formation, mitochondria fragmentation and mitochondria-endoplasmic reticulum tethering. Using biochemical and bioinformatics approaches, I have demonstrated that TCHP is a novel target of miR-503. The data presented in this thesis will address the hypothesis that TCHP regulates endothelial function through the control of autophagy. TCHP expression in ECs was reduced when cultivated in high glucose in combination with low growth factors condition. Notably, ECs sorted from mice with Type-1 diabetes showed low levels of Tchp compared to non-diabetic mice. Knockdown of TCHP in ECs in vitro was characterized by marked changes in the tubulin cytoskeleton organization, resulting in an impaired microtubule network, reduced EC migration, reduced barrier function and impaired EC sprouting. Loss of TCHP function in ECs in vitro led to a defective autophagy, resulting in accumulation of SQSTM1/p62 and unfolded protein aggregates. Therefore, blocking autophagic flux in ECs resulted in premature cellular senescence as demonstrated by the appearance of β- galactosidase staining, the senescence-associated secretory phenotype (SASP) and increased expression of p16INK4A. This phenotype was associated with activation of the mTOR pathway and could be rescued using mTOR inhibitors. Thus, Torin-1 improved the migratory capacity of TCHP knockdown cells and rescued their senescent phenotype. In addition, ultrastructure analysis, immunofluorescence and epidermal growth factor pulse chase experiments revealed substantial abnormalities in the endosome and lysosome compartments in TCHP knock down cells. Remarkably, Tchp knockout mice exhibited a decreased cardiac vascularization and a significant accumulation of SQSTM1/p62 in cardiac vessels and cardiomyocytes, as a demonstration of defective autophagic flux. Finally, I observed loss of TCHP and an increase of SQSTM1/p62 and aggregates in the vessel wall derived-ECs, from patients with vascular dysfunction and premature coronary artery disease. Remarkably ECs from patients exhibited impaired migration and increased expression of SASP, resembling the phenotype of TCHP knockdown cells. In line with this, function in EC from patients was rescued by Torin-1 treatment. Taken together, these results reveal for the first time the pivotal role played by TCHP in the vascular endothelium and identify a new mechanism by which pathological conditions, through the silencing of TCHP, lead to the endothelial dysfunction. Importantly, this study highlights a fundamental link between EC function and cellular proteostasis, through the control of autophagy, and suggests a new therapeutic direction in cardiovascular disease.

Senescence is a highly efficient mechanism that provides an irreversible barrier to cell cycle progression to prevent undesired proliferation. However, under pathological circumstances, senescence can adversely affect organ function, viability and regeneration. In the context of biliary disease, we hypothesize that senescence is initiated in the bile ducts and spreads to the liver parenchyma, impairing the liver’s regenerative capacity and aggravating the condition. We have developed a mouse model of biliary senescence, based on the conditional deletion of Mdm2 in bile ducts, that mimics clinical features of biliary disease. Using this model, we studied the underlying mechanisms that characterize biliary disease, and established an essential role of TGFβ in paracrine senescence-associated regeneration. Lastly, we disrupted TGFβ signalling to therapeutically rescue this phenotype in our model of biliary senescence. These results reveal the detrimental role of senescence in biliary disease, and a TGFβ- dependent mechanism for dissemination of senescence from the biliary epithelium to the parenchyma, impairing liver function. Finally, we have identified TGFβ signalling disruption as a potential therapeutic target to limit senescence-dependent aggravation in human cholangiopathies.

The individual unit of computation in the human brain is the neuron. To advance our understanding of the human brain, we need to generate relevant cellular models of human neurons in vitro. Direct conversion of human somatic fibroblasts into induced neurons (iNs) allows for the generation of functional neurons while bypassing any stem cell intermediary stages. Although iN technology has an enormous potential for modeling age-related diseases, as well as therapeutic approaches, the technology faces limitations due to variable conversion efficiencies and a lack of thorough understanding of the signaling pathways directing iN conversion. Here, I introduce a new all-in-one inducible lentiviral system that simplifies fibroblast transgenesis for the two pioneer transcription factors, Ngn2 and Ascl1, and a small molecule cocktail that markedly improves iN yields and sheds new insight into the mechanisms influencing direct iN conversion. Next, I will apply the iN protocol on a cohort of Alzheimer’s disease (AD) and healthy age matched control donor samples to model age-related molecular phenotypes in AD neurons. I previously found that AD brains have significantly higher proportions of neurons that express senescence markers, and their distribution indicates bystander effects. AD patient-derived iNs exhibit strong transcriptomic, epigenetic, and molecular biomarker signatures that illuminate a specific human neuronal senescence-like state. AD iN single-cell transcriptomics revealed that senescent neurons face oncogenic challenges, and metabolic dysfunction, and they display a proinflammatory signature. Integrative profiling of the inflammatory secretome of AD iNs and patient cerebral spinal fluid revealed a neuronal senescence-associated-secretory-phenotype, that can trigger astrogliosis in human astrocytes. Finally, I show that targeting senescence-like neurons with senotherapeutics could be a novel strategy for preventing or treating AD.

Cellular senescence is considered a signal transduction process where damaged or stressed cells arrest proliferation. Exogenous stressors including bacterial infection, chemotherapy, and irradiation can drive normal cells to senescence. Sodium orthovanadate (Na3VO4) is a phosphatase inhibitor known to mediate the release of the proinflammatory cytokine, TNF-a. We hypothesized that NaVO4 treatment will reduce proinflammatory expression from senescent cells. In our study we illustrate that prolonged exposure to LPS caused RAW 264.7 cells induced morphological changes indicative of senescent cells including the formation of giant multinucleated cells (GMCs). Furthermore, we found that RAW 264.7 GMC cells stain positive for senescence and increase expression of key proinflammatory cytokines associated with the senescent phenotype including IL-10 and TNF-α. Our data demonstrate that 100uM and 1000uM sodium orthovanadate treatment reduced cell proliferation. The reduction in proliferation was concomitant with changes in pro-inflammatory cytokines, IL-10, and TNF-α (Figure 9B &C at 24hrs). IPA analysis confirmed connections between these proinflammatory cytokines, senescence, and disease progression.

Telomere dysfunction plays a complex role in tumorigenesis. While dysfunctional telomeres can block the proliferation of incipient cancer clones by inducing replicative senescence, fusion of dysfunctional telomeres can drive genome instability and oncogenic genomic rearrangements. Therefore, it is important to define the regulatory pathways that guide these opposing effects. Autophagy is a highly conserved stress response pathway that degrades cytoplasmic components and, in recent work, has been shown to regulate both senescence and genome instability in various contexts. In this study, we apply models of acute telomere dysfunction to determine whether autophagy modulates the resulting genome instability and senescence responses. While telomere dysfunction rapidly induces autophagic flux in human fibroblast cell lines, inhibition of the autophagy pathway does not have a significant impact on the transition to senescence, in contrast to what has previously been reported for oncogene-induced senescence. Our results suggest that this difference may be explained by disparities in the development of the senescence-associated secretory phenotype as a result of different levels of DNA damage. We also show that chromosome fusions induced by telomere dysfunction are comparable in autophagy-proficient and autophagy-deficient cells. Altogether, our results highlight the complexity of the senescence-autophagy interface and indicate that autophagy induction is unlikely to play a significant role in telomere dysfunction-driven senescence and chromosome fusions. Chapter 1 provides an overview of the autophagy and senescence pathways as well as the research available to date linking autophagy to senescence and genome instability with a focus on the major open questions. In Chapter 2, I will present data identifying the autophagy-independent nature of senescence triggered by targeted telomere dysfunction. In Chapter 3, I will present data defining the autophagy-independent nature of genome instability that results from telomere dysfunction. Finally, Chapter 4 is a discussion of the implications of this work for the fields of telomere dysfunction, senescence, and autophagy in a basic biology and clinical context.