Medical Biology - Theses
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ItemAnalysing the impact of the absence of CARD containing caspases on different forms of cell death( 2018)Cell death is an important process during embryogenesis as well as tissue homeostasis in the adult. Apoptosis, pyroptosis and necroptosis are three of the major programmed cell death pathways. Dysregulation of either of these cell death pathways can promote the development of a variety of diseases, such as cancer or autoimmune pathologies. Cysteine-dependent aspartate-specific proteases, known as caspases, exert key functions in all of these cell death pathways. Of note, certain caspases have been shown to play a role in more than one cell death pathway. This thesis presents the functional analysis of different caspases, in particular caspase activation and recruitment domain (CARD) containing caspases and their contributions to the pyroptotic, apoptotic and other cell death pathways. We have generated a novel triple knockout mouse strain deficient for the CARD containing caspases-1, -11 and -12. We initially used this strain to improve our understanding on the contributions of caspases-1, -11 and-12 to sepsis and different forms of cell death. Previous studies have suggested a role for caspase-12 in endoplasmic reticulum (ER) stress-induced cell death. However, we were not able to attribute a role of caspase-12 to sepsis or ER stress-induced apoptosis in vitro and in vivo. In Chapter 4 we present a study on the roles of different caspases as well as RipK3 during Salmonella infection in vitro and in vivo. There is evidence for a substantial functional overlap between different cell death pathways in the cellular response to pathogens, such as Salmonella. We examined this functional overlap of different cell death processes in the organismal and cellular response to infection by generating mice deficient for multiple caspases and also RipK3, an essential mediator of necroptotic cell death. Upon infection with S. Typhimurium SL1344 strain, primary myeloid cells from caspase-1/11/12/8 RipK3-/- mice showed marked resistance to cell death and survived even at high bacterial loads for up to 24 hours. When infecting the caspase-1/11/12/8 RipK3-/- mice with the vaccine Salmonella Typhimurium strain, they were not able to clear the bacteria from primary organs. Collectively, these findings provide evidence that there is substantial functional overlap between the different cell death pathways and hence the caspases involved in these processes in the cellular as well as organismal response to infection with S. Typhimurium and possibly other pathogens. Lastly, I generated mice lacking all murine CARD containing caspases, i.e. caspase-1, -11, -12, -2 and -9. These preliminary analyses revealed no major defects when comparing the embryonic development of mice lacking caspases-1, -11, -12, -2 and -9 to wildtype. Furthermore, we isolated haematopoietic stem and progenitor cells (HSPCs) from foetal livers derived from caspase-1/11/12/2/9 deficient mice and reconstituted lethally irradiated wildtype mice. Surprisingly, we did not find notable defects in the lymphoid and myeloid compartments in the caspase-1/11/12/2/9 deficient mice at steady state. In thymocyte cell death assays, cells from the quintuple caspase knockout mice still could undergo cell death, induced by the cytotoxic agent ionomycin, albeit at a delayed rate.
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ItemApoptotic caspases: silencing the mitochondrial danger within( 2017)Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that, upon activation, oligomerise in the mitochondrial outer-membrane to induce its permeabilisation (MOMP). This event allows cytochrome-c efflux from the mitochondria subsequently triggering a family of cysteine-dependent, aspartic-specific proteases, the caspases. These caspases, once initiated, cleave hundreds of substrates to bring about global cellular demolition. In doing so, they mediate many key characteristics of apoptosis, such as DNA laddering, membrane blebbing, and phosphatidylserine exposure. For many years caspases were thought to be essential for death, but it is increasingly apparent that they do not instigate the killer event, instead acting to accelerate cellular demise. Furthermore, there exists a burgeoning literature suggesting apoptotic caspases may have functions beyond cell death. One such study, reported apoptotic caspases also control hematopoietic stem cell (HSC) proliferation and function, and recent work from our lab suggested this phenotype was driven by increased levels of circulating Type I interferons (IFNs). Yet the molecular pathway responsible for the increased Type I IFN secretion, and how the apoptotic caspases fit into this response, was unknown. The work presented here, characterizes the mechanism by which caspase blockade drives an IFN response. It shows that during intrinsic apoptosis, BAK/BAX-mediated damage to the mitochondria not only triggered cytochrome-c release but also, the subsequent efflux of mitochondrial DNA (mtDNA) into the cytoplasm. In the absence of caspase activation, mtDNA activated the innate anti-viral cGAS/STING-signaling pathway to induce IFN production. To further this investigation, a live-cell imaging assay was developed, which utilized lattice light-sheet microscopy, to document mitochondrial morphology and behavior during apoptosis. The resulting images showed mtDNA was delivered to the cytoplasm via an orchestrated process involving mitochondrial fragmentation and inner membrane herniation through large BAK/BAX pores. This event was downstream of BAK/BAX activation, occurred independently of caspases, and was assisted by, but not reliant on, DRP1-mediated fission. Thus, mtDNA release is a common consequence of BAK/BAX-mediated MOMP, however subsequent caspase activation prevents mtDNA-triggered IFN production from dying cells, thereby maintaining the immunological silence of apoptosis. Despite the extensive literature implicating mtDNA in disease, the images presented in this thesis represent the very first demonstration of mtDNA release in real-time, in any setting. Thus, this live-cell imaging assay presents an exciting opportunity to further our understanding of mtDNA release in a wide range of human pathologies. Furthermore, this thesis also presents preliminary data demonstrating that pharmacological caspase inhibition is capable of driving apoptotic-IFN production in vivo, and suggests that caspase-inhibitors may have thus-far unappreciated potential as anti-viral and anti-cancer therapies.
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ItemFunctional characterisation of Caspase-9 in haematopoiesis( 2012)Caspases are a family of cysteine-aspartic proteases that play essential roles in programmed cell death (apoptosis), programmed necrosis (necroptosis), and inflammation. This work aims to clarify additional reported functions of caspases, and to enhance our understanding of the functional roles of caspases in the blood (haematopoietic system). By genetically dissecting the apoptotic pathway, I show that caspase activation is not required for megakaryocytes to form platelets from their cytoplasm. Rather the opposite is true, apoptotic caspase activation must be restrained for megakaryocytes to survive and produce platelets. In addition, platelets are fully functional without the initiator Caspase-9. Caspase-9-deficient platelets maintain blood clotting (hemostasis), and are capable of facilitating thrombin generation via the exposure of membrane phospholipid phosphatidylserine – supporting the notion that platelet apoptosis and platelet activation are biochemically distinct processes. Herein, I also show that the Bcl-2 regulated caspase cascade is critical for haematopoietic stem cell maintenance. A novel relationship between apoptotic caspase activation and type-1 interferon production – a cytokine known to regulate ‘stem-ness’ – is established. Together, this research refines previously described biological functions for caspases, and provides new insight into the role of caspases in cell death and the physiological consequence of their genetic or pharmacological inhibition.