Apoptotic caspases: silencing the mitochondrial danger within
Document TypePhD thesis
Access StatusOpen Access
© 2017 Dr Kate McArthur
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.
Keywordsmitochondria; mtDNA; apoptosis; caspases; DAMPs; interferon; microscopy; lattice light-sheet microscopy
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