Involvement of mitochondria and peroxiredoxin in TNF-mediated necroptosis
Necroptosis is a form of regulated necrosis considered to be involved in several pathophysiological conditions such as inflammation and ischaemia-reperfusion injury. The cellular events involved in the initiation of necroptosis are unclear, but recent studies indicate that the pseudokinase mixed lineage kinase domain like (MLKL) plays an important role. Understanding the molecular mechanisms regulating necroptosis is therefore an important priority that may lead to the development of new therapies. Mitochondria and reactive oxygen species generation has been reported in necroptosis, however it is not clear whether these play a direct role in the death process. These changes were investigated in a TNF-dependent model of necroptosis using mouse dermal fibroblasts (MDF) from wild-type and MLKL knock-out animals. Viability analysis by FACS and live cell imaging indicated early signs of cell death by 60 min and by three hours about half of the population were executed by necroptosis in an MLKL dependent manner. TMRE labelled cells showed a loss of mitochondrial membrane potential before cell death. Rapid increase in oxygen consumption 30 min after the injection of TNF indicated a direct link between the necrosome and downstream signalling to mitochondria for efficient necroptosis to occur. This is the first report of MLKL dependant effects on mitochondrial function. Western blot analysis indicated a rapid accumulation of oxidized mitochondrial peroxiredoxin in an MLKL dependent manner. Timing of mitochondrial changes and oxidation of peroxiredoxin suggest they may be early markers or even executors of cell death rather than a consequence of cellular damage. However further investigation is required to determine the importance of these changes in cell death.
Advisor: Hampton, Mark
Degree Name: Bachelor of Biomedical Sciences with Honours
Degree Discipline: Pathology
Publisher: University of Otago
Keywords: Redox; changes in TNF; mediated; necroptosis
Research Type: Thesis