Genetic elucidation of the roles of apoptosis-inducing factor (AIF) in mitochondrial respiration and programmed cell death.

摘要

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which, following an apoptotic stimulus, translocates to the nucleus and mediates chromatin degradation. The physiological functions of AIF within the mitochondrion and in programmed cell death (PCD), however, remain unknown. In this thesis, I report the targeted deletion of the gene orthologs encoding AIF in mice and Drosophila. Embryoid bodies differentiated from Aif -/Y mouse embryonic stem (ES) cells fail to cavitate due to a cell-autonomous defect in PCD of inner core cells. In contrast, embryoid bodies differentiated from ES cells deficient in Apaf-1 or caspase-9, two key effectors of PCD, cavitate normally, and AIF translocates to the nuclei of dying cells. Furthermore, Apaf-1 and caspase-9 are dispensible for some morphological features of apoptosis including partial chromatin condensation and membrane blebbing, indicating a potential role for AIF in these apoptotic manifestations. These results indicate an essential requirement for AIF in PCD during cavitation of embryoid bodies. I next analyzed mice which carry a gene-targeted conditional allele of Aif. Mice in which Aif has been inactivated specifically in muscle develop dilated cardiomyopathy, heart failure, skeletal muscle atrophy and lactic acidosis. These pathologies are accompanied by a severe defect in respiratory chain complex I activity and a significant reduction in the level of complex I proteins in mutant tissues. These data point to an essential requirement for AIF in mitochondrial respiration and energy metabolism essential for normal tissue function. In the final section of this thesis, we identify and genetically characterize the Drosophila Aif ortholog DmAif. Like mammalian AIF, DmAIF is a mitochondrial protein that can induce PCD when overexpressed in cells. Transgenic flies which misexpress in the eye an N-terminally-truncated DmAIF lacking the presumptive mitochondrial import sequence (DeltaN-DmAIF) exhibit severely reduced eye size due to ectopic PCD. This cell death can occur in the absence of caspase function. Unlike mammalian AIF, however, DmAIF does not translocate from the mitochondrion to the nucleus following a death-inducing stimulus, as shown in an insect cell line. Finally, we generated and analyzed a Drosophila mutant carrying a loss-of-function mutation in DmAif. Loss of zygotic expression of DmAIF results in growth arrest during early larval stages and lethality. DmAIF mutant animals exhibit severe defects in respiratory complex I and complex IV function, accompanied by diminished levels of cellular ATP. (Abstract shortened by UMI.)