Title

Characterization of the regulation of mitochondrial events during apoptosis

Date of Award

2003

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology

First Committee Member

Lawrence H. Boise, Committee Chair

Abstract

Apoptosis is genetically encoded program that maintains tissue homeostasis in multicellular organisms. Dysregulation of the apoptotic machinery can lead to disease states such as cancer, autoimmunity and neurodegeneration. The Bcl-2 and the caspase family are two of the gene families involved in the regulation and execution of apoptosis. Efficient destruction and phagocytosis of the cell ensures that apoptotic cells do not initiate an immune response. Several biochemical "hallmarks" distinguish apoptosis from alternate forms of death, such as necrosis, among these cellular shrinkage, chromatin condensation and loss of the mitochondrial membrane potential (Deltapsi m). The mitochondria play an essential role during apoptosis by releasing apoptogenic factors that initiate a caspase cascade. These changes are well described, however the effects of caspases on the mitochondrial function remain poorly characterized. The focus of this dissertation is to characterize the changes that occur in mitochondrial physiology during apoptosis. We have shown that the release of cytochrome c and the loss of Deltapsim are separate and independent events. Using TNFalpha-induced apoptosis, as a model of the extrinsic cell death pathway, we show that overexpression of Bcl-xL does not inhibit TNFalpha-induced caspase-3 activation or apoptosis in the IL-3 dependent cell line FL5.12 cells. While ectopic expression of Bcl-xL fails to inhibit TNFalpha-induced apoptosis, Bcl-x L retains its ability to prevent cytochrome c release; however, it cannot prevent loss of the Deltapsim. These data suggest that cytochrome c release and loss of the Deltapsi m are two separate and independent events and that loss of the Deltapsi m occurs independent of Bcl-xL and the permeability transition pore, as the inhibitor cyclosporin A has no effect. Additionally, loss of the Deltapsim was prevented only when effector caspase activity was inhibited, suggesting that effector caspases may cause the loss of the Deltapsi m. Taken together, mitochondria can occupy two positions in the death pathway, as initiators of apoptosis and as targets of the caspase cascade. To further characterize the effects of caspases on mitochondrial function, we investigated the consequences of caspase-9 and effector caspase inhibition on mitochondrial physiology in the intrinsic cell death pathway. Inhibition of either caspase-9 or effector caspases prevents the complete loss of the mitochondrial membrane potential without affecting cytochrome c release. When effector caspases are inhibited, mitochondria release cytochrome c, are uncoupled from ATP production and produce reactive oxygen species. Interestingly, the effector caspase-mediated depolarization of the mitochondria occurs independent of the activity of complexes I--IV of the electron transport chain, suggesting that effector caspases do not target these complexes. In contrast, when caspase-9 is inactivated mitochondria remain coupled to ATP production and fail to produce ROS, despite the release of cytochrome c. Take n together these data suggest that caspase-9 prevents the accessibility of cytochrome c to complex III, resulting in the increased production of reactive oxygen specie and that effector caspases may depolarize mitochondria to terminate ROS production and preserve an apoptotic phenotype.

Keywords

Biology, Molecular

Link to Full Text

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