Abstract

As the terminal enzyme in the mitochondrial respiratory chain, cytochrome c oxidase (COX) couples the translocation of protons across the inner mitochondrial membrane to the transfer of electrons from reduced cytochrome c to molecular oxygen. COX thereby metabolizes oxygen and contributes to the proton gradient that drives phosphorylation of ADP by ATP synthase.

Deficiency of COX activity is associated with significant pathology in humans. While tissues with high metabolic activity are generally affected, there is much heterogeneity in disease presentation and symptoms. This phenotypic diversity has prompted investigations into the effects of COX deficiency on early development, sources of tissue specific pathology, and correlations between degree of COX impairment and severity of pathology.

In this dissertation I present zebrafish, Danio rerio, as a new model system with which to address these issues. I describe the use of morpholino antisense oligonucleotides targeting Surf1, a COX assembly factor, and CoxVa, a structural subunit, that effectively induce COX deficiency in developing zebrafish. The resulting COX deficient phenotype is analyzed in terms of COX subunit levels, DNA transcription, cellular responses, organ formation and function, and organism behavior and lifespan. This multilevel analysis provides insights into the different pathophysiologic mechanisms underlying COX deficient cardiac and nervous system pathology.

By inducing different levels of COX deficiency in developing zebrafish and performing simultaneous biochemical and phenotypic analyses I have identified a critical range of COX activity below which very few animals develop normally. Additionally, measurements of COX activity in control and COX deficient fish over the first five days of development give a temporal dimension to this critical range and allow for a description of developmental pathology in terms of biochemical fluctuations. Finally, simultaneous reduction of Surf1 and p53, a mediator of cell death, impairs COX activity to a greater degree than reduction of Surf1 alone. These data imply that traditionally non-mitochondrial factors can augment COX deficiency and may be important in generating the heterogeneity of disease phenotypes.

These results establish zebrafish as a valuable model of COX deficiency and expand our understanding of the role of COX deficiency in developmental pathologies.

This dissertation contains co-authored material.