Date of Award

Summer 1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Frank J. Castora

Committee Member

Laura K. Moen

Committee Member

Bruce W. Tedeschi

Committee Member

William J. Wasilenko

Abstract

Alzheimer's Disease (AD) is a complex neurodegenerative disorder that affects a significant portion of the human population regardless of ethnicity or gender. A mitochondrial hypothesis of AD has been proposed based on a number of studies which establish altered oxidative phosphorylation (OXPHOS) and ATP synthesis in AD tissue. ATP demand is most prevalent in the brain; damage to OXPHOS could severely impair brain metabolism, thereby leading to a decline in cognitive function. Four out of five complexes in the OXPHOS pathway are partly encoded by mitochondrial DNA (mtDNA); thus, this may be a crucial site of lesions that alter brain activity. Within the last decade a number of neuromuscular disorders have been found to be associated with mutations in mtDNA. AD patients share a number of similarities with the mitochondrial encephalomyopathies such as reduced oxidative metabolism, delayed onset of neurological symptoms, and pathological changes that damage tissues with high ATP demand. For these reasons we have been studying AD brain tissue for known, as well as any uncharacterized, mtDNA mutations.

We examined brain autopsy tissue for deleted mtDNA by PCR-based methods and Southern analysis. AD brain tissue was obtained from autopsy-confirmed cases. Using a rat brain model system to examine postmortem effects, we found no mtDNA degradation after 30 hours at RT by Southern analysis. We then assessed brain tissue for the 5 kb deletion ($\rm mtDNA\sp{\Delta 4977})$ by PCR-based methods. While optimizing quantitative techniques we found that serial dilution PCR and kinetic PCR yielded different deletion levels although both methods indicated a greater ratio of $\rm mtDNA\sp{\Delta 4977}$ in the caudate than in the parietal cortex of a cognitively intact control. By serial dilution PCR we determined that AD temporal cortex had a 12 fold greater frequency of $\rm mtDNA\sp{\Delta 4977}$ than controls (0.0628% vs 0.0053%).

Using diagnostic restriction enzyme analysis, we detected the previously described point mutation, $\rm tRNA\sp{gln4336},$ in one Caucasian AD patient and in none of the controls. Biochemical studies indicated that there is a significant decrease in cytochrome c oxidase (COX) activity in platelets and brain tissue of AD patients as well as perturbed COX I, II and III mRNA levels. We examined the mitochondrially encoded COX subunits by single strand conformation polymorphism (SSCP) and DNA sequencing and identified thirty two variants. SSCP efficiency was estimated at 80%. Sixteen of the mutations are new mtDNA variants including a moderately conserved phe- $>$ leu missense change in COX III at np 9861 which was observed in 4.2% (1/24) of the AD patients and in 0% (0/16) of the controls. Further studies will define if this mutation plays any pathogenic role in AD or in COX activity suppression.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree ofDoctor of Philosophy in Biomedical Sciences.

DOI

10.25777/32nm-gt46

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