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Access Type

WSU Access

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Molecular and Cellular Toxicology

First Advisor

Ruden Douglas

Second Advisor

Victoria Meller

Abstract

Friedreich’s ataxia (FRDA) is an inherited autosomal recessive neurodegenerative disease. It affects 1 in every 50,000 people in central Europe and North America. FRDA is caused by deficiency of Frataxin, an essential mitochondrial iron chaperone protein, and the associated oxidative stress damages. Autophagy, a housekeeping process responsible for the bulk degradation and turnover of long half-life proteins and organelles, is featured by the formation of double-membrane vacuoles and lysosomal degradation. Previous researches indicate that Danon’s disease, the inherited neural disorder disease that shares similar symptoms with FRDA, is due to the malfunction of autophagy. Based on this, we raise the question whether the autophagy activity is modified and what is its role in FRDA. Study has shown that oxidative stress may play a major role in the progression of neurodegenerative diseases by attacking the cytoplasmic molecules and organelles, and autophagy is the major pathway in reducing oxidative stress and removal of malfunctioned organelles. Additionally, autophagy has been closely related to cell apoptosis and organism remodeling. Mitochondrial Autophagy (mitophagy) is also the major turnover pathway for damaged mitochondria. Therefore, the dysfunctional autophagy in removing the malfunctioned mitochondria in FRDA may responsible for its pathogenesis. Since the mechanism of autophagy in the development of FRDA is still largely unknown, a systematic analysis of the status and function of autophagy pathway is needed.

My thesis is targeting at four goals: (1) to construct FRDA fly model and characterize autophagy expression pattern in each stage; (2) to determine the effect of autophagy modification on the symptoms of the FRDA flies; (3) to identify the potential downstream events of autophagy; (4) to explore the possible upstream activities by examine whether the AMPK or SAPK stress response pathway is involved in FRDA flies. Our hypothesis is the up-regulation of autophagy occurs in the early stage of FRDA and may induce apoptosis or mitophagy. We identified that autophagy level is up-regulated in FRDA flies at both transcriptional and translational levels. Moreover, the overexpression of Frataxin also increases autophagy activity. The comparable Atg5 mRNA level in both Frataxin deficiency and overexpression flies indicates this induction of autophagy in FRDA flies is Atg5 independent. Autophagy inducer Methylene blue and rapamycin could partially prolong the longevity and restore the fertility of FRDA flies, but could not rescue the pupae lethal phenotype. When treated with the autophagy inhibitor chloroquine, FRDA KD flies showed reduced longevity and locomotor activity, implying the beneficial effect of autophagy in certain development stages. The FRDA KD flies also showed up-regulated caspases-3 and cytochrome C level, indicating enhanced apoptosis in cells with reduced Frataxin. We also attempt to apply the heart pacing assay to evaluate the FRDA Drosophila cardiac function. Although the results are inconclusive, the heart pacing assay appears to be a valuable tool for future research.

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