Date of Award

January 2013

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Ron A. Adelman

Second Advisor

Wei Li

Subject Area(s)

Ophthalmology, Molecular biology, Medicine

Abstract

This project aimed to clarify the role that mitophagy and autophagy play in the 13-lined ground squirrel's adaptations during hibernation. We studied the structure, function, and dynamics of the mitochondria in this animal model, with an emphasis on the mitochondria in the liver and retina. The work was designed to evaluate systemic rates of autophagy and mitochondrial fusion and fission and to classify the shape, volume and orientation of the mitochondria found within the photoreceptors of the retina.

A variety of techniques including confocal microscopy, western blot analysis, electron microscopy and mitochondrial enzyme assays were utilized in this project. We hypothesized that there would be more mitochondrial-specific autophagy, or mitophagy, in the tissue of hibernating ground squirrels as compared to awake animals and that the mitochondria in hibernating retinas would have a greater rate of fusion with one another.

Western blot analysis demonstrated a significant (p<0.05) increase in autophagy during hibernation in the liver and retina. Two-dimensional difference gel electrophoresis showed notable differences in mitochondrial protein expression between the awake and hibernating states in both the liver and retinal tissue despite a nearly equivalent mitochondrial enzyme activity level between the two states. Electron microscopy revealed an increased number of "cylindrical" mitochondria in the hibernating retinas (p < 0.05) with a trend toward an increased total number of mitochondria in the hibernating animals. However, the average total surface area of each mitochondria was greater in the awake animals (p < 0.05), likely due to "kissing" and "funnel cake" mitochondria with an average greater surface area.

Our data suggests that there is increased autophagy in hibernating animals and that the amount of autophagy differs by organs, with the liver demonstrating the greatest change in autophagy levels. The variation in mitochondrial protein expression is possibly secondary to modifications in fission or fusion proteins. This is supported by the finding that the hibernating mitochondria in the retina are more likely to be unfused, and that the fused mitochondria in hibernating animals are themselves smaller than their awake counterparts. Further study will be important in confirming the trends identified in this project.

Share

COinS