Date of Award

8-2007

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Ronald Wetzel

Committee Members

Jonathan S. Wall, Stephen J. Kennel, Brynn H. Voy

Abstract

The role of various polyglutamine (polyGln) aggregated states in the disease mechanism of expanded CAG repeat disorders continues to be a perplexing subject. We are interested in learning more about these species and the relationship between the aggregation pathways. We have developed an innovative staining technique that allows, for the first time, the visualization of small polyGln aggregates, which we refer to as aggregation foci (AF), that are functionally distinguished by their ability to serve as seeds for amyloid growth. The aggregation foci stain should prove useful in the Huntington’s disease research as a tool for the identification of a functional class of aggregates that was previously undescribed. The ability to account for different aggregates may provide a better correlation with neurodegeneration. Using this method we were able to detect at least four phenotypic classes of aggregates in stable PC12 cells engineered for inducible expression of different polyGln repeat length forms of a green fluorescent protein-fused huntingtin (htt) exon-1 fragment: 1) small, recruitment-competent, cytoplasmic AF, 2) large, cytoplasmic, perinuclear, green-fluorescent, recruitment-inert htt aggregates, 3) cytoplasmic, perinuclear, green-fluorescent, recruitment-competent aggregates, and 4) small, nuclear, recruitment-inert, green-fluorescent aggregates. Each species can be found either in isolation or co-existing within the same microscope field depending on the time post-induction and Gln repeat-length. In our studies, AF tend to be the earliest species that appear in the cells. The PC12 cells generally did not exhibit indications of toxicity due to the polyGln species, and thus provided a good model to study the aggregation pathway without the bias of cell death. Biochemical data were gathered to understand the properties of each distinct aggregate specie. We also examined the ability of two separate compounds (curcumin and demecolcine) to alter the aggregation pathway. Each experiment was designed to obtain knowledge about possible precursor/product relationships that exist among the species, with the ultimate goal of gaining insight into the aggregation pathways responsible for the formation of the amyloid-like AF and larger inert aggregates that arise in these the cell lines expressing various polyGln repeat-lengths. We interpret this data as indicating that the two species are on competing assembly pathways.

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