Date of Award

5-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Valerie Berthelier

Committee Members

Elizabeth Howell, Christopher Stanley, Jonathan Wall

Abstract

Huntington’s disease (HD) is a genetic neurodegenerative disorder, associated with the mutant huntington (Htt) protein, containing an abnormally long stretch of glutamine (polyQ) residues. Upon proteolityc cleavage, this protein forms htt-exon1 fragment and aggregates into highly stable and organized beta-sheet structures. Currently, no clear evidence exists to determine if Htt protein aggregates are epiphenomena, if they are beneficial to or pathogenic for neurons. Although the correlation between the length of polyQ repeats, their propensity for aggregation, and disease is undeniable – the longer the polyQ region, the earlier the onset of HD and its symptoms are more severe – several cell and HD animal models studies demonstrated an absence of a link between aggregate presence and neuronal toxicity. It was proposed that neuronal toxicity is associated with early stages of protein fibril formation and that mature aggregates actually represent an inert end stage, serving as a rescue mechanism. At present, a detailed understanding of the structures of different intermediate species, formed both on- and off-pathway to Htt fibril formation, is not established. Molecular insights in amyloid research and protein aggregation suffer from fundamental difficulties in controlling the formation of early intermediate assemblies and characterizing them. Within the scope of this research project we performed a comprehensive analysis on the aggregation of the htt-exon1 fragments containing normal (22Gln) and pathological (42Gln) length of polyglutamine repeats. To unravel their aggregation pathways, we performed time-resolved small angle neutron scattering (TR-SANS) with ab-initio reconstruction approaches, we obtained the 3-D structures of the earliest intermediates, followed their progression into protofilbrills and obtained the internal composition of the mature fibrils. Using SANS and other biophysical techniques we found that the length of polyGln repeat within the htt-exon1 fragment does not only affect the kinetics of aggregate formation, but also drastically influence the structural dynamics and mechanism of aggregation. We also performed osmotic stress experiments coupled with contrast variation techniques to determine quantitatively the differences in the internal structures of the mature fibrils. The described results illustrate the utility of SANS for identification of various intermediates associated with amyloid and neurodegenerative diseases.

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