Date of Award

1-1-2013

Document Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Martin Margittai

Keywords

Alzheimer's disease, amyloid, fibrils, filaments, protein misfolding, tau

Abstract

In various neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, Pick's disease, and corticobasal degeneration, the deposition of fibrils composed of misfolded tau protein is observed. Recent evidence suggests that tau fibrils transfer between cells and spread throughout the brain, underscoring the significance of fibril propagation.

Six tau isoforms exist in the adult human brain that can be grouped into 4-repeat (4R) tau and 3-repeat (3R) tau based on the presence or absence of the second of four microtubule binding repeats. We demonstrate in vitro that seeded fibril growth, a prerequisite for the spreading of the tau pathology, is crucially dependent on the isoform composition of individual seeds. Seeds of 3R tau and 3R/4R tau recruit both types of isoforms. Seeds of 4R tau recruit 4R tau, but not 3R tau, establishing an asymmetric barrier. Conformational templating of 4R tau onto 3R tau seeds eliminates this barrier, giving rise to a new type of tau fibril.

Tau fibrils formed in vitro routinely utilize polyanionic molecules as cofactors to stimulate nucleation. A broad set of negatively charged cofactors, including nucleic acids, polypeptides, and glycosaminoglycans were applied to induce fibril assembly. Utilizing electron paramagnetic resonance (EPR) spectroscopy, we found that the core structure of the fibril is conserved, regardless of cofactor used. Additionally, we assessed whether a cofactor provides a role in tau aggregation beyond inducing the initial nucleation events, and observed that the presence of a cofactor is needed for fibril propagation to be sustained. The cofactor-fibril interaction was investigated, revealing that cofactors are bound to the fibril and that the basis of the interaction is electrostatic. Cofactor binding is dynamic, as introduction of an alternative cofactor was shown to result in exchange with the bound cofactor.

Protein misfolding cyclic amplification (PMCA) is a tool used for the detection of dilute concentrations of prion fibrils. We have successfully applied this concept towards the amplification of tau fibrils for the first time and demonstrated that fibrils can be amplified, even when diluted by several orders of magnitude.

These findings provide basic mechanistic insights into the seeding, propagation, and diversification of tau fibrils. Importantly, we demonstrate that cofactors are not only of consequence for inducing nucleation events, but decorate the fibril and provide a critical role in fibril propagation.

Provenance

Recieved from ProQuest

Rights holder

Paul David Dinkel

File size

148 p.

File format

application/pdf

Language

en

Discipline

Biochemistry, Biophysics

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