Date of Award

2010

Document Type

Campus Access Dissertation

Department

Chemical Engineering

First Advisor

Melissa A. Moss

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder currently afflicting five million Americans. Recent work suggests that pathogenesis can be explained through the `amyloid cascade hypothesis'. This hypothesis states that as the peptide amyloid-β (Aβ) aggregates to form insoluble plaques which deposit in the brain tissue of AD patients, it triggers a sequence of events culminating in neuronal cell death. It has been shown that smaller, soluble intermediate aggregates are the more toxic than mature insoluble fibrils. However, the kinetics governing the growth of intermediate aggregates and effect of important physiological variables on this growth have not been established.

A quartz crystal microbalance (QCM) is employed to isolate specific growth pathways of the peptide and quantify kinetic steps of Aβ aggregation. The ability of the technique to selectively observe aggregate growth via monomer addition to soluble intermediate aggregate (elongation) is established. Elongation is found to be reversible and linear, and the data fit a first order model from which kinetic parameters are derived. The observance of a single growth phase in conjunction with the dissociation constant calculated from these parameters indicate that growth is governed by a mechanism similar to the reversible `docking' of monomer to mature fibrilar aggregate. The rate intermediate growth is further found to be increase with solution ionic strength and as solution pH becomes less basic.

As QCM is well suited for surface measurements, the technique is employed here to quantify the effect of cellular membranes on elongation. Intermediate aggregate affinity for supported phospholipid bilayers as well as the ability of these surfaces to support intermediate growth via monomer addition is evaluated as a function of phospholipid fatty acid saturation. Both unsaturated bilayers as well as those which incorporate saturated fatty acids are found to bind intermediate aggregate and support elongation. A kinetic analysis of this process reveals that while monomer binds to intermediates on both surfaces with a similar rate, this interaction is less reversible over membranes with a higher degree of phospholipid saturation, leading to faster assembly on those surfaces.

The QCM is capable of isolating elongation in a wide range of surface and solution conditions. As such it is used to determine effect of pertinent physiological variables on small molecule aggregation inhibitors that have been shown to slow Aβ aggregation. Ionic strength has little affect on the degree of inhibition while pH plays a larger role, actually causing the compound to promote elongation at pH 8.0. This indicates that hydrophobic, rather than electrostatic interactions are responsible for the interactions between the compound and Aβ. It further suggests that elongation could occur through multiple mechanisms in a pH dependent manner. The compound also inhibits elongation of intermediates bound both to unsaturated and partially saturated bilayers, showing a higher degree of inhibition over fully unsaturated membranes.

These results establish the ability of QCM to detect Aβ aggregate growth and derive quantitative kinetic data. The technique has further evaluated the dependence of growth on pertinent in vivo parameters, as well as the ability of compounds to affect elongation as a function of these conditions.

Rights

© 2010, Joseph Andrew Kotarek

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