Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Zilm, Kurt

Abstract

Alzheimer’s Disease (AD) is a neurodegenerative disease with significant societal impacts. AD is the only top ten leading cause of death that cannot be prevented, cured, slowed, or even diagnosed before symptoms are present. AD, similar to other neurodegenerative diseases, has been linked to protein aggregation as the key factor in the disease pathology. However, the mechanism by which AD proliferates is poorly understood. Recent advancements in techniques and methods in structural biology, including nuclear magnetic resonance (NMR) and electron microscopy (EM), have opened a new era of studies into protein aggregates implicated in neurodegenerative diseases, including AD. Therefore, this thesis utilizes these techniques in order to study two important proteins identified in the proliferation of AD. It is now well established that in AD, cellular prion protein (PrPC) binds with high affinity to amyloid-b oligomers (Abo) and that this binding plays a role in pathological symptoms in animal models of AD. However, the mechanism and interactions by which this binding occurs remain unresolved. In vitro, PrP and Abo combine to form a stoichiometric insoluble aggregate, making it an ideal candidate for structural studies via solid state NMR (ssNMR). In the structural study of insoluble protein aggregates, EM is also typically considered a complementary technique to ssNMR as ssNMR provides detailed structural information as well as information on specific atomic interactions, whereas EM can provide an overall image of the protein structure and/or architecture of larger assemblies. In this thesis, structural studies of PrPC and Abo are undertaken with two overarching objectives: (1) to gain insight into how the complex may be formed in vitro and how that may relate to in vivo interactions and (2) to measure the mobility and structural perturbations that may be important for complexation and how that may relate to toxicity. Utilizing ssNMR, it is found that PrPC remains highly mobile when complexed with Abo, whereas Abo shows significantly slower dynamics in the same complex. Additionally, 15N ssNMR studies showed that there are two distinct groups of binding residues on PrPC. From these results, a picture of the complex emerged, which pointed towards a network polymer with a highly mobile PrPC. Further, studies of the immobile Abo with multidimensional ssNMR reveal that its conformation is distinctly changed by the interaction with PrPC and is structurally different than both Abo alone and fibrillar Ab. TEM and Cryo-EM studies of the complex reveal that the overall structure of the complex is a well-defined dumbbell shape. It is hypothesized that, supported by the ssNMR mobility studies of PrP, that PrP is a linker between the lobes of separate Abo aggregates, leading to a hydrogel complex. This theory agrees with the model of the overall structure proposed in Chapter 2. These results, which help gain insight into the toxic interaction between PrP and Abo, may be key in understanding the AD cascade.

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