Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Chemistry, Biochemistry

Date of Defense

8-5-2015

Graduate Advisor

Michael R. Nichols, PhD

Committee

James K. Bashkin

Chung F. Wong

Keith J. Stine

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the accumulation and deposition of aggregated amyloid-β protein (Aβ). Most of the studies indicate that Aβ1-42) and Aβ(1-40) play an important role in the neurodegenerative pathology in AD. A critical component of AD pathology is inflammation, which results from the activation of glial cells around the Aβ deposits. The goal of my dissertation research was to determine the role of Aβ aggregation in microglia-triggered inflammation and identify cellular components that mediate the inflammatory response. CD47 is a cellular receptor found on the microglia surface and it has been proposed to have a role in Aβ-induced inflammation. I utilized three separate strategies including the CD47 antagonist peptide 4N1K, anti-CD47 neutralizing antibodies, and CD47 knockout mouse to conclusively demonstrate that CD47 is not involved in Aβ(1-42) protofibril-triggered microglial production of cytokines tumor necrosis factor-α and interleukin-1β. In support of my cellular work, I conducted additional studies aimed at determining differences in early aggregation events and stability between Aβ(1-42) and Aβ(1-40). I used a conformation-specific antibody (OC anti-serum) to determine the presence of nascent elements of fibrillar structure in Aβ(1-42), but not Aβ(1-40) monomers. Once aggregated, Aβ(1-40), but not Aβ(1-42), aggregates were sensitive to chaotropic agents indicating the former was much less stable than the latter. Interestingly, nascent Aβ(1-42) aggregates were sensitive to harsh detergents, but became resistant over time. The stability changes that were monitored by OC immunoreactivity occurred before more commonly-used methods were capable of detecting the nascent aggregate. Finally, I developed new in situ cell aggregation assays to investigate changes in Aβ(1-42) conformation in the presence of a cellular environment. Preassembled Aβ(1-42) protofibrils induced cytokine production between 4-6 hours. However, Aβ(1-42) monomers displayed a delayed response and this response corresponded to changes in β-sheet structural content and surface hydrophobicity, which are indicative of aggregate formation.

OCLC Number

936470189

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