Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biomedical and Chemical Engineering and Sciences

First Advisor

Shaohua Xu

Second Advisor

Alan C. Leonard

Third Advisor

David Carroll

Fourth Advisor

Samuel Durrance


Alzheimer’s disease (AD) is a neurodegenerative disorder affecting close to 5.1 million Americans and its incidence is expected to rise with the higher number of people in the aging population. A hallmark of the disease is the production of the amyloid-beta (Aß) peptides and eventual self-assembly of these peptides into fibrils and extracellular Aß plaques. Both plaques and oligomers are proposed to be the direct cause of AD, but it remains unclear how these structures affect neuronal function and pathogenesis. While a direct toxic effect is currently the favored hypothesis, biomolecule aggregation is also known to play a role in the pathogenesis of numerous diseases by restricting diffusion and bulk flow. The same restriction could occur in the brain due to the dense amyloid plaques forming in the extracellular space, preventing proper flow and diffusion of essential nutrients and cellular waste removal. Unfortunately, limited model systems are available to test this possibility, and molecular tools need to be developed for the analysis of diffusion and bulk flow in relation to neural function and the physical presence of amyloid plaques. The project presented here is focused on two important areas. The first is to develop a nerve fiber-based model system, coupled with molecular rotor tools, which will allow gel-dependent changes in diffusion to be quantified and correlated with the loss of neuronal function. The second is to evaluate methods appropriate to measure diffusion in tissues derived from normal and AD animal models. Using Xenopus laevis (African clawed frog) sciatic nerves and fibrin to simulate the physical presence of amyloid fibril gels, we measured a 40% reduction of the compound action potential when fibrin gel was formed outside frog sciatic nerve fiber epineurium and a 70% reduction of the action potential was recorded when fibrin was formed inside the epineurium. Fibrin encapsulating individual axons was verified using fluorescein-labeled fibrinogen. Measurements using the molecular rotor 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ) revealed a 42% increase in microviscosity during fibrin gelation. Glycerol also caused a concentration dependent reduction of the compound action potential. However, Aß (1-40) oligomers and fibrils had no effect on action potential. Measurements of diffusion in fixed tissue samples was performed by fluorescence recovery after photobleaching (FRAP), a method typically used on live tissues to analyze protein binding and cell membrane mechanics. Our results indicate that diffusion coefficients can be determined in fixed tissues, but methods of sample preparation affected these values. The successful use of FRAP on fixed tissues broadens the scope of the method and potential research areas; however, great care must be taken during sample preparation to ensure accurate comparisons. Diffusion coefficients of small and large molecules were then determined through Aß plaques in mouse/human amyloid precursor protein and mutant human presenilin 1 (APP/PS1) transgenic mice and human AD brain samples. Diffusion was similar through plaques compared to the surrounding tissues of APP/PS1 mice for small molecules like rhodamine B; however, overall tissue diffusion was 0.5- fold faster through transgenic mice plaques and tissues than through wild-type mice tissues. In human AD brain samples, rhodamine B diffused 0.5-fold faster through the tissue than through the plaques, suggesting significantly restricted diffusion through the core of the plaque. This restriction correlates to a theoretical 17-fold reduction in bulk flow through the plaque compared to the surrounding tissue. It remains unclear why mouse and human plaques behave differently, but physical differences in plaque size or structure may play a role. A large molecule, bovine serum albumin conjugated with fluorescein isothiocyanate (BSA-FITC), was also analyzed through mouse and human plaques and tissues but diffused too slowly for accurate measurements. Plaques in tissue slices were further examined by fluorescence microscopy using thioflavin S (ThS) and thioflavin T (ThT), two known molecular rotors which increase fluorescence upon binding to Aß fibrils and plaques. These rotors were compared to the CCVJ rotor used for previous studies. Tissues incubated with CCVJ revealed plaque specificity when compared to ThS stained tissues; however, some CCVJ binding to plaques was indicated causing possible interference with accurate viscosity measurements. An amyloid binding assay comparing ThT with CCVJ in the presence and absence of increased viscosity indicated that CCVJ did possess minimal binding to amyloid fibrils; however, a majority of the fluorescence was due to viscosity. Since the amyloid plaques expressed a greater CCVJ fluorescence than the surrounding tissues, it was likely that plaques represent regions of higher viscosities, but the amyloid fibril binding ability of CCVJ made the plaque viscosity difficult to quantify using the molecular rotor method. The new approaches described here suggest that amyloid plaques may represent regions of increased viscosity, and the impact of diffusion and bulk flow on AD pathology should not be ignored. Hopefully, with further refinement of this methodology, it will be possible to better understand the complex pathology of AD and to develop rational treatments that are more effective than those currently available.


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