Alzheimer's disease (AD) is a common neurodegenerative disorder marked by fibrillar aggregates of misfolded Aβ peptides and tau protein in the brain. Misfolded Aβ peptides form extracellular senile plaques and cerebral amyloid angiopathy (CAA) in brain blood vessels. On the other hand, misfolded tau protein accumulates in intracellular tau tangles. Although the disease-causing protein shares the same primary sequence, its tertiary and quaternary fibrillar structures can exhibit poly-morphism. Previous studies suggest that this structural polymorphism may be linked to distinct AD clinical phenotypes. Thus, understanding structural polymorphism is crucial to acquire insight into the disease mechanism.   

In this thesis, I examined the variation in Aβ fibril morphology within amyloid plaques in AD mouse models carrying familial mutations in the AβPP gene. A com-bination of amyloid binding conformation-sensitive fluorescent dyes and Aβ-specific antibody staining reveals that the AβPP processing genotype influences the structure of Aβ fibrils within Aβ plaques. Plaques from APP23 mice with Swedish AβPP mutation (KM670/671NL) exhibit two distinct fibril polymorphic regions: a core and a corona. The plaque core has tightly packed Aβ40 fibrils, while the corona has diffusely packed Aβ40 fibrils. AppNL-F mice with the AβPP Iberian (I716F) and the Swedish mutation have tiny plaque cores of compact Aβ42 fibrils.

I also examined the seeding activity of recombinant Aβ fibrils. The Aβ pathology in the brain propagates through a process called seeding, where preformed fibrils, known as seeds, promote fibril formation by bypassing the nucleation step. Previous research demonstrated that injecting brain extracts rich in Aβ (seeds) from transgenic mice and AD patients can induce AD pathology in transgenic mice. While research on recombinant seeds is still limited, we focused on investigating the seeding activity of pure recombinant Aβ fibrils of different compositions. Seeds were inoculated into APP23 mouse brains at 3 months and were analyzed after 6 months of incubation. We observed that recombinant seeds (fibrils from Aβ1-42, Aβ1-40, and Aβ1-40+Aβ1-42) accelerated plaque formation compared to non-inoculated transgenic control mice. In addition, all seeds induced profound CAA in young APP23 mice (9 months). Interestingly, pure Aβ1-42 seeds produced significantly more CAA and amyloid plaques than seeds containing Aβ1-40, which is surprising given that APP23 mice produce up to five-fold more Aβ1-40 than Aβ1-42. 

I furthermore examined the seeding activity of Aβ1-42 aggregates isolated from neurons and glial cells from Drosophila melanogaster. Aβ peptides were expressed in neurons and glia by nsyb-Gal4 and repo-Gal4, respectively. Seeds from neuron and glial cells were again inoculated in APP23 mice and incubated for six months. We found that both the neuronal and glial seeds were not potent in inducing seeding. However, both the seeds became potent when fibrils were first amplified in vitro with recombinant Aβ1-42 before inoculation. These active seeds originating from neuronal expression produced more CAA and plaques than seeds from glial cells in terms of the number of aggregates per section, strongly suggesting that the amyloid fibril polymorphs are replicated into two distinct amyloid strains with different seeding efficiency.  

In the last study of the thesis, we developed a multiple-ligand fluorescence micros-copy approach to detect diverse pathological Aβ fibrils. Since Aβ amyloid plaques pose various fibrillar structures, using a single ligand is not enough to detect all these pathological aggregates. This study used both AD mouse models and AD patient’s brain samples.  It was shown that ligand binding in mice is dependent on mutation and age. Thus, combining different ligands enhances the possibility of detecting various types of Aβ amyloid aggregates.  

In summary, this thesis provides an understanding of the diversity of structural variations of amyloid fibril aggregates in Alzheimer’s disease, which will help to identify disease-relevant fibril polymorphs and provide insight for designing molecules for diagnostics and therapeutics.