Abstract:
Alzheimer’s disease (AD) is a progressive neurodegenerative condition characterized by behavioral changes and memory impairments, predominantly observed in the elderly population. As the most common form of dementia worldwide, AD has a large socioeconomic burden that is expected to grow in the next decades, given that no potent therapeutic strategy exists to treat the increasing population of aged individuals. The deposition of misfolded β-amyloid (Aβ) within extracellular senile plaques is a pathological hallmark of disease. The appearance of Aβ plaques has been identified as one of the earliest events in AD, and the prevailing amyloid cascade hypothesis suggests that the abnormal cleavage and misfolding of Aβ is the trigger of disease. The focus on misfolded Aβ as a central agent in AD has drawn parallels to the infectious prion protein and the protein-only hypothesis of disease transmission for prion disease. A number of studies have demonstrated that Aβ assembles into amyloid fibrils and that misfolded species can seed the aggregation of monomeric Aβ both in vitro and in vivo. Although the seeding properties of aggregated Aβ are robust, a more recent line of investigation aimed to characterize how variations in Aβ aggregate assembly influence the potency of seeding and also the progression of disease. In this doctoral dissertation, the structural features of amyloid plaque cores within a diverse cohort of 40 patients, with either sporadic or familial AD, were assessed using a unique class of conformation sensitive amyloid binding dyes referred to as luminescent conjugated oligothiophenes or LCOs. The fluorescence spectral signature of LCO stained plaque cores was strikingly different between familial AD and sporadic AD, and subtle differences were also identified between the typical and posterior cortical atrophy variants of sporadic AD. This demonstrates that the amyloid structure is distinct between AD subtypes, an observation not explained by Aβ biochemical features or clinical data. Surprisingly, within a single AD brain, multiple spectral signatures for amyloid were present and are referred to as clouds of conformational Aβ variants. The structural features of human AD-derived Aβ aggregates were also preserved upon transmission to human APP transgenic mice. The direct analysis of Aβ conformations within post-mortem human tissue provided insight into the spectrum of species present within a brain, but the presence of such Aβ variation at earlier stages of disease is unknown. In a second study, the change in conformational and biological Aβ features with aging was studied in APP transgenic mouse models with either slow or rapid cerebral β-amyloidosis. Both histological and biochemical levels of Aβ increased with aging, but the ratio of specific Aβ species, namely Aβ42/40, surprisingly peaked at the early stage of plaque appearance in the two models. An in vivo bioassay was then used to show that different aged brain extracts had increasing seeding activities, or seeding dosages (SD50), that plateaued with advanced age when injected into a transgenic host. Interestingly, when seeding activity was normalized to the amount of Aβ within the extracts, a peak in specific activity became apparent at the age when Aβ deposition first appears and Aβ42/40 was highest. This study provides further evidence that treatment of AD should be initiated early, i.e. at the time point when these potent seeds are present and before a cascade of neurodegeneration can occur. Finally, in a third study, a novel methodology capable of investigating various native Aβ assemblies in the brain was described. Here, the use of agarose electrophoresis facilitated the separation of Aβ aggregation states by size, and demonstrated that transgenic mouse brain extracts harbor Aβ aggregates with a different size distribution than in vitro Aβ fibrils. Agarose fractions were collected and enzymatically digested to produce a liquid sample, which could be used for further analysis. Immunoprecipitation with an amyloid-conformation-specific antibody confirmed that Aβ migrating with a high molecular weight had a preserved quaternary structure after the enrichment protocol. Further evidence that the structure was preserved was demonstrated when a high molecular weight fraction induced Aβ deposition when injected into transgenic mice. This novel tool provides the opportunity to screen potential therapeutic antibodies or compounds against native in vivo aggregates, while generating samples that can be further analyzed to determine the relationship between aggregate size and structure with biological features such as seeding activity. The original research within this dissertation has provided a significant contribution to the knowledge of Aβ structural features within AD, and the seeding properties over the course of disease. Additionally, the establishment of a new method for isolating in vivo seeds using agarose fractionation will allow for further basic investigations of these findings and aid in the development of novel therapeutics. Specifically targeting the earliest generated seeds within an AD subtype using immunotherapies could enhance the removal of pathogenic Aβ and provide a viable strategy to prevent AD.
Alzheimer's disease