Polymorphic protein aggregation in tauopathies

Sammanfattning: Alzheimer’s disease(s) comprises one of the most common and costly neurodegenerative diseases. With a larger population and an increasing life expectancy, amyloid diseases (with age as one of the most prominent risk factors) will generate an even larger burden on healthcare. We know that protein misfolding is involved in the disease process but lack a complete understanding of the mechanism behind these diseases, both the sporadic and hereditary variants. It is not always known whether it is a gain-of-toxic function or loss‐of‐function that causes the neurodegeneration. To determine the correct diagnosis is a major challenge. If diagnosed, only a few amyloid diseases can be treated today.Amyloids are highly ordered filamentous protein aggregates with a β‐sheet structure. From identical or similar amino acid sequences, a large variety of structures can be formed by different secondary and tertiary structures and by different packing of the individual filaments. This is known as fibril polymorphism.This work focuses on characterization on two proteins involved in Alzheimer’s disease and other neurodegenerative diseases, namely Amyloid‐β (Aβ) and microtubule associated protein tau (tau). In order to investigate the properties of these proteins in vitro it is important to have protocols for production of recombinant protein that enables characterization of these aggregation prone proteins. We present protocols for recombinant expression, purification and non‐denaturing fibrillation assays used in our lab to produce and analyze Aβ, tau and the prion protein.Development of new ligands for characterization of fibrils is an important way of investigating different fibrillary structures and characterizing and distinguishing between the different polymorphs of aggregates. We showed that the central benzene ring of the amyloid ligand X‐34 can be exchanged for other heterocyclic motifs and still retain targeting of the “Congo red” binding site. The compounds do not compete with the Pittsburgh compound B (PiB) binding site on recombinant Aβ fibrils.We also characterized tau fibrils formed from seeding with tau aggregates from patients diagnosed with different neurodegenerative tauopathies. We use aggregation kinetics to test the seeding activity on two different sequence isoforms of tau, 0N3R and 0N4R. Fibrillation kinetics, an array of recently developed ligands (including the X‐34 analogs) and electron microscopy were used to characterize different polymorphs of the tau aggregates formed by seeded templating from patient derived seeds. Our data showed that brains contain seeds with different morphologies even with in patients diagnosed with the same disease.Investigations of the rare tau mutant G273R found in a patient with a presumed tauopathy also highlights the problem with proper diagnostics. Our results reveal that in vitro this mutation change the binding properties of 0N4R tau to the cytoskeletal proteins microtubule and F‐actin. Furthermore, we could show that when seeded, the fibril formation seeding activity followed a sequence similarity dependent manner. In fibrils formed during heparin-induced aggregation we can be distinguished between wild type and mutant tau as they form fibrils with different thickness. Our in vitro biophysical data support that the G237R mutant is causing a 4R tauopathy.The work in this thesis increase our knowledge in the field of tau aggregation and tau fibril polymorphism.