Alzheimer’s Disease (AD) was first diagnosed over 100 years ago, and much progress has been made in the understanding of this neurodegenerative disease. However, the causative factors have not been definitively identified, strategies to postpone disease progression are limited, and strategies to prevent the occurrence are completely lacking.

Many neurodegenerative diseases, including AD, are linked to the misfolding and fibrillar aggregation of protein. In AD, self-replicating “prionoid” fibrillization of β-amyloid and tau proteins are hallmarks of the disease. While β-amyloid aggregation leads to extracellular plaques, tau aggregation produces intracellular neuro-fibrillar tangles. Today, the pathological aggregation of tau is widely thought to be transmitted from cell-to-cell by a seeded template-dependent prion-like spreading mechanism. What causes the first seed, how aggregation proceeds, and how propagation takes place is poorly understood. The prion-like fibrils are known to have different, self-replicating structural conformations, which are here termed prionoid “strains”. It is now suspected, that these strains may define the different phenotypes of tauopathies, and thereby define the disease from which the patient suffers.

This project will study the aggregation process of tau and generate libraries of structural tau strains, which we will classify according to their conformation and biophysical characteristics, and their cell-biological impact. We will search for “seeding effectors” present in brain extracts obtained from late patients afflicted by different tauopathies, and use structural strains obtained in vitro or produced in cellular cultures with human brain extracts to seed tauopathy in cell cultures and to assess the tau strain toxicity.

A combination of related methods will be employed, such as light microscopy (LM), transmission electron microscopy (TEM), and a novel method called Single Cell Visual Proteomics, which is a microfluidic cytosol extraction technology, combined with affinity isolation, interaction-labeling, and loss-less visualization by TEM. This unique technology that was developed by the team of Thomas Braun in the Stahlberg lab with funding from SystemsX.ch, enables us to visualize the entire cytosol of a single cell at sufficient resolution to recognize tau fibrils by automated TEM imaging and computer image analysis. Human brain samples (received from Annemieke Rozemuller from the Netherlands Brain Bank, Amsterdam, The Netherlands, see accompanying letter) will be analyzed directly, used to seed cell culture systems, or used to obtain effectors to test their ability to induce tau-aggregation.

We will employ capillary electrophoresis to segregate the different brain extract components and obtain possible effectors. The ability of these to induce aggregation will then be assessed in the microfluidic aggregation test chips. Here, the tau aggregation is induced in 100 nanoliter volume droplets that contain tau protein, buffer and effector sample. The tau aggregation will be optically monitored without the use of labels. Selected test-droplets will be prepared for imaging by TEM to generate a visual library of different prionoid tau strains, and to seed cell cultures for later examination by Single Cell Visual Proteomics. Fibril-fragment seeding will also be a convenient way to the amplify tau strain effectors for subsequent experiments.

Today’s biophysical and biochemical methods can trace the presence of proteins, but do not allow their structural arrangement in the complexes involved or in prionoid strains to be detected and monitored. Single Cell Visual Proteomics is tailor-made to study prionoid strains, complementing classical analysis methods. The loss-less, visual inspection of the cell's entire cytosol and the ability of TEM to detect very few single molecules of a given component, provide the extreme sensitivity important for the single-cell analysis. The characteristics of neuronal cells and the stochastic nature of transmission processes make an analysis at both, the single-cell and single-molecule level essential.