Amyloid seeding assays for the selective amplification of tau aggregates from human brain homogenates

The microtubule-stabilising protein tau misfolds and accumulates into amyloid inclusions in a diverse class of diseases called tauopathies. A growing body of biochemical and structural literature has now confirmed that tau takes different disease-specific aggregate conformations, which then propagate throughout the brains of afflicted patients. The disease-specific aggregate conformers are classically divided between isoforms of tau resulting from splicing events wherein tau expresses either three or four microtubule binding repeat domains. Thus, tauopathies are defined biochemically by the aggregates themselves either containing three repeats (3R, Pick's disease, PiD), four repeats (4R, Progressive supranuclear palsy, PSP; corticobasal degeneration, CBD; others), or a combination of 3R/4R tau aggregates (Alzheimer disease, AD; chronic traumatic encephalopathy, CTE; primary age-related tauopathy, PART). This work details several methods for the selective detection and amplification of tau aggregates from human brain homogenates. This approach uses the self-propagating properties of amyloid aggregates to sensitively amplify ex vivo seeds using careful selection of recombinant tau proteins and optimisation of aggregation conditions, in the presence of the amyloid-sensitive fluorescent dye Thioflavin T (ThT). Recombinant tau fragments for seed amplification were designed based on structural and biochemical literature to include or exclude essential residues along the tau sequence to promote propagation of one class of tau aggregates over another. The optimisation of assay conditions focused on three principles: 1) sensitive amplification wherein aggregates could be amplified from very dilute (million- to billion-fold diluted) samples; 2) selective amplification wherein target aggregates are amplified, while off-target, control aggregates e.g. α-synuclein are not amplified; 3) where applicable, strain discrimination is possible through assay outcomes including fluorescence amplitudes and aggregate structural properties. Beyond the use of these assays for potential diagnostic purposes, the properties of the amplified aggregates suggest propagation of at least some of the original conformers as they occur in the brain. Thus, the final chapter of this work details the structural characterisation of tau aggregates amplified from AD and PiD brain homogenates, as well as kinetic analysis which suggests distinct propagation mechanisms of the aggregates in vitro. This final chapter suggests a framework for studying the kinetics of aggregation of other protein misfolding diseases wherein a single protein can adopt multiple conformations.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Metrick, Michael

Advisor dc:contributor.advisor

• Vendruscolo, Michele

• Amyloid
• Tau
• Protein
• Aggregation
• Seeding
• Neurodegeneration
• Alzheimer
• Prion
• RT-QuIC
• Kinetics

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0002-9998-4630

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/341009