Visualisierung der transzellulären Ausbreitung von Tau-Aggregationskeimen bei der Alzheimer-Krankheit mit Einzelaggregatauflösung
Biophysik
Zellbiologie
Zusammenfassung der Projektergebnisse
The objective of the proposed work was to study the trans-cellular propagation of tau aggregation, its aggregation profile and its phosphorylation status. To achieve this goal, high resolution microscopy methodologies and two different cell culture systems were employed. Well established antibodies were used for the detection of pathological tau assemblies and they were combined with Single-Molecule Localization Microscopy [direct stochastic optical reconstruction microscopy (dSTORM)]. This allowed the detailed nanoscopic characterization of tau assemblies formed upon seeding in HEK cells and murine primary neurons with a resolution of 40 nm. During this project we were given access to high-resolution microscopy equipment and worked in close collaboration with the McEwan group in the UK Dementia Research Institute, who provided access to samples for imaging. Using a simple experimental tau seeding biosensor cell culture system, I initially validated the established imaging probes and determined the oligomerisation and phosphorylation status of the formed assemblies. I found that the initial formation of fibril-like tau assemblies takes place in close proximity to the microtubule cytoskeleton. I further analysed the kinetics of seeded P301S tau aggregation and the results suggested that small globular aggregates form as early as 4 hours after seeding, before fibrillar-shaped aggregates are generated. Interestingly, the doubling time of the aggregates was determined to be four hours in HEK cells and one day in primary neurons. Small aggregated species also form spontaneously in non-seeded primary neurons, suggesting that spontaneous aggregation of the tau protein can occur in the cellular environment. I moreover examined the presence of tau assemblies in the extracellular space. Tau aggregates were released into the media after seeding, in both the HEK cells and the primary neurons, and the released assemblies were very short, similarly to what has been reported in the past. Furthermore, the seeded tau aggregation of the wt tau protein was studied, but I only identified the formation of short fibrillar structures. Finally, I also investigated the potential involvement of the degradation machineries in the process. I found that inhibition of the proteasome decelerates the amplification rate in the templated seeded aggregation of tau, in contrast to autophagy, which did not impact the process. Overall, the results of this study show that tau aggregation proceeds rapidly in a cellular environment. The critical step appears to be the initial formation of aggregates in the cytosol, whose rapid growth and replication can then overcome the protective cellular machinery. The results of this work have been included in a manuscript which has been submitted for publication and will be available to the public.
Projektbezogene Publikationen (Auswahl)
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An economic, square-shaped flat-field illumination module for TIRF-based super-resolution microscopy. Biophysical Reports, 2 (1), 100044
Lam JYL, Wu Y, Dimou E, Zhang Z, Cheetham MR, Körbel M, Xia Z, Klenerman D, Danial JSH,
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Constructing a cost-efficient, high-throughput and high-quality single molecule localization microscope for super resolution imaging. Nature Protoc. 17(11):2570-2619
Danial JSH, Lam JY , Wu Y, Woolley M, Dimou E, Cheetham MR, Emin D, Klenerman D
