De La-Rocque, Samantha;
(2022)
Monitoring and investigating the process for cytosolic translocation after tau endocytosis.
Doctoral thesis (Ph.D), UCL (University College London).
Preview |
Text
SDLR_PostViva_Monitoring and investigating the process for cytosolic translocation after tau endocytosis.pdf Download (5MB) | Preview |
Abstract
Tau pathology is well documented in Alzheimer's disease and its build up follows a stereotypical spread correlating with disease severity. This reveals an apparent anatomically linked spread of tau, supporting the hypothesis that tau can propagate via cell-to-cell transfer. However, a direct visualisation of tau entering the cytosol of a recipient cell and a full understanding of the mechanism at the basis of this process are still lacking. I have setup and optimised a strategy based on split-green fluorescent protein (GFP) to conclusively demonstrate cytosolic translocation of tau. Split-GFP is derived from the superfolder beta-barrel structure of GFPs truncated between 10th and 11th beta strands generating two non-fluorescent fragments named GFP1-10 and GFP11. In this project, the GFP11 is used to tag tau whereas the GFP1-10 is cytosolically expressed and acts as a sensor to detect the presence of tau in the cytoplasmic environment; only when the two GFP fragments associate will they reconstitute the fluorophore, thus maximising the signal to noise ratio. I have demonstrated the ability of the split-GFP assay to detect cellular uptake and release of tau into the cytosol in living cells. With this technique I have observed the real-time uptake dynamics of all six tau isoforms and the disease relevant P301S mutant and investigated potential mechanisms underpinning this process, including pH sensitivity and Rab GTPase activity. This assay has been tested both in cell-lines and primary neuronal cultures grown in custom-made microfluidic chambers, with the aim to improve the tools available to study tau propagation in vitro and in vivo.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Monitoring and investigating the process for cytosolic translocation after tau endocytosis |
| Event: | University College London |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10142332 |
Archive Staff Only
 |
View Item |
