Exploring Endoplasmic Reticulum as a Therapeutic Target Site to Antagonize Amyloid Production.

The excessive release of aggregation prone, toxic Amyloid-β (Aβ) from the Amyloid Precursor Protein (APP) by secretases has been genetically and biochemically established as central to Alzheimer’s Disease (AD) pathogenesis. Therapeutic strategies targeting extracellular deposits of Aβ, or inhibiting enzymes releasing Aβ from APP, have not been as successful as first envisioned and motivate alternative approaches, such as reducing the production of Aβ by modifying APP localisation. This requires a better understanding of the APP intracellular processing events which liberate Aβ peptide. In my thesis, I mapped the kinetics of APP cleavage to produce Aβ with subcellular resolution. To visualize APP’s processing with organellar resolution, I developed a dual-tagged APP reporter system combined with Retention Using Selective Hook (RUSH), which enabled the controlled localisation of the protein in the organelles of the secretory pathway whilst monitoring its cleavage status. Using pulse-chase long-term fluorescence imaging and analysing the label-signal decay patterns, I quantified APP cleavage within each of these compartments. The measurements demonstrate that APP undergoes cleavage in endosomes and Golgi, and to a lesser extent in the Endoplasmic Reticulum (ER) or plasma membrane. Biochemical analysis was performed using Single-Molecule Pulldown and Single Molecule Array to detect monomeric and aggregated Aβ, which demonstrated that retaining APP in the ER reduced amyloid production. Based on these insights, I explored a therapeutic strategy focused on redirecting APP to the ER, to reduce amyloidogenic processing. I developed an intrabody-based system to control localisation of endogenous (untagged) APP in the ER. This strategy proved effective in an immortalised cell line, human cortical neurons, and in vivo. A rationally designed ER-targeted intrabody that targets the APP luminal domain caused ER enrichment of endogenous APP in the mouse brain, leading to a substantial decrease in Aβ plaque formation and associated neuropathological features. The manipulation appeared inert in terms of viability and ER stress in cells and in vivo. These findings suggest that modulating APP early secretory pathway localisation leverages the organelle’s high proteostasis capacity and presents a promising therapeutic avenue that avoids the adverse effects associated with direct secretase inhibition or complete loss of APP expression.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Gupta, Karnika

Advisor dc:contributor.advisor

• Avezov, Edward

• Aggregates
• amyloid
• Endoplasmic Reticulum
• Nanobody

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/7661b35b-8efb-47ec-9f4d-2d4f0791d964/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.113696

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/376472