âDementia and Alzheimerâs diseaseâ is the leading cause of death in the UK, so new therapeutic options are of critical importance. One possible therapeutic avenue could be to protect neurons from damage through the use of neuroprotective molecules. The initial cleavage of the amyloid precursor protein (APP) by either Î±- or Î²-secretase produces the soluble amyloid precursor protein alpha (sAPPÎ±) or beta (sAPPÎ²) fragments, respectively. sAPPÎ± binds to neuronal cells in a receptor-dependent manner and is 100-times more neuroprotective than sAPPÎ², despite differing by just 16 amino acids at their C-termini. The receptor(s) to which sAPPÎ± binds to facilitate neuroprotection are unknown. Therefore, the identification of new binding partners for sAPPÎ± could have therapeutic relevance. This thesis used two unbiased receptor identification methods to identify sAPPÎ± binding partners in neuronal cells. HEK cell lines stably overexpressing His-tagged sAPPÎ± and sAPPÎ² were generated. Both recombinant proteins were purified and their identities were confirmed through immunoblotting and GeLC-MS/MS. sAPPÎ± and sAPPÎ² were shown to bind to induced pluripotent stem cell (iPSC)-derived neurons and SH-SY5Y cells. sAPPÎ± increased the expression of synaptophysin in iPSC-derived neurons. Pre-treatment of SH-SY5Y cells with sAPPÎ± significantly reduced the production of reactive oxygen species following toxic insult in a time-dependent manner. Calsyntenin-1 and calsyntenin-3, type-I transmembrane proteins expressed on the post-synaptic membrane of neurons, were identified in both unbiased receptor identification methods as novel binding partners for sAPPÎ±. Knockdown of calsyntenin-1 did not affect the binding of sAPPÎ± to SH-SY5Y cells; however, knockdown of calsyntenin-3 significantly reduced the binding of sAPPÎ± to SH-SY5Y cells. Overall, the work described in this thesis demonstrated the beneficial effects of sAPPÎ± to reduce oxidative stress in neuronal cells in vitro and identified calsyntenin-3 as a novel binding partner which was required for the binding of sAPPÎ± to neuronal cells. The identification of calsyntenin-1 and calsyntenin-3 as neuronal binding partners for sAPPÎ± may provide new therapeutic targets for neurodegenerative diseases such as Alzheimerâs disease.
|Date of Award
|31 Dec 2023
- The University of Manchester
|Martin Humphries (Supervisor) & Nigel Hooper (Supervisor)
- Amyloid Precursor Protein