Investigating Secreted Amyloid Precursor Protein-alpha and its Functional Domains in Stimulating Hippocampal Protein Synthesis

Abstract

Secreted amyloid precursor protein alpha (sAPPα) is produced by α-secretase cleavage of amyloid precursor protein (APP). Not only is sAPPα of interest because its production competes with amyloid beta (Aβ) production which is involved in Alzheimer’s disease (AD), but it is also neuroprotective and neurotrophic. Due to these positive effects on the brain, it has been suggested as a potential therapeutic agent for AD treatment. Past studies have shown sAPPα to increase protein synthesis, which is thought to be one of the underlying mechanisms of its beneficial effects, but its course of action is still not fully understood. Group 1 mGluRs also have been found to increase protein synthesis. Due to the action of both sAPPα and Group 1 mGluRs converging at this point, it raises the question of whether they are acting together or separately. Two amino acid sequences within sAPPα; RER (peptide in the E2 region) and CTα16 (peptide chain that differentiates sAPPα from sAPPβ, which is the precursor of Aβ) have also shown promise in being used as a therapeutic agent. Smaller peptides are usually preferred over larger proteins in therapeutic agents and therefore they are of great interest. The aim of this thesis was to investigate a possible interaction between Group 1 mGluRs and α-secretase and to test the effects both RER and CTα16 have on protein synthesis. Exogenous sAPPα and several agonists/antagonists were applied (30 minutes) in vitro to investigate protein synthesis in CA1 hippocampal mini-slices from 6-8-week-old male Sprague Dawley rats. The surface sensing of translation (SUnSET) technique was used to measure changes in global protein synthesis across groups. Application of sAPPα (10 nM) and Group 1 mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 20 μM) both significantly increased protein synthesis. Furthermore, α-secretase antagonist TAPI-1 (20 μM) inhibited the increase caused by DHPG while the Group 1 mGluR antagonist LY341495 (100 μM) did not inhibit the increase caused by exogenous sAPPα. These results indicate that Group 1 mGluRs were working upstream of α-secretase in a serial pathway to produce an increase in protein synthesis. Application of RER at both 1 nM and 10 nM concentrations significantly increased protein synthesis in a similar manner to full length sAPPα. Using a modified sAPPα chain in which the RER site was replaced with three alanine (1 nM), the increase was inhibited. Similarly, CTα16 at both 1 nM and 10 nM concentrations significantly increased protein synthesis in a sAPPα like manner. A scrambled variant of sAPPα (10 nM) showed no significant increase compared to control. These results show support for RER and CTα16 being not only required but sufficient to mimic sAPPα in increasing protein synthesis. Overall, the present study shows support for a novel serial pathway of action between sAPPα and Group 1 mGluRs as well as evidence for two functional domains of sAPPα being able to increase protein synthesis. These findings have implications for understanding the mechanisms of sAPPα that could lead to its use as a potential therapeutic agent against Alzheimer’s.