How does neuroprotective sAPPα initiate its signalling cascade?
Prendergast, Joshua Roshan
AbstractThe occurrence of Alzheimer’s disease (AD) is expected to quadruple by 2050. Currently, there is no treatment that cures or prevents the disease, thereby highlighting the necessity of developing a prophylactic and/or restorative therapeutic. Secreted amyloid precursor protein alpha (sAPPα) elicits neuroprotection, neurotrophism and enhances neuroplasticity, and so is an ideal candidate for development as an AD therapeutic. A number of membrane bound receptors for sAPPα have been suggested, however they have not been definitively validated. Furthermore, whether sAPPα is internalised upon binding is uncertain.This study aimed to express, purify, functionally validate and analyse cell surface binding of the fluorescent tagged sAPPα, EGFPsAPPα, and a number similarly tagged variant constructs of sAPPα. Enhanced green fluorescent protein (EGFP) was conjugated to the N-terminus of sAPPα, sAPPβ and sAPPα variants Δ602-612, Δ608-612, K612A and RER328AAA to create a databank of variants which in the future could be used to investigate the critical active regions of sAPPα. The internal tripeptide motif (RER328) and the final C-terminal residues of sAPPα (595-612) have been proposed to be important in the protein’s function. As GFP is a large molecule, a variety of methods were used to assess that EGFPsAPPα maintained the functional properties of sAPPα.Glucose starved SH-SY5Y cells displayed an 18% increase in cell viability when incubated with EGFPsAPPα (10 nM) under optimised conditions, mirroring sAPPα’s neuroprotective capacity. EGFPsAPPα matched sAPPα’s ability to enhance long-term potentiation (LTP) in rat hippocampal slices, thereby validating that sAPPα function is not structurally perturbed by having EGFP conjugated at the N-terminus. Some evidence was obtained that these neuroprotective and LTP enhancing properties are due to EGFPsAPPα interacting with the plasma membrane. Total internal reflection fluorescence microscopy (TIRFM) analysis displayed single EGFPsAPPα molecules binding to the cell surface of SY-SY5Y cells.iiThis study therefore supports the theory of sAPPα initiating its functions by binding to the cell surfaces, but rapid internalisation has not been excluded and is suggested from the data.
Advisor: Tate, Warren; Ryan, Margaret
Degree Name: Master of Science
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: Alzheimer's disease; sAPPα; secreted amyloid precursor protein alpha; membrane binding
Research Type: Thesis