Development and validation of cellular models for studying amyloid precursor protein isoforms.

The development and validation of cellular model for studying amyloid precursor protein isoforms. Amyloid beta peptide (Abeta) is the major constituent of neuritic plaques in the brains of patients with Alzheimer's disease (AD). Abeta is a small insoluble 39-43 amino acid peptide derived from the Amyloid Precursor Protein (APP) by proteolytic cleavage. Different gene splicing produces variant isoforms ranging from 365 to 770 amino acids in length. The main three isoforms are: APP695, APP751 and APP770 and all are potentially sources of Abeta.The project aimed to investigate the hypothesis that one of these APP isoforms (APP695, APP751 and APP770) is more likely to be the source of Abeta in Alzheimer's disease under normal and stress-induced conditions. The clones of HEK293 cells stably expressing human APP695, APP751 and APP770 at comparable levels were put under stress inducing conditions: Serum alteration and energy deprivation.By altering FBS concentration in culture medium, more APP751 was secreted than APP695 and APP770 at all concentrations of FBS. The serum alteration in culture medium had no significant effect on cell number, secreted APP and APP gene expression. Energy deprivation was achieved using 2-deoxy-D-glucose (2DG). There was a significant reduction in cell number to a similar level for all three clones while the level of secreted APP and APP gene expression increased significantly. Also, the trend of APP secretion from each clone under the same concentration of 2DG was the same: more APP751 was secreted than APP695 and APP770.In summary, this project has suggested that serum in culture medium has no effect on cell number, APP secretion and APP gene expression between isoforms while energy deprivation using 2DG affected cell number, APP gene expression and APP production significantly. Not only does this confirm the importance of glucose as a source of energy but has also revealed the potential relationship between glucose metabolism and pathogenesis of AD. Ultimately, glucose metabolism could be the predominant factor in relation to Abeta peptide production.