Protective and Toxic Proteins in Alzheimer’s Disease

Student thesis: Master Thesis and HD Thesis

  • Maj Schneider Thomsen
Background: Secreted amyloid precursor protein alpha (sAPPα) is a neurotropic and neuroprotective protein released after cleavage of amyloid precursor protein (APP). Cleavage of APP can also lead to the release of secreted amyloid precursor protein beta (sAPPβ) and amyloid beta (Aβ), which is postulated to be the primary neurotoxin in Alzheimers Disease (AD). The single difference between sAPPα and sAPPβ is 16 extra amino acids at the C-terminus of sAPPα. These 16 amino acids are of special interest since they also form the N-terminus of Aβ. Therefore, it was hypothesized that sAPPα and Aβ might interact.
Method/Results: A GST pull-down assay was performed with bait GST-sAPPα or GST-sAPPβ immobilized to glutathione Sepharose 4B beads and Aβ1-42 added as prey. Subsequent, Western blot and densitometry analysis revealed significantly more binding of Aβ1-42 by GST-sAPPα compared to GST-sAPPβ. This indicates that sAPPα interacts with Aβ1-42 through the 16 amino acids at the C-terminus. To further characterize the interaction, five GST tagged sAPPα variants with mutations in the critical 16 amino acid region (K612A, K612V, D608-612, D602-612, and H609/10A) were constructed as well as one within a motif (RER328-330AAA) of much functional interest. The pull-down assay was repeated with the GST tagged sAPPα variants as bait and Aβ1-42 as prey. All the sAPPα variants except K612A sig-nificantly reduced the binding of Aβ1-42 compared to sAPPα. This further implies that the in-teraction between Aβ1-42 and sAPPα is through the 16 amino acids at the C-terminus of sAPPα.
Discussion/Conclusion: sAPPα has previously been shown to protect cell cultures against Aβ induced toxicity and the finding that sAPPα interacts with Aβ may contribute to the neuroprotective effect of sAPPα by keeping sAPPα and Aβ in equilibrium.
Publication date1 Jun 2012
Number of pages55
ID: 63500516