P3‐039: Aβ binding properties of a synthetic peptide corresponding to an amino acid sequence in human pericentriolar material 1 (PCM‐1) protein

Abstract

We have isolated a ∼ 20kDa polypeptide (termed PK-4) from a T-7 Phage Display system expressing human brain cDNA library (Novagen) using polyclonal antibodies against a protein kinase C (PKC) inhibitor as bait. In addition to having PKC inhibitory activity, we serendipitously discovered that this polypeptide also bound Aß 1–42 with high affinity. The amino acid sequence of this polypeptide has 89 percent homology to an amino-acid sequence in human pericentriolar material-1 (PCM-1) protein. We have mapped the Aß-binding motif on this polypeptide, and have synthesized short (∼4 kDA; 40 amino acids) amyloid-binding peptides (ABPs) corresponding to this region. We have determined the Aß 1–42 binding properties of one of these ABPs, ABP-p4–5, using ELISA, dot blot and poly-acrylamide gel electrophoresis, and immunohistochemistry with brain sections. Cell toxicity assays were done with SH-SY5Y human neuroblastoma cells and Hoechst stain. We have found that ABP-p4–5 binds Aß 1–42 in the nM range. Thioflavin-binding assay and Western blot analysis indicate that ABP-p4–5 inhibits Aß 1–42 aggregation. Interestingly, ELISA studies indicated that ABP-p4–5 bound aggregated Aß1–42 preparations (37°C, 24 hrs) better than non-aggregated preparations, suggesting that it may have higher affinity towards Aß oligomers. In cell toxicity assays using human SH-SY5Y neuroblastoma cells, ABP-p4–5 inhibited Aß1–42-induced apoptotic cell death. Furthermore, we have found using fluorophore labeled p4–5 (FITC-p4–5) that it binds amyloid deposits in brain sections from Alzheimer's disease (AD)-transgenic mice and human AD patients in vitro and also when microinjected into AD-mouse brain. These results indicate that the ABP-p4–5 peptide can potentially be used therapeutically to reduce Aß burden and also to detect Aß deposits in the brain. Most importantly, since PCM-1 and the Aß 1–42-binding ABP-p4–5 have a high-sequence homology it would be interesting to determine whether PCM-1 has a role in the development of AD. Thus, for example, PCM-1 is known to be required for ciliogenesis which in turn is known to be required for neurogenesis and memory formation. So the question remains, would the accumulating oligomeric Aß 1–42 bind PCM-1 and prevent it from promoting ciliogenesis and supporting neurogenesis in an AD-developing brain?