Identification and characterization of modulators of Alzheimer's disease pathogenesis

Coordinator:    Prof. Dr. Erich Wanker
Institution: Max-Delbrück-Centrum für Molekulare Medizin (MDC) Berlin-Buch
The objective of the subproject was the identification of previously unknown molecular connections between therapeutic compounds, proteins and protein networks in Alzheimer's disease (AD).
To create an interaction network for the disease-relevant amyloid precursor protein (APP) and to predict potential modulators of the production and aggregation of amyloid-β (Aβ), the peptide that is derived from APP, a genome-wide yeast-two-hybrid (Y2H) screen was performed with approximately 14,000 proteins and protein fragments, from which nine potential modulators of APP could be identified.
In a subsequent, focussed yeast-two-hybrid screening, 800 expression plasmids were generated for genes that, according to public databases, potentially play a role in AD. The nine potential modulators from the genome-wide screen were integrated. 666 protein-protein interactions (PPIs) involved in AD were identified. Combined with publicly available PPI data, we generated a comprehensive network of 20,231 PPIs between 6,396 proteins interacting directly or indirectly with APP.
To find proteins or small molecules that modulate the production or aggregation of Aβ, we established several cell models. Aβ aggregates were introduced in human neuroblastoma cells (SH-SY5Y, SH-EP) and in APP-overexpressing CHO and HEK293 cells. An Aβ ELISA with APP-overexpressing SH-SY5Y-APP695 cells was developed. The cell models have been successfully used to study the influence of two compounds, EGCG and O4, on Aβ aggregation and toxicity. (Bieschke et al, PNAS, 2010; Bieschke et al, NatChemBiol, 2011).
Based on data of known modulators of Aβ production, on gene expression data of AD patients and on complex network analyses, we identified 200 potential modulators of APP processing. Knock-down experiments of 84 of the most promising ones resulted in 65 hits in an SH-SY5Y-APP695 cell model, which significantly altered the extracellular Aβ42 and Aβ40 levels.
383 small molecules were investigated in SH-EP cells for their effect on the aggregation. The quantification of Aβ aggregates by high-throughput microscopy showed that 43 compounds significantly decreased aggregation, while six increased it. Together with 31 structurally related substances, two promising drugs were selected and systematically analysed in terms of their effect on Aβ aggregation and toxicity. The two most interesting of them were characterized further.

Figure 1. Aβ fibrils visualized by atomic force microscopy.
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