P2–041: Investigating the role of the proteasome in prion infection

cell-permeable and drug-like compounds. Objective(s): Here we report the development of cellular assays in primary neuronal cultures and characterization of cell-permeable BACE-1 inhibitors. Methods: We used a novel electroporation-based method (Nucleofection’) for transfection of murine cerebellar granule cell (CGC) cultures with human APP harboring the Swedish mutation (huAPPswe). This method allowed high transfection efficiency of 40% in neurons. Endogenous murine secretases cleaved huAPPswe, and human A 1-40/1-42 was secreted into the culture medium. In addition, we established cortical cultures and CGC from transgenic mice co-expressing huAPPswe and mutated presenilin 1 (huAPPswe/PS1 E9) to test cell-permeable small molecule secretase inhibitors. Results: We characterized the properties of two different inhibitors with low and high affinity, which inhibited recombinant human BACE-1 in vitro in the low nM and low M range, respectively. In neurons, these compounds displayed a dose-dependent reduction of A generation with an IC50 of approximately 10 nM and 2.5 M. Furthermore, we observed reduction in sAPP (N-terminal product of APP following BACE-1 cleavage) and elevation in APP. In contrast to -secretase inhibitors, BACE-1 inhibitors increased the amount of sAPP (N-terminal product of APP following -secretase cleavage). When using transgenic huAPPswe/PS1 E9 mice as model system, robust reduction of A in brain homogenates was observed upon BACE-1 and -secretase inhibition within 3 h. Conclusions: These studies confirm pharmacologically, that BACE-1 plays the predominant role in the -site cleavage of APP and that inhibition of -secretase activity could potentially redirect APP-processing via the non-amyloidogenic -secretase pathway.