O2–04–01: Combined amyloid and tau–pathology in APPV717I X taup301L double transgenic mice: Increased survival correlates with less hindbrain pathology, despite dramatic forebrain tauopathy

interacting protein(s). Our objectives are to identify such proteins by proteomic analysis and to elucidate the regulation mechanisms of BACE1 activity. Methods: Human neuroblastoma SH-SY5Y cells overexpressing BACE1 with a C-terminal tag were used as a source material. Possible BACE1-interacting proteins were separated by affinity purification and further analyzed by mass spectrometry. Protein-protein interaction was analyzed by co-immunoprecipitation. A levels were measured using sandwich ELISA kits. Results: We identified Nogo-B (reticulon 4-B; RTN4-B) as a BACE1-binding protein. Co-immunoprecipitaion experiments confirmed a physical association between BACE1 and Nogo-B, Nogo-C (RTN4-C), and their homologue reticulon 3 (RTN3). Overexpression of these RTN proteins resulted in a 30-50% reduction in the secretion of both A 40 and A 42 from human embryonic kidney 293 cells expressing Swedish mutant amyloid precursor protein (APP). These proteins, however, did not influence A secretion from cells expressing the APP Cterminal fragment. In addition, immunohistochemical analysis showed that RTN3 is predominantly expressed by pyramidal neurons in the mouse cerebral cortex. Conclusions: These data collectively indicate that NogoB/C and RTN3 can negatively modulate BACE1 cleavage of APP, suggesting that they play important roles in the regulation of A production in the brain.