Synaptotagmins interact with APP and promote Aβ generation

Background: Accumulation of the beta-amyloid peptide (A beta) is a major pathological hallmark of Alzheimer's disease (AD). Recent studies have shown that synaptic A beta toxicity may directly impair synaptic function. However, proteins regulating A beta generation at the synapse have not been characterized. Here, we sought to identify synaptic proteins that interact with the extracellular domain of APP and regulate A beta generation. Results: Affinity purification-coupled mass spectrometry identified members of the Synaptotagmin (Syt) family as novel interacting proteins with the APP ectodomain in mouse brains. Syt-1, -2 and -9 interacted with APP in cells and in mouse brains in vivo. Using a GST pull-down approach, we have further demonstrated that the Syt interaction site lies in the 108 amino acids linker region between the E1 and KPI domains of APP. Stable overexpression of Syt-1 or Syt-9 with APP in CHO and rat pheochromocytoma cells (PC12) significantly increased APP-CTF and sAPP levels, with a 2 to 3 fold increase in secreted A beta levels in PC12 cells. Moreover, using a stable knockdown approach to reduce the expression of endogenous Syt-1 in PC12 cells, we have observed a similar to 50 % reduction in secreted A beta generation. APP processing also decreased in these cells, shown by lower CTF levels. Lentiviral-mediated knock down of endogenous Syt-1 in mouse primary neurons also led to a significant reduction in both A beta 40 and A beta 42 generation. As secreted sAPP beta levels were significantly reduced in PC12 cells lacking Syt-1 expression, our results suggest that Syt-1 regulates A beta generation by modulating BACE1-mediated cleavage of APP. Conclusion: Altogether, our data identify the synaptic vesicle proteins Syt-1 and 9 as novel APP-interacting proteins that promote A beta generation and thus may play an important role in the pathogenesis of AD.