We MS 275 found that endocytosis of APP is essential for the activity-dependent convergence of APP/BACE-1 in neurons (Figure 6). Specifically, experimental paradigms blocking clathrin-mediated

endocytosis (or APP endocytosis) also abrogated APP/BACE-1 convergence (Figures 6C and 6D), and such conditions led to expected stalling and clustering of APP and clathrin (Figure 6E), suggesting that a recycling-dependent pathway (as opposed to homotypic fusion) is responsible for this convergence. What is the relevance of our findings to human disease? Studies show that amyloid plaque deposition is most conspicuous in the “default mode network”—a circuit that is metabolically active during unidirected mentation (Buckner et al., 2009)—leading to the hypothesis that activity-dependent amyloidogenesis may play a role in AD (Bero et al., 2011). Though our experiments do not address this directly, our finding that APP/BACE-1 convergence is exaggerated in stimulated neurons as well as AD brains is consistent with this idea. However, other studies implicate defective Aβ clearance (and not increased Aβ production) as the primary pathologic event in AD (Mawuenyega et al., 2010), and further work is needed to clarify these issues. In summary, our studies uncover fundamental trafficking strategies

by which neurons largely restrict APP (substrate) and BACE-1 (enzyme) in distinct organelles—thus limiting Aβ biogenesis in physiologic states (Figures 1 and 2); define a trafficking CHIR-99021 cost pathway by which APP and BACE-1 converge upon induction of neuronal activity (Figures 3, 4, 5, and 6); and, finally, our data from human brains (Figure 7) suggest potential relevance of these mechanisms in human disease. Several constructs old were obtained from other laboratories, as mentioned in the Acknowledgments. The CFP/YFP tags in the BACE-1:CFP/APP:YFP constructs were replaced by GFP or mCherry, cloned in-frame, and confirmed by sequencing (see Figure S1).

The promoter in the NPYss construct (Banker laboratory) was swapped with CMV. The clathrin:GFP and Rab-5:mCherry constructs were obtained from Addgene. Antibodies used for biochemistry were the following: APP (1565-1; Epitomics), BACE1 (MAB931; R&D), TfR (clone H68.4; Invitrogen), Tubulin (clone DM1A, Sigma), anti-pan cadherin (ab22744, Abcam), KDEL (Ab12223, Abcam), Rab11 (71-5300, Invitrogen), GM130 (610822, BD Transduction), and Rab5 (108011, Synaptic Systems). D-AP5, dynasore, picrotoxin, and memantine were from Sigma and Alexa 488 Transferrin was from Molecular Probes. Beta-secretase Inhibitor II (Calbiochem) was prepared in DMSO and neurons were treated with final 0.5 μM inhibitors for 24 hr. Primary hippocampal neurons were obtained from postnatal (P0–P1) CD-1 mice and cells were transfected using Lipofectamine-2000 (Invitrogen) as described previously in Roy et al. (2012). Briefly, dissociated neurons were plated at a density of 50,000 cells/cm2 in poly-D-lysine-coated (1.