Visualizing APP and BACE-1 approximation in neurons yields insight into the amyloidogenic pathway

Extracellular Aβ deposition is a pathologic hallmark of Alzheimer’s disease and a central tenet of the decades-old amyloid cascade hypothesis positing that neuronal dysfunction, synapse loss, neurofibrillary degeneration and the full manifestation of Alzheimer neuropathology is initiated by Aβ. Aβ is generated by sequential cleavage of APP by the enzymes βand γ-secretase, with BACE-1 cleavage being the rate-limiting step in this pathway1. Thus physical approximation of APP and BACE-1 is a required cell biological event in Aβ generation, and precise understanding of APP–BACE-1 interaction sites as well as pathways leading up to this approximation is vital. Where do wild-type (WT) APP and BACE-1 meet, and what is the site of β-cleavage in neurons? Despite much research, the answer remains unclear1. A model favored at present is that after biogenesis in the Golgi both APP and BACE-1 recycle with the plasma membrane and converge in early endosomes, and that β-cleavage of APP occurs in early endosomal compartments2–4. However, several observations are inconsistent with this view, and it is unclear how the model applies to neurons—the relevant cell type in this case. First, at steady state, much of the WT APP and BACE-1 in neuronal and non-neuronal cell lines is located at the Golgi5, and it is unclear why β-cleavage would be precluded there. Furthermore, APP and BACE-1 colocalize in multiple endocytic compartments in these cells, including both early and recycling endosomes6. Notably, existing models of the amyloidogenic pathway are almost entirely based on experiments in cells that lack the anatomy of neurons, which have elongated processes orders of magnitude longer than microscopic cell bodies. Anatomically and functionally distinct neuronal microdomains such as dendritic spines and presynaptic specializations are not considered in canonical models, and it is difficult to imagine that trafficking concepts that have emerged from studies in non-neuronal cells can be simply transferred to neurons. In previous studies we visualized the trafficking of WT APP and BACE-1 in cultured hippocampal neurons using minimal and transient expression of fluorescently tagged proteins7. We found that in dendrites, BACE-1 is largely localized to recycling endosomes, where APP and BACE-1 can colocalize. However, neuronal sites of APP–BACE-1 interaction and β-cleavage have remained unknown. Moreover, putative APP–BACE-1 interactions in axons and presynaptic sites were also not addressed. Here we report a tool we call the optical convergence of APP and BACE-1 (OptiCAB) assay that allows one to directly visualize APP–BACE-1 interactions in cells. Combining this with other assays that track the fate of internalized APP from the plasma membrane, as well as two-color high resolution live imaging, our experiments offer concrete insights into longstanding questions and controversies in the field.