Many studies point to endosomes as the sites of Aβ production, but the big picture may be more complicated, according to a paper in the December 7 Nature Neuroscience. Researchers led by Subhojit Roy at the University of California, San Diego, detected the convergence of APP and BACE1 in hippocampal neurons using protein chimeras that fluoresced only when the two proteins came together. Because BACE1 begins the process of snipping APP into Aβ, the results point to potential sites of Aβ production. Surprisingly, the researchers saw many encounters between APP and BACE1 in the Golgi apparatus, as well as during axonal transport. In dendrites, the researchers localized APP and BACE1 meetings specifically to recycling endosomes, which shuttle internalized proteins back to the cell surface. In addition, a rare APP variant that lowers the risk of Alzheimer’s disease associated more weakly with BACE1 in these assays, suggesting a mechanism for its protective effect. The study provides perhaps the first direct look at how BACE1 interacts with APP in neurons, the authors said.
Roy believes this fluorescence complementation assay may aid preclinical research. “I hope we can use it to discover new targets or drugs that interfere with the APP/BACE1 interaction, and thus inhibit Aβ production,” Roy wrote to Alzforum.
Other scientists liked the method as well. “This is a rigorous study with fantastic imaging,” Claudia Almeida at NOVA Medical School, Lisbon, Portugal, wrote to Alzforum. Gunnar Gouras at Lund University, Sweden, found the differences between axons and dendrites intriguing. “This is elegant neuroscience,” he said. Nonetheless, commenters noted that the study does not detect Aβ production, nor did it examine a time course of APP internalization, which might provide more clues to where Aβ is most likely to form. More work will be needed to pin down the dynamics of APP movements and Aβ generation, they added.
Researchers have long implicated endosomes as the primary sites of Aβ generation (for review, see Nixon, 2005; Rajendran and Annaert, 2012). There are many types of endosomes, however, and the details of Aβ production were hazy. Previously, Roy and colleagues labeled APP and BACE1 and found that synaptic activity stimulated the two proteins to come together in recycling endosomes of dendrites (see Aug 2013 news). Nonetheless, this type of co-localization did not prove a direct interaction, which would be needed for Aβ production.
Cozying Up in the Golgi. APP and BACE chimeras bind to reconstitute Venus protein fluorescence (green) in the Golgi (red; overlay appears yellow) in a primary hippocampal neuron (left); blocking export from the Golgi strengthens this association (right). [Courtesy of Das et al., Nature Neuroscience.]
To demonstrate a physical association, first author Utpal Das used a fluorescence complementation assay, a well-established tool of cell biologists. He conjugated one-half of Venus fluorescent protein to wild-type human APP, and the other half to mouse BACE1. The authors then transfected both constructs into mouse primary hippocampal neurons. The first fluorescence appeared about five hours after transfection, at which point APP and BACE1 protein levels remained low, so results were likely not an artifact of overexpression, the authors noted. Cells fluoresced only when the two proteins bound each other. They remained dark if the authors expressed an APP variant that did not bind BACE1.
About half of all APP-BACE1 interactions in these neurons occurred in dendritic spines. Some fluorescence also appeared in cell bodies around the nuclei, and along axons. To determine what organelles were involved, the authors co-labeled cultures with various vesicle markers. They found that the cell-body fluorescence associated mainly with the Golgi apparatus, where proteins are processed. Cooling the cultures to 20°C, which blocks export from the Golgi, enhanced this localization (see image above). In axons, Venus fluorescence also occurred in Golgi-derived vesicles. Most of these vesicles associated with presynaptic boutons, implicating synaptic compartments as the main site for Aβ production. The authors also saw movement of these vesicles along axons, suggesting that APP and BACE1 remain in close contact during axonal transport. Previously, data conflicted on whether APP and BACE1 were transported together (see Dec 2001 news; Lazarov et al., 2005; May 2006 conference news).
In dendritic spines, 60 percent of Venus fluorescence occurred in vesicles that contained the GTP-binding protein Rab11 and the transferrin receptor. These markers distinguish recycling endosomes. Only about 30 percent of fluorescence co-labeled with the early endosomal marker Rab5, and even less occurred in late endosomes. Together, these data suggest that recycling endosomes might be more important sites of synaptic β-cleavage than early endosomes, the authors noted. Recycling endosomes are acidic, and thus have the right pH for BACE1 activity.
The authors wondered how much APP from the cell membrane ends up in recycling endosomes. They labeled cell-surface APP and measured where it appeared 30 minutes later. About half of it was in vesicles that contained transferrin receptor, supporting the idea that a large fraction of APP shuttles through recycling endosomes. However, 70 percent of cell-surface APP ended up in vesicles containing the late endosome marker LAMP1. The percentages add up to more than 100 because some vesicles may have both markers, Roy noted. Because few APP-BACE1 interactions occurred in late endosomes or lysosomes, this degradative pathway may protect APP from cleavage, the authors speculated.
The Icelandic A673T mutation in APP is known to stymie BACE1 cleavage and lower the risk of Alzheimer’s (see Jul 2012 news). The authors transfected this APP variant, conjugated with the N-terminal end of Venus fluorescent protein, into hippocampal neurons along with a BACE1-Venus C-terminal chimera. They saw much less fluorescence than in assays using wild-type APP, although APP trafficking was unaffected. These findings suggest that the protective mutation lessens the association of BACE1 with APP, the authors noted.
Some commenters had reservations about the study’s conclusions, however. Almeida pointed out that in the authors’ earlier study, relatively little APP ended up in recycling endosomes, belying the new findings. In the Venus fluorescence assay, once APP and BACE1 bind, they remain tethered, and Almeida wondered if this artificially stable interaction could override the normal sorting of APP. “It remains unclear how much the irreversible binding of APP and BACE1 in the Golgi altered their trafficking and their sites of physiological interaction,” she wrote to Alzforum.
Roy agreed that the irreversibility of the APP/BACE1 interaction does limit this assay’s ability to detect normal trafficking. However, he noted that the irreversibility is also a strength of the assay. APP and BACE1 normally associate very briefly, and so their encounters would be difficult to detect in living cells. The fluorescence assay captures these transient interactions and makes them visible, Roy wrote.
In future work, Roy plans to dissect the trafficking pathways that precede the convergence of APP and BACE1, and look for small molecules that inhibit their interaction. Such molecules might have therapeutic potential for lowering Aβ levels. “We hope that our approach will bypass some of the toxicity issues associated with enzymatic BACE inhibitors,” he wrote to Alzforum.—Madolyn Bowman Rogers.
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- Das U, Wang L, Ganguly A, Saikia JM, Wagner SL, Koo EH, Roy S. Visualizing APP and BACE-1 approximation in neurons yields insight into the amyloidogenic pathway. Nat Neurosci. 2016 Jan;19(1):55-64. Epub 2015 Dec 7 PubMed.