Das U, Scott DA, Ganguly A, Koo EH, Tang Y, Roy S.
Activity-Induced Convergence of APP and BACE-1 in Acidic Microdomains via an Endocytosis-Dependent Pathway.
Neuron. 2013 Aug 7;79(3):447-60.
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This is a very nice study using high-level microscopy to provide evidence for BACE1 and APP converging in dendritic endosomes of cultured neurons by live cell imaging following synaptic activation. It is less clear what this work means for Alzheimer’s disease. Synaptic activity is of course fundamental to brain function. The modulation of APP processing and Aβ by synaptic activity is of considerable interest, although there are many questions. One concern is how physiological the experimental stimulation is to what occurs in brain; physiological stimulation is more rapid than what is typically employed experimentally (including in our own studies in primary neurons). A major question is why a normal process (modulation of APP trafficking and processing with synaptic stimulation) becomes abnormal with age and AD. Some of these issues could have been discussed a bit more. It is also quite interesting that this study shows greater enrichment of BACE1 with APP in human AD brain fractionation studies.
There are some interesting aspects of this manuscript that the authors might have addressed more, for example in Fig. 3B one observes movement of APP into spines upon synaptic activation, which is not commented on. In Fig. 4D, while BACE and APP are said to come together with activity, in the kymograph BACE1 and APP actually appear together from the get-go. The authors could consider adding glycine directly during the imaging (as we have done), to provide live imaging evidence of dynamic convergence. One also wonders if transferrin receptor trafficking might be altered by activity.
In their Neuron paper, Roy and co-workers used a combination of polarized neurons and in vivo imaging to scrutinize the transport routes utilized by APP and BACE1. Their rationale is that substrate and enzyme are best spatially separated to avoid default processing leading to aberrant production of proteolytic fragments. I am enthusiastic about the work, not only because of the high quality and live imaging approaches but also because the work beautifully confirms our initial proposal that transport regulation is likely to be more central in the pathology than anticipated, as we outlined some years ago in a review ((Sannerud and Annaert, 2009; and updated in (Rajendran and Annaert, 2012) and reported that for APP and BACE1 that this is indeed the case (Sannerud et al., 2011). Therein we demonstrated that APP majorly follows along a clathrin-dependent internalization route while BACE1 reaches early endosomes via a clathrin-independent route governed by the small GTPase ARF6. The authors now extend this towards neurons and support our findings that APP and BACE1 separately get internalized to endosomal compartments.
What I find surprising is that the authors only inquired about APP's internalization, essentially confirming its dependence on clathrin. On the other hand, only one group, ours, found evidence that BACE1 and APP follow distinct internalization routes. Moreover, we identified the major route of BACE1 internalization, namely a clathrin-independent route regulated by the small GTPase ARF6. I would have expected that in this paper the authors would have confirmed this. It is reassuring that in their view much of the transport regulation and processing appears to occur in the dendritic compartments, confirming and extending our data in SCG neurons in a compartmentalized culture system.
Although I fully agree that more cell biology is needed in neurons, at the end of the day, the major conclusions relate to transport regulations that are no different from neuronal mechanisms. A major added value is that the authors studied the effect of neuronal activity on APP-BACE1 convergence, which nicely complements earlier studies and increased awareness of the importance of activity in amyloidogenic processing.
Much work remains to be done and further optimization of the cellular models is required to fully understand how transport regulation is intermingled with APP processing. For instance, although in vivo imaging is appealing, it requires overexpression of fluorescently labeled proteins. To achieve stable physiologically relevant levels of reporter proteins, we likely have to use viral vectors. Also the location of reporter tags can jeopardize the readout because the C-terminus of BACE1 harbors sorting motifs that may not work properly with a large tag next to it; hence live imaging should go along with confirmations of the location of endogenous proteins. Another intriguing aspect is the abundant co-localizations that the authors observe in endosomes, particularly in activity-induced circumstances. From our experience, if one co-expresses APP with BACE1 there processing occurs rapidly, as becomes evident when incubating cells with BACE1 inhibitors. Hence, the authors cannot rule out that a major part of the colocalizations are caused by the APP-CTF (hooked up to GFP) rather than full-length APP, arguing that maybe shedding has occurred earlier, closer to, or in early endosomes. Although the authors controlled this by using BACE1 inhibitors, their western blots demonstrate α-CTF as the overwhelming proteolytic fragment and hence inhibiting the small contributions of BACE1 shedding may not affect the extent of colocalization significantly. It would be interesting to repeat some of the key experiments with a dual-tagged APP to monitor in real time in neurons the actual shedding events.
Finally, the extrapolation to AD brain needs further scrutiny. It is questionable whether endosomal (and other subcellular) compartments remain properly preserved in frozen brain sections. In summary, this paper, overall, supports the growing appreciation that endosomal compartments, and not, for instance, Golgi, are the primary sites of BACE1-mediated APP processing and that enzyme and substrate remain separated until they find each other in the “shedding compartments”.
Sannerud R, Annaert W.
Trafficking, a key player in regulated intramembrane proteolysis.
Semin Cell Dev Biol. 2009 Apr;20(2):183-90.
Rajendran L, Annaert W.
Membrane trafficking pathways in Alzheimer's disease.
Traffic. 2012 Jun;13(6):759-70.
Sannerud R, Declerck I, Peric A, Raemaekers T, Menendez G, Zhou L, Veerle B, Coen K, Munck S, De Strooper B, Schiavo G, Annaert W.
ADP ribosylation factor 6 (ARF6) controls amyloid precursor protein (APP) processing by mediating the endosomal sorting of BACE1.
Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):E559-68.