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.
PubMed.
The study by Das et al. was meticulously conducted and provides an important platform for further work in understanding how Aβ production normally occurs. It has been known for some time that Aβ secretion is activity-dependent, but the precise mechanisms are only now starting to be delineated. The prevailing model for APP processing was that it took place in axons/synaptic boutons and was in some way related to synaptic vesicle recycling. This study and a few other studies from the Holtzman, Malinow, and Worley groups (Verges et al., 2011; Wu et al., 2011; and Kamenetz et al., 2003) now highlight the role of APP processing and Aβ production in dendrites. Indeed, one of the putative physiological functions proposed for Aβ is to regulate postsynaptic AMPA type glutamate receptor levels (Kamenetz et al., 2003) in a negative feed-back cycle. Synaptic dysfunction in Alzeheimer’s disease thus may result from poor neuronal homeostasis.
References:
Verges DK, Restivo JL, Goebel WD, Holtzman DM, Cirrito JR.
Opposing synaptic regulation of amyloid-β metabolism by NMDA receptors in vivo.
J Neurosci. 2011 Aug 3;31(31):11328-37.
PubMed.
Wu J, Petralia RS, Kurushima H, Patel H, Jung MY, Volk L, Chowdhury S, Shepherd JD, Dehoff M, Li Y, Kuhl D, Huganir RL, Price DL, Scannevin R, Troncoso JC, Wong PC, Worley PF.
Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent β-amyloid generation.
Cell. 2011 Oct 28;147(3):615-28.
PubMed.
Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R.
APP processing and synaptic function.
Neuron. 2003 Mar 27;37(6):925-37.
PubMed.
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”.
References:
Sannerud R, Annaert W.
Trafficking, a key player in regulated intramembrane proteolysis.
Semin Cell Dev Biol. 2009 Apr;20(2):183-90.
PubMed.
Rajendran L, Annaert W.
Membrane trafficking pathways in Alzheimer's disease.
Traffic. 2012 Jun;13(6):759-70.
PubMed.
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.
PubMed.
This paper by Das et al. extends previous studies that indicated that endosomes—specifically, the early and recycling endosomes—are major sites of cleavage of Aβ precursor protein (APP) by β-secretase. It also provides additional support for the notion that the amyloidogenic processing of APP is potentiated by neuronal activity. Using live imaging of fluorescently tagged APP and BACE-1 (the major β-secretase), the authors also provide data supporting the hypothesis that APP is processed by diverting it to recycling endosomes, where BACE-1 normally accumulates. We agree that this is one of several ways by which amyloidogenic processing of APP could occur, and wholeheartedly recommend this paper. Here, we would like to comment on the intraneuronal sites, other than the synapse, where AD-relevant APP processing could occur.
Das et al. focus on the processing of APP in dendrites, and the clear suggestion is that APP cleavage by β-secretase occurs within the dendrites, at postsynaptic sites. While there is no doubt that processing of APP in neuronal processes is robust, we would like to stress that the endosomes localized in neurites are not the exclusive sites of intraneuronal APP processing, and that compartments in the soma also contribute to the cleavage of APP by β-secretase. Newly synthesized APP is extensively transported to the plasma membrane, and is thereafter retrieved by endocytosis and delivered to early and recycling endosomes in the soma that contain large amounts of β-secretase. In our studies, we also find that the levels of BACE1 in the soma by far exceed those present in neurites (see Muresan and Mursean, 2006). In addition to endosomes, β-secretase is present throughout the early secretory pathway: in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC), the Golgi apparatus, and in the trans-Golgi network (TGN) (see Thinkaran and Koo, 2008, Vassar et al., 2009). Das et al. also find that APP and β-secretase colocalize at the level of ER and Golgi. Although the general view is that β-secretase activity in these less-acidic compartments would be low, it is hard to believe that it is completely suppressed. In fact, earlier studies have reported APP processing in the ER and ERGIC (see Cook et al., 1997).
Those of us who listened to Charles Glabe’s talk at the AD/PD Conference in Florence earlier this year should have been impressed with the strong data suggesting that significant proteolytic processing of APP in Alzheimer’s disease could in fact occur in the neuronal soma, rather than at the synaptic terminal. To us, the interpretation that Aβ-loaded cell bodies could also be responsible for the initiation of the neuritic plaques is likely correct. Our own immunohistochemical data on paraffin sections from AD brain and on cryosections from a mouse model of AD (Bruce Lamb’s R1.40 mouse) support this interpretation. This is not to say that significant APP cleavage, and generation of Aβ, does not also occur at the synapse. We only want to draw attention to the possibility that the processing of APP, and the generation of AD-relevant APP fragments (CTFβ, sAPPβ, in addition of Aβ), also occur—perhaps to a large extent—in the neuronal soma. Although the synapse is a major site of the neuronal pathology in AD, and the processing of APP (with generation and subsequent secretion of Aβ) is likely regulated by synaptic activity (see Cirrito et al., 2008, Tampellini and Gouras, 2010), the neuronal activity could also affect processing of APP in the soma, via retrograde signaling, for example. It would be naive to rule out this latter possibility.
References:
Glabe, C., Conformational Diversity of Amyloid in Human AD Brain. The 11th International Conference on Alzheimer’s & Parkinson’s Disease, Florence, Italy, March 6-10, 2013. ARF related story
References:
Muresan Z, Muresan V.
Neuritic deposits of amyloid-beta peptide in a subpopulation of central nervous system-derived neuronal cells.
Mol Cell Biol. 2006 Jul;26(13):4982-97.
PubMed.
Thinakaran G, Koo EH.
Amyloid precursor protein trafficking, processing, and function.
J Biol Chem. 2008 Oct 31;283(44):29615-9.
PubMed.
Vassar R, Kovacs DM, Yan R, Wong PC.
The beta-secretase enzyme BACE in health and Alzheimer's disease: regulation, cell biology, function, and therapeutic potential.
J Neurosci. 2009 Oct 14;29(41):12787-94.
PubMed.
Cook DG, Forman MS, Sung JC, Leight S, Kolson DL, Iwatsubo T, Lee VM, Doms RW.
Alzheimer's A beta(1-42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells.
Nat Med. 1997 Sep;3(9):1021-3.
PubMed.
Cirrito JR, Kang JE, Lee J, Stewart FR, Verges DK, Silverio LM, Bu G, Mennerick S, Holtzman DM.
Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo.
Neuron. 2008 Apr 10;58(1):42-51.
PubMed.
Tampellini D, Gouras GK.
Synapses, synaptic activity and intraneuronal abeta in Alzheimer's disease.
Front Aging Neurosci. 2010;2
PubMed.
Comments
University of Utah School of Medicine
The study by Das et al. was meticulously conducted and provides an important platform for further work in understanding how Aβ production normally occurs. It has been known for some time that Aβ secretion is activity-dependent, but the precise mechanisms are only now starting to be delineated. The prevailing model for APP processing was that it took place in axons/synaptic boutons and was in some way related to synaptic vesicle recycling. This study and a few other studies from the Holtzman, Malinow, and Worley groups (Verges et al., 2011; Wu et al., 2011; and Kamenetz et al., 2003) now highlight the role of APP processing and Aβ production in dendrites. Indeed, one of the putative physiological functions proposed for Aβ is to regulate postsynaptic AMPA type glutamate receptor levels (Kamenetz et al., 2003) in a negative feed-back cycle. Synaptic dysfunction in Alzeheimer’s disease thus may result from poor neuronal homeostasis.
References:
Verges DK, Restivo JL, Goebel WD, Holtzman DM, Cirrito JR. Opposing synaptic regulation of amyloid-β metabolism by NMDA receptors in vivo. J Neurosci. 2011 Aug 3;31(31):11328-37. PubMed.
Wu J, Petralia RS, Kurushima H, Patel H, Jung MY, Volk L, Chowdhury S, Shepherd JD, Dehoff M, Li Y, Kuhl D, Huganir RL, Price DL, Scannevin R, Troncoso JC, Wong PC, Worley PF. Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent β-amyloid generation. Cell. 2011 Oct 28;147(3):615-28. PubMed.
Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R. APP processing and synaptic function. Neuron. 2003 Mar 27;37(6):925-37. PubMed.
View all comments by Jason ShepherdLund University
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.
KULeuven & VIB
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”.
References:
Sannerud R, Annaert W. Trafficking, a key player in regulated intramembrane proteolysis. Semin Cell Dev Biol. 2009 Apr;20(2):183-90. PubMed.
Rajendran L, Annaert W. Membrane trafficking pathways in Alzheimer's disease. Traffic. 2012 Jun;13(6):759-70. PubMed.
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. PubMed.
Rutgers - New Jersey Medical School
This paper by Das et al. extends previous studies that indicated that endosomes—specifically, the early and recycling endosomes—are major sites of cleavage of Aβ precursor protein (APP) by β-secretase. It also provides additional support for the notion that the amyloidogenic processing of APP is potentiated by neuronal activity. Using live imaging of fluorescently tagged APP and BACE-1 (the major β-secretase), the authors also provide data supporting the hypothesis that APP is processed by diverting it to recycling endosomes, where BACE-1 normally accumulates. We agree that this is one of several ways by which amyloidogenic processing of APP could occur, and wholeheartedly recommend this paper. Here, we would like to comment on the intraneuronal sites, other than the synapse, where AD-relevant APP processing could occur.
Das et al. focus on the processing of APP in dendrites, and the clear suggestion is that APP cleavage by β-secretase occurs within the dendrites, at postsynaptic sites. While there is no doubt that processing of APP in neuronal processes is robust, we would like to stress that the endosomes localized in neurites are not the exclusive sites of intraneuronal APP processing, and that compartments in the soma also contribute to the cleavage of APP by β-secretase. Newly synthesized APP is extensively transported to the plasma membrane, and is thereafter retrieved by endocytosis and delivered to early and recycling endosomes in the soma that contain large amounts of β-secretase. In our studies, we also find that the levels of BACE1 in the soma by far exceed those present in neurites (see Muresan and Mursean, 2006). In addition to endosomes, β-secretase is present throughout the early secretory pathway: in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC), the Golgi apparatus, and in the trans-Golgi network (TGN) (see Thinkaran and Koo, 2008, Vassar et al., 2009). Das et al. also find that APP and β-secretase colocalize at the level of ER and Golgi. Although the general view is that β-secretase activity in these less-acidic compartments would be low, it is hard to believe that it is completely suppressed. In fact, earlier studies have reported APP processing in the ER and ERGIC (see Cook et al., 1997).
Those of us who listened to Charles Glabe’s talk at the AD/PD Conference in Florence earlier this year should have been impressed with the strong data suggesting that significant proteolytic processing of APP in Alzheimer’s disease could in fact occur in the neuronal soma, rather than at the synaptic terminal. To us, the interpretation that Aβ-loaded cell bodies could also be responsible for the initiation of the neuritic plaques is likely correct. Our own immunohistochemical data on paraffin sections from AD brain and on cryosections from a mouse model of AD (Bruce Lamb’s R1.40 mouse) support this interpretation. This is not to say that significant APP cleavage, and generation of Aβ, does not also occur at the synapse. We only want to draw attention to the possibility that the processing of APP, and the generation of AD-relevant APP fragments (CTFβ, sAPPβ, in addition of Aβ), also occur—perhaps to a large extent—in the neuronal soma. Although the synapse is a major site of the neuronal pathology in AD, and the processing of APP (with generation and subsequent secretion of Aβ) is likely regulated by synaptic activity (see Cirrito et al., 2008, Tampellini and Gouras, 2010), the neuronal activity could also affect processing of APP in the soma, via retrograde signaling, for example. It would be naive to rule out this latter possibility.
References:
Glabe, C., Conformational Diversity of Amyloid in Human AD Brain. The 11th International Conference on Alzheimer’s & Parkinson’s Disease, Florence, Italy, March 6-10, 2013. ARF related story
References:
Muresan Z, Muresan V. Neuritic deposits of amyloid-beta peptide in a subpopulation of central nervous system-derived neuronal cells. Mol Cell Biol. 2006 Jul;26(13):4982-97. PubMed.
Thinakaran G, Koo EH. Amyloid precursor protein trafficking, processing, and function. J Biol Chem. 2008 Oct 31;283(44):29615-9. PubMed.
Vassar R, Kovacs DM, Yan R, Wong PC. The beta-secretase enzyme BACE in health and Alzheimer's disease: regulation, cell biology, function, and therapeutic potential. J Neurosci. 2009 Oct 14;29(41):12787-94. PubMed.
Cook DG, Forman MS, Sung JC, Leight S, Kolson DL, Iwatsubo T, Lee VM, Doms RW. Alzheimer's A beta(1-42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nat Med. 1997 Sep;3(9):1021-3. PubMed.
Cirrito JR, Kang JE, Lee J, Stewart FR, Verges DK, Silverio LM, Bu G, Mennerick S, Holtzman DM. Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron. 2008 Apr 10;58(1):42-51. PubMed.
Tampellini D, Gouras GK. Synapses, synaptic activity and intraneuronal abeta in Alzheimer's disease. Front Aging Neurosci. 2010;2 PubMed.
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