Introduction

The amyloid-β-yielding extracellular domain of APP has gotten most of the attention over the last decade or so, but inside the cell there is a large cytoplasmic fragment that might be the key to understanding the true nature of APP.
 

Sanjay Pimplikar led this live discussion on 3 October 2006. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Transcript:
Participants: Sanjay W. Pimplikar (Case Western Reserve University), Tom Fagan (Alzforum), Bill Rebeck (Georgetown University), Lucia Pastorino (Beth Israel, HMS, Boston), Veronica Galvan (Buck Institute), Virgil Muresan (Case Western Reserve University), Ed Ickert (Case Western Reserve University, Cleveland), Wendy Wang (NTU, Taiwan), Rupert Egensperger (Essen, Germany), Randall Lauffer (professional student of APP), Thorsten Müller (Proteome Center, Brain Proteomics, Bochum, Germany), Tommaso Russo (University of Napoli, Italy), Bernadette.Allinquant (INSERM, France), Suzanne Guenette (Harvard University), Sebastien Hebert (KU Leuven, Belgium), Lucia Pastorino (Beth Israel Deaconess, Boston), Qubai Hu (University of Washington, Seattle).

Note: Transcript has been edited for clarity and accuracy.

Sanjay Pimplikar
Not much to say in this august company. First of all, thank you all for joining us. I think we should start with something we all agree on, that is, the importance of APP and Fe65. We can end this discussion with something on which we have differing opinions, such as, does AICD play a role in gene transcription? So, to get the ball rolling, I am really impressed with the phenotypes that Suzanne Guenette gets with her Fe65 knockout (KO) mice (see Guenette et al., 2006) and Ulrike Muller showed with her APP triple KO (see Herms et al., 2004). What do you think—and I will start with Tomasso—is the role of APP and Fe65 in brain? What's the mechanism? Suzanne can follow up on that.

Sebastien Hebert
Sanjay, for APP, I would maybe consider broadening the subject to the APP family instead?

Sanjay Pimplikar
Sure, good point, Sebastian!

Suzanne Guenette
Sanjay, the thing we still need to find out is whether loss of the Fe65 proteins is allowing other APP-binding proteins to bind APP family members.

Tommaso Russo
Suzanne, competition is a good point, but we actually don't know the dissociation constants and the concentrations of the competing proteins, the availability for binding, etc.

Sanjay Pimplikar
Yes, Suzanne, that does complicate the interpretation.

Bill Rebeck
Suzanne makes a good point about competition with other adaptor proteins. Dab binds the same regions as Fe65 on APP and on ApoE receptors, and Dab certainly has an effect on neuronal migration.

Virgil Muresan
Yes, Fe65 is surely binding APP in vivo. What can we say about the other APP binders? How many of them are confirmed to bind APP in vivo?

Tommaso Russo
Fe65 and APP are expressed also outside the brain. The phenotype observed by Suzanne does not exclude that APP and Fe65 could also have housekeeping functions.

Veronica Galvan
APP is also highly expressed in cells of the immune system. A common motif of adhesion/migration/synaptic contact is likely.

Sanjay Pimplikar
Nonetheless, is it not striking that loss of APP and Fe65 show a similar phenotype? And, Tomasso, it is so often that we forget that both of these proteins are also expressed non-neuronally.

Veronica Galvan
I don't think it's striking since they are pretty well-defined interactors. There are at least 10 proteins (I can't remember the precise number) described that have been shown to bind to the GYENPTY motif on APP's C-terminus.

Virgil Muresan
Yes, Veronica, but do all of them also bind in vivo? In the presence of Fe65?

Veronica Galvan
All of them are adaptors in signaling pathways—kinases or other proteins involved in intracellular transduction. I don't see the necessity for requiring the presence of Fe65.

Sanjay Pimplikar
Virgil and Veronica raised a good point that I once discussed with Suzanne and Tommaso—how can we find out, at any given time in vivo, what fraction of APP is bound with all the known interactors (Fe65, X11, Dab, Shc, Numb, etc.)? Any insight, folks?

Virgil Muresan
Do people agree that Fe65 is the major binding protein for APP (or AICD)?

Veronica Galvan
No. There are other very important signaling proteins that have been confirmed in a variety of systems like JIP-1b, Dab, X11.

Sebastien Hebert
Virgil, yes, I agree, in my experience, among all "candidate" AICD-binding proteins, Fe65 is one, if not the strongest, interactor under endogenous conditions. But then again, this is only monitored using co-IPs....

Bernadette Allinquant
Other adaptors have not been studied enough.

Sanjay Pimplikar
True, Bernadette. If you look for it, you will find it...just like those oligomers, I guess.

Bill Rebeck
We find that with ApoE receptors, when we add extracellular ligands, we get more Dab binding the cytoplasmic domain and less Fe65. That's co-IPs, but it suggests that these interactions are regulated (see Hoe et al., 2006).

Tom Fagan
All, is it just a case that people are not looking for other interactions or is there a technical difficulty that needs to be overcome?

Virgil Muresan
We find indeed an interaction of JIP-1 with APP. However, most of it is competed off in the presence of Fe65. In the presence of Fe65, we find that JIP-1 binds only to pAPP, which makes sense, since Fe65 has higher affinity for non-phospho-APP (see Muresan and Muresan, 2005).

Qubai Hu
I agree that Fe65 is the major AICD binding protein and we have shown genetic evidence that the interaction may be selected during animal evolution.

Veronica Galvan
We have confirmed the interaction of APP with dynamin and a-b-crystallin (a chaperone) in neuronal projections in brain sections of APP transgenic animals.

Virgil Muresan
The fact that APP binds to so many different proteins will make it very difficult to interpret results from experiments where one of the proteins is overexpressed. Binding equilibriums for all other proteins interacting with APP are perturbed.

Suzanne Guenette
Luciano D'Adamio has identified many other interactors for APP-CTF. In terms of the relative importance of the various adaptors, it will depend on the cellular pathway studied, but it is remarkable that loss of Fe65 proteins reproduces the phenotype of the APP triple KO, and now we have to figure out the underlying molecular mechanism.

Sanjay Pimplikar
I agree with Suzanne's views. One last comment, Tommaso?

Veronica Galvan
I agree. Data from transgenic/knockout/knock-in mice or other complete organisms is most compelling.

Tommaso Russo
I agree with Suzanne that the phenotypes of gene KO are of help. For example, in C. elegans we found a close relationship between the phenotypes of Fe65 and APP KO.

Veronica Galvan
Tommaso, Suzanne, I think that in vivo data coming from different species is most compelling.

Randall Lauffer
Suzanne, if Ulrike Muller's latest data holds up, showing that the soluble form of APPs rescues various APP KO phenotypes, does that mean that Fe65 KO simply causes lack of APP α cleavage and secretion?

Tom Fagan
Randall, interesting point. I'm not familiar with that data. Are they published?

Randall Lauffer
Tom, she had the data in Spain, showing that deletion of the last couple of exons of APP did not hurt the animal.

Suzanne Guenette
Randy, we did not find any differences in APP processing in whole brain lysates of our KO mice except for a decrease in Aβ42 in brains of male mice.

Thorsten Müller
Indeed, we found differential regulation of cytoskeletal genes after AICD/Fe65 induction in neuroblastoma cells.

Veronica Galvan
That would make perfect sense with a role for APP in synaptic plasticity and in neuronal migration during development. Data from the Selkoe lab presented in Madrid (electroporation in utero of rat pups with RNAi) very nicely showed that you require APP for migration through the marginal zone during development--as they go into the cortical plate (see ARF related conference story).

Thorsten Müller
Veronica, that also fits to first results with phalloidin staining (actin disorganization).

Virgil Muresan
So, this shifts the role of APP/Fe65 from the nucleus to the cell periphery....

Lucia Pastorino
Suzanne, have you checked the processing separating the soluble from the insoluble fraction?

Suzanne Guenette
Lucia, we looked at Aβ in diethylamine extracts of brain.

Sanjay Pimplikar
Next topic. Is AICD a transcriptional regulator like NICD or it is not? I will start with Sebastian Hebert and then perhaps Rupert or Thorsten could follow up.

Sebastien Hebert
Well, we started to work with this hypothesis (AICD in gene regulation) many years ago. When I arrived at the lab, we had many contradictory results which needed to be worked out. Basically, in overexpression paradigms, AICD can do (a little) something. However, all our genetic evidence (among many biochemical) does not point to a role for AICD in gene transcription. I would also like to point out that Qubai's group has observed basically the same things we did. So now, we feel that the endogenous way is the (although hard) way to go and wait for other labs to confirm or infirm our observations in vivo.

Tom Fagan
Thanks Sebastien. So a bigger question, then, for all. If we assume AICD is not involved in transcriptional regulation, what's the role of γ-secretase cleavage of APP? (Sorry if I'm jumping the gun, Sanjay.)

Sanjay Pimplikar
No problem Tom; good steering!

Veronica Galvan
Sebastien, there was this publication that I thought was pretty impressive when people doing chromatin immunoprecipitations (completely unrelated to APP itself) happened to find the AICD on top of their favorite promoter (see Baek et al., 2002 and ARF related news story).

Sebastien Hebert
Well, here again, only overexpression data. We do not exclude that in certain circumstances AICD can find itself on DNA, but does this have a physiological meaning? It should be remembered that AICD, in binding to so many proteins, can perhaps "piggyback" into the nucleus.

Veronica Galvan
Well, if you find it in physiological conditions, I wouldn’t worry about what happens with overexpression that much. People have found AICD on complexes with chromatin.

Tommaso Russo
Chromatin IP with anti-APP antibodies demonstrated that fragments of APP (probably C-terminal) are present in chromatin complexes together with Fe65. However, Fe65 could also reach the nucleus as a consequence of APP phosphorylation.

Rupert Egensperger
Has someone done ChIP on ChIP with AICD or Fe65?

Virgil Muresan
Could any role of Fe65/APP in regulating transcription be indirect? Could the effects start at the cell periphery and any effect that some people see on transcription is due to something else (triggered by Fe65/APP)?

Tommaso Russo
Sebastien, if we assume that AICD is not involved in any nuclear function, do you think that, independently from APP or AICD, Fe65 has some role in the nucleus?

Tom Fagan
All, there's a paper coming out in this week's Cell from Gabriel Corfas's lab showing that the ICD of another γ-sec substrate, ErbB4, is involved in transcriptional regulation (see ARF related news story).

Qubai Hu
We have tried ChIP on Fe65. It did not work well.

Tommaso Russo
Qubai, Fe65 ChIP works very well, but the antibody used is critical.

Qubai Hu
We used tandem affinity purification.

Sebastien Hebert
Tommaso, Fe65 has, as I think we could all agree, potent transcriptional properties and can be found in the nucleus under endogenous conditions. So transcription regulator, why not?

Thorsten Müller
In part, in our cell culture model, Fe65 alone was able to induce differential gene expression (without AICD overexpression).

Sebastien Hebert
Veronica, I don't want to seem pessimistic, of course. Say a small fraction of endogenous AICD is in the nucleus; does this really mean it participates in gene transcription events?

Veronica Galvan
If you find it in a transcriptional complex with a bunch of other proteins involved in transcription, well, it's a suggestion.

Virgil Muresan
Veronica, no, there are many other things that happen in the nucleus besides transcription.

Veronica Galvan
Absolutely. Not that many in association with open chromatin, but there could be other reasons to find AICD with chromatin, for sure.

Tommaso Russo
Virgil, I agree with you: there are many, many things happening in the nucleus, not only transcription.

Sanjay Pimplikar
Veronica, I personally am a bit skeptical, though that work was well done and has received most attention. Perhaps, I am wrong, but I am expressing my opinion.

Suzanne Guenette
Sebastien, transcription factors/regulators are not often that abundant. An interesting question is whether there is a basal function or whether AICD and its putative role in the nucleus can be regulated.

Sebastien Hebert
Suzanne, I agree, I think it would be important to show a physiological regulation of AICD in the nucleus. Perhaps insights from a paper from Selkoe's group on neuronal differentiation could help us understand.

Sanjay Pimplikar
Tom F., I think the question you posed is one of the most important unanswered issue—why the heck APP gets processed? What's the biological meaning?

Virgil Muresan
Sanjay, I think that the purpose of processing APP is to generate separate proteins, each with its own, independent function. This is one point of view.

Veronica Galvan
The major cleavage, by far, is the α to release sAPP. Cleavage by γ releases Aβ—a regulator of synaptic function—Kamenetz et al. and other very thorough publications (see ARF related news story).

Sebastien Hebert
Sanjay, quick answer on γ-secretase of APP. Interestingly, we could observe APP CTFs binding quite strongly to Fe65 in the absence of PS/γ-secretase.

Sanjay Pimplikar
Thanks, Sebastian—I think the future experiments need to address the cleavage issues.

Qubai Hu
To me, AICD seems to be not very important, which is why it is quickly degraded.

Sanjay Pimplikar
Qubai, so are the cell cycle regulator proteins! I mean, you could be right, but that reasoning is probably not enough. Everyone, last five minutes!

Randall Lauffer
I think the release of C31 shown by Galvan and coworkers is very important—that aspartate is conserved to the lowest of animals! But, did anyone see the recent Martens paper showing that in brain only, the transcription of APP in Xenopus can start with methionine-677, creating a tiny cytosolic construct of the GYENPTY motif? This is a paper in Brain Research (see Collin and Martens, 2006).

Veronica Galvan
Randall, thanks for the tip. Sounds very interesting.

Tom Fagan
Randall, do you remember what fraction of translation starts at Met 677?

Sanjay Pimplikar
Randy, we will end our discussion with that observation, but I want to take the next 10 minutes to talk about phosphorylation of APP and what it means biologically. Virgil, your turn!

Virgil Muresan
What about the necessity of membrane anchoring of APP for transactivation of transcription (see Cao and Sudhof, 2004)?

Bill Rebeck
I just want to remind everyone that Fe65 also binds those ApoE receptors, so we need to consider the cleavage and phosphorylation of them when we are considering whether Fe65 is tethered to the cell membrane.

Sanjay Pimplikar
Good point, Bill!

Sanjay Pimplikar
Next question: why is APP getting phosphorylated at T668? What's the biological significance? Virgil, do you want to comment?

Virgil Muresan
To regulate interactions with other proteins.... This phosphorylation changes the conformation of APP.

Lucia Pastorino
From our point of view, it could be that T668, when phosphorylated, changes its conformation, thus probably altering the interactions with other protein partners, and this could lead to altered intracellular localization of APP and processing.

Sanjay Pimplikar
Thanks Lucia, I was about to ask you to comment!

Sebastien Hebert
I agree with Virgil concerning the modulation of binding affinities.

Sanjay Pimplikar
Sebastian (and everybody), then could phosphorylation be an important factor concerning the very first point we had in this discussion: how can so many proteins bind APP and when do they do it?

Sebastien Hebert
Sanjay, I guess everyone would agree on that comment. We cannot exclude, of course, the impact of phosphorylation on APP trafficking (which also includes these and likely other proteins).

Lucia Pastorino
In my opinion, yes, APP phosphorylation may affect and maybe also end up to "select" the kind of binding partners that will bind to APP.

Virgil Muresan
The problem with APP phosphorylation is that several proteins may bind preferentially to the phosphorylated or to the non-phosphorylated forms. These interactions may lock APP in either phosphorylated or non-phosphorylated form for quite some time.

Tommaso Russo
Of course, APP phosphorylation could regulate Fe65 nuclear availability.

Qubai Hu
This is just guessing: AICD is important before it has been cleaved off from APP. But after that, its job is done.

Virgil Muresan
What about APP dephosphorylation? Did anyone look at dephosphorylation of APP?

Sanjay Pimplikar
Last comment and then we move on to Veronica's story and Bernadette's work. What are the smaller AICD (or APP-CTF) fragments doing? This was raised earlier by Randy.

Bernadette Allinquant
About the smaller APP-CTF, it seems that they are neurotoxic in vitro when overexpressed.

Sebastien Hebert
Sanjay, as discussed in our recent review, we think that AICDs/CTFs are mainly APP metabolites.

Sanjay Pimplikar
Sebastian, are they just the end products without any biological meaning?

Sebastien Hebert
Sanjay, I have the impression that answering "yes" would be dangerous....

Sanjay Pimplikar
Sebastian, very clever.

Lucia Pastorino
Has anyone seen these smaller C-terminal fragments with mass spectrometry, that is, in the brain?

Thorsten Müller
Lucia, do you think this will work without immunoprecipitation in brain tissue?

Lucia Pastorino
Maybe, I don't know. It’s probably hard to have a good separation from all the other C-terminal fragments, which are certainly abundant in the brain. I mean, if you then separate them in SDS-PAGE.

Sanjay Pimplikar
Lucia, funny you should ask. I just saw a paper from Kume (Kume and Kametani, 2006). They have done mass spectrometry and see AICD59 after α cleavage. Go figure!

Sebastien Hebert
Sanjay, an interesting hypothesis would be that γ-secretase cleavage, for example, would lead to a disruption of AICD-binding complex of protein(s).

Virgil Muresan
Sebastien, it is possible. The CTFs and AICD may have no meaning when separated from APP, but a lot of meaning when present in APP. This is another point of view.

Bernadette Allinquant
Smaller fragments have been observed going from α-secretase site until caspase site (see Lu et al., 2000 and ARF related news story) and different fragments have been found by others (Passer et al., 2000).

Thorsten Müller
Lucia, I agree; we had similar problems. Have you tried different antibodies, and do you have a good one?

Lucia Pastorino
No, I actually have never tried immunoprecipitating these smaller fragments. I was just wondering whether they are produced in the brain.

Sebastien Hebert
Virgil, I'm surprised no one has "publicly" tackled this question, and I would be interested to know whether these (or similar) experiments are going on somewhere.

Sanjay Pimplikar
Last question: what's with C31 and caspase cleavage? Please enlighten us. Veronica, your turf!

Veronica Galvan
Following on Randall's comment, the Asp at 664 is indeed conserved to the "lowest" species, and we have evidence that it is used in non-diseased conditions in adult mouse and human brain; the cleavage at 664 peaks at ~25-30 years of age in humans (coincident with end of myelination). We stabilized the interaction region of APP and got a profound change in the phenotype of FAD-APP transgenic mice, without changes in Aβ production or deposition. That is suggestive of a role of Asp664 in Aβ-mediated pathogenesis, or of the activation of APP signaling as compensatory, or both. We are trying to figure it out with transgenic models.

Bernadette Allinquant
It goes from the membrane to the caspase site. This is unmasked after caspase cleavage. In addition, the fragment going from the γ-secretase site until caspase cleavage is neurotoxic, too (for Veronica).

Sanjay Pimplikar
My impression was that the cleavage at D664 was mediated by caspases. The caspases get activated because of some emergency or something bad happening. Do you think this cleavage is occurring normally, under physiological in vivo conditions?

Bernadette Allinquant
Sanjay, caspase cleavage may be induced by amyloid peptide.

Lucia Pastorino
Sanjay, could this be due to oxidative stress?

Veronica Galvan
We don't know what cleaves in vivo. It could be a calpain. In any case, you can find activated caspases in vivo in cells that are not dying (particularly in neurons; there was a very interesting talk in Madrid) and they have been implicated in synaptic remodeling, which would make sense. In any case, it could be a calpain. What we know is that the cleavage occurs in physiological, non-diseased conditions pretty abundantly in neuronal terminals.

Sanjay Pimplikar
Hmmm...so Aβ induces caspases, which further cleave CTF to C31. Is that the story?

Veronica Galvan
No, I don't think so. Aβ is very likely a synaptic modulator. It may bind APP (see Lu et al., 2003 and Shaked et al., 2006) as part of this function.

Sanjay Pimplikar
Veronica, I was not aware of that. Interesting point.

Suzanne Guenette
Veronica, I was wondering if you had examined whether the binding of APP D664 to CTF binding proteins was altered relative to Tg controls?

Veronica Galvan
Yes. In our PDAPP(D664A) mice we have altered signaling from the C-terminus. We are looking as close to in vivo as possible. The data on APP being a synaptic modulator is pretty solid by now (see Kamenetz et al., 2003 and others). Moreover, Aβ is produced as a result of synaptic activity and in turn it inhibits synaptic activity in a feedback. Those are papers from Kamenetz and from David Holtzman's lab (see Cirrito et al., 2005 and ARF related news story).

Sanjay Pimplikar
Uh oh, Veronica...looks like I am wrong on Aβ. Well…not for the first time but still, it doesn't make sense to me.

Suzanne Guenette
Veronica, what do you mean by altered signaling?

Veronica Galvan
Suzanne, we are looking at signaling pathways downstream (for instance, with activated-specific antibodies for JNK or things like that) to see if we see a difference in signaling in PDAPP(D664A) brains versus PDAPP brains, since we have stabilized the signaling portion of APP.

Virgil Muresan
I was wondering how many other proteins get "chopped off" into fragments, and no one is worried about these "remnants" of the proteins. With APP, we wonder about every tiny fragment.

Sebastien Hebert
Virgil, good point. I guess this would be a good time to put into perspective again my "metabolite" hypothesis about AICDs.

Virgil Muresan
It's been a great discussion. To put it in Einstein's words, I am still confused about the AICD, but I am confused on a higher level. Thank you.

Veronica Galvan
Virgil, that's a very nice quotation. Thanks! I will need to go, too, in a few minutes. Thanks a lot to everybody for a very interesting discussion.

Sanjay Pimplikar
I would like everybody to be able to say one last thing. What is the most pressing issue that the field is not addressing.

Suzanne Guenette
Sanjay, I was wondering if you have or are planning to examine hippocampal-dependent behavior in your Fe65/AICD Tg mice. This gets back to what AICD might be doing.

Sanjay Pimplikar
Suzanne, yes!

Sebastien Hebert
I guess the last words from our group (mainly on AICD signaling) would be to "keep it physiological!" This has been fun and I hope to find an answer to this sometimes frustrating story.

Sanjay Pimplikar
Thanks, Lucia.

Veronica Galvan
Sanjay, would you mind letting me know what your Fe65/AICD Tg mice are? What background?

Sanjay Pimplikar
C57Bl6, Veronica.

Veronica Galvan
And what is the construct and promoter?

Sanjay Pimplikar
CamKII.

Tommaso Russo
I should leave.

Tom Fagan
Okay, Tommaso, thanks for joining. Ciao!

Lucia Pastorino
Thanks, Sanjay! I am running gels and have to run to them now. Thanks to everybody, and I would like to conclude by saying not to forget APLP1 and APLP2: in the end they get cleaved by the same secretases; the ICD fragments were observed to have this here "questioned" transcriptional activity; they have the same residue as the T668 motif in APP; they are very abundant in the brain, but just missing the Aβ sequence. Maybe they are worth studying, as something that collaborates or buffers the effects produced by APP. Thanks again to everybody.

Tom Fagan
Everyone is beginning to drift off so before it is too late, I just wanted to thank everyone for coming and making this a very lively discussion. There's obviously so much we have learned but too many outstanding questions. So in the last few minutes, maybe we could address what would be the most revealing experiment, assuming that there was no technical or financial limitations.

Veronica Galvan
I need to go. Thanks again to everybody. Thanks Sanjay and Tom for organizing the discussion.

Sanjay Pimplikar
Thanks for your time and insight; I learned a lot, Veronica. And good luck with C31—nice story!

Suzanne Guenette
Yes, I agree. Nice story!

Randall Lauffer
As for the most revealing experiment, I wonder what Suzanne would think of deleting APP or Fe65 in meningial fibroblasts alone (as for focal adhesion kinase).

Suzanne Guenette
Yes, I would love to do that; human power needed!

Randall Lauffer
…and money, too!

Sanjay Pimplikar
What do you predict, Randy?

Randall Lauffer
I think, Sanjay, that APPs will rescue all, and perhaps we will be injecting ourselves with it in the not-too-distant future!

Sanjay Pimplikar
Randy....I will bet a beer that you are wrong! Coming back to your question, Tom- I think there are two issues we must address. 1) What's the biological significance of APP processing, be it α or β or γ? 2) In vivo, which protein binds APP CTF at what time and where and what is the biological consequence of this binding or prevention of binding because the site is already occupied?

Thorsten Müller
And what about the binding partners to N-terminal domain of APP and their influence on secretase activity (remember the F-Spondin story, for example) —a lot of work to do.

Sanjay Pimplikar
Yes, Thorsten. I wonder if Tom Sudhof is following up on that one.

Suzanne Guenette
I have to go. Thanks a lot for the interesting discussion. Hope to see you in Atlanta.

Sanjay Pimplikar
Thanks for joining us, Suzanne. What do you think, Virgil? What about the JIPs and kinesin receptor story?

Thorsten Müller
Sanjay, Virgil, indeed, another interesting point: the axonal transport story.

Sanjay Pimplikar
This was my first live discussion appearance and I want to thank you all for coming and sharing your views. This discussion also tells us that we need to focus more on the cytoplasmic domain, especially for the biology part.

Thorsten Müller
That was really interesting and we should repeat such a discussion. It was my first discussion, too. Thank you.

Virgil Muresan
Yes, let's have another live discussion on axonal transport.

Bernadette Allinquant
Thank you, Sanjay, very exciting discussion.

Sanjay Pimplikar
Thanks to our European friends; it is late for them. Thank you!

Tom Fagan
Yes, and I'd like to thank Sanjay for volunteering to host this discussion and doing the background work for it. Thanks, Sanjay. You made our job easier!

Rupert Egensperger
Sanjay, this was a very interesting, fruitful discussion; thanks a lot.

Bill Rebeck
Thanks, Sanjay. I enjoyed it.

Virgil Muresan
Thanks again to everyone.

Sanjay Pimplikar
Bill, thanks for joining us. Hope to see you soon and learn more about ApoE!

Randall Lauffer
Thanks.

Thorsten Müller
It was a pleasure. All the best for you and your work. Good night!

Background

Background Text
By Sanjay W. Pimplikar

The amyloid-β precursor protein (APP) cytoplasmic domain exists in three forms—as a part of uncleaved APP, as a part of the membrane-associated C-terminal fragments, and as a free, soluble fragment termed the APP intracellular domain (AICD). Though the cytoplasmic domain has lived in relative obscurity compared to its better-known half, the Aβ peptide, interest in the domain has been aroused by recent reports that addressed its biological roles. The APP cytoplasmic domain interacts with Fe65 (1), along with several other proteins, and the significance of this domain (and that of its interaction with Fe65) was revealed when APP double or triple (APP, APLP1, and APLP2) knockout mice (2) were found to have a similar phenotype as the Fe65 double knockout animals (3). Fe65 associates with both intact APP and free AICD, and the exact mechanism underlying cortical dysplasia seen in these knockout animals poses an exciting challenge in the field.

The APP cytoplasmic domain attracted immense attention with the observations that AICD regulates gene expression (4, 5, and ARF related news story), and a number of studies have since come to similar conclusions. Because it has been difficult to identify the gene targets of AICD, and a recent study has cast doubts about its transcriptional role (6), the debate over AICD’s role in transcriptional regulation is likely to continue in the near future. However, the biological effects of the APP cytoplasmic domain and/or its proteolytic fragments are less uncertain. The observation that a point mutation in the APP cytoplasmic tail (that potentially abolishes the generation of C31 fragment by caspases) rescues memory deficits (7 and ARF related news story) in a mouse model of AD is a significant observation that should prompt re-evaluation of how we look at the etiology of the disease.

It is likely that the APP cytoplasmic domain exerts biological effects as a part of APP-CTFs (such as C99 and C83), although this area of research remains less studied. Another area of interest is the role of phosphorylation of the APP cytoplasmic domain on the T668 residue, its effect on AICD-mediated transcription, and its association with interacting proteins such as Fe65, Dab1, and x11-α. Two recent papers studied the role of T668 phosphorylation and came to opposite conclusions regarding nuclear translocation of AICD and its role in transcription (8,9). In summary, as with other areas of AD research, the studies on APP cytoplasmic domain have generated conflicting and sometimes contradictory views.

The aim of this live discussion is to bring researchers together to discuss the issues related to the APP cytoplasmic domain as a prelude to a mini-symposium on the same topic at the forthcoming SfN meeting in Atlanta (Tuesday, October 18, 8:30 a.m.). Some of the items up for discussion will include the following:

• Does AICD play a role in gene expression? If so, how?
• What is the role of the APP cytoplasmic domain and Fe65 in cortical dysplasia or neuronal migration?
• How does phosphorylation of T668 regulate APP function? What role does Pin1 play, since it binds to the phosphorylated form of APP?
• How does a mutation in the APP cytoplasmic domain (D664A) reduce the memory deficits in a mouse model of AD?

References:
1. Zambrano N, Buxbaum JD, Minopoli G, Fiore F, de Candia P, De Renzis S, Faraonio R, Sabo S, Cheetham J, Sudol M, Russo T. Interaction of the phosphotyrosine interaction/phosphotyrosine binding-related domains of Fe65 with wild-type and mutant Alzheimer's β-amyloid precursor proteins. J Biol Chem. 1997 Mar 7;272(10):6399-405. Abstract

2. Herms J, Anliker B, Heber S, Ring S, Fuhrmann M, Kretzschmar H, Sisodia S, Müller U. Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members. EMBO J. 2004 Oct 13;23(20):4106-15. Abstract

3. Guénette S, Chang Y, Hiesberger T, Richardson JA, Eckman CB, Eckman EA, Hammer RE, Herz J. Essential roles for the Fe65 amyloid precursor protein-interacting proteins in brain development. EMBO J. 2006 Jan 25;25(2):420-31. Abstract

4. Cao X, Südhof TC. A transcriptionally [correction of transcriptively] active complex of APP with Fe65 and histone acetyltransferase Tip60. Science. 2001 Jul 6;293(5527):115-20. Abstract

5. Gao Y, Pimplikar SW. The gamma -secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus. Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14979-84. Abstract

6. Hébert SS, Serneels L, Tolia A, Craessaerts K, Derks C, Filippov MA, Müller U, De Strooper B. Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes. EMBO Rep. 2006 Jul ;7(7):739-45. Abstract

7. Galvan V, Gorostiza OF, Banwait S, Ataie M, Logvinova AV, Sitaraman S, Carlson E, Sagi SA, Chevallier N, Jin K, Greenberg DA, Bredesen DE. Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664. Proc Natl Acad Sci U S A. 2006 May 2;103(18):7130-5. Abstract

8. Nakaya T, Suzuki T. Role of APP phosphorylation in Fe65-dependent gene transactivation mediated by AICD. Genes Cells. 2006 Jun ;11(6):633-45. Abstract

9. Chang KA, Kim HS, Ha TY, Ha JW, Shin KY, Jeong YH, Lee JP, Park CH, Kim S, Baik TK, Suh YH. Phosphorylation of amyloid precursor protein (APP) at Thr668 regulates the nuclear translocation of the APP intracellular domain and induces neurodegeneration. Mol Cell Biol. 2006 Jun ;26(11):4327-38. Abstract

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References

News Citations

1. Long-elusive Function for APP Cleavage Product Comes into View: It's Gene Transcription 22 Nov 2002
2. Paper Alert: Pathology Reversed by Abolishing APP Caspase Site 19 May 2006
3. Just How Does APP Build a Better Brain? 26 May 2006
4. Aβ's Shadowy Sibling—What Becomes of the Intracellular Domain? 4 Feb 2003
5. Squelching Gene Expression Controls Development, Neurodegeneration 6 Oct 2006
6. Does Aβ Normally Rein in Excited Synapses? 26 Mar 2003
7. Another Fatal Peptide from APP 22 Nov 2002
8. Paper Alert: Synaptic Activity Increases Aβ Release 22 Dec 2005

Webinar Citations

1. The Functional Roles of APP Cytoplasmic Domain—Conflicts and Consensus 21 Dec 2006

Paper Citations

1. Zambrano N, Buxbaum JD, Minopoli G, Fiore F, de Candia P, De Renzis S, Faraonio R, Sabo S, Cheetham J, Sudol M, Russo T. Interaction of the phosphotyrosine interaction/phosphotyrosine binding-related domains of Fe65 with wild-type and mutant Alzheimer's beta-amyloid precursor proteins. J Biol Chem. 1997 Mar 7;272(10):6399-405. PubMed.
2. Herms J, Anliker B, Heber S, Ring S, Fuhrmann M, Kretzschmar H, Sisodia S, Müller U. Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members. EMBO J. 2004 Oct 13;23(20):4106-15. PubMed.
3. Guénette S, Chang Y, Hiesberger T, Richardson JA, Eckman CB, Eckman EA, Hammer RE, Herz J. Essential roles for the FE65 amyloid precursor protein-interacting proteins in brain development. EMBO J. 2006 Jan 25;25(2):420-31. PubMed.
4. Cao X, Südhof TC. A transcriptionally [correction of transcriptively] active complex of APP with Fe65 and histone acetyltransferase Tip60. Science. 2001 Jul 6;293(5527):115-20. PubMed.
5. Gao Y, Pimplikar SW. The gamma -secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus. Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14979-84. PubMed.
6. Hébert SS, Serneels L, Tolia A, Craessaerts K, Derks C, Filippov MA, Müller U, De Strooper B. Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes. EMBO Rep. 2006 Jul;7(7):739-45. PubMed.
7. Galvan V, Gorostiza OF, Banwait S, Ataie M, Logvinova AV, Sitaraman S, Carlson E, Sagi SA, Chevallier N, Jin K, Greenberg DA, Bredesen DE. Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664. Proc Natl Acad Sci U S A. 2006 May 2;103(18):7130-5. PubMed.
8. Nakaya T, Suzuki T. Role of APP phosphorylation in FE65-dependent gene transactivation mediated by AICD. Genes Cells. 2006 Jun;11(6):633-45. PubMed.
9. Chang KA, Kim HS, Ha TY, Ha JW, Shin KY, Jeong YH, Lee JP, Park CH, Kim S, Baik TK, Suh YH. Phosphorylation of amyloid precursor protein (APP) at Thr668 regulates the nuclear translocation of the APP intracellular domain and induces neurodegeneration. Mol Cell Biol. 2006 Jun;26(11):4327-38. PubMed.
10. Hoe HS, Tran TS, Matsuoka Y, Howell BW, Rebeck GW. DAB1 and Reelin effects on amyloid precursor protein and ApoE receptor 2 trafficking and processing. J Biol Chem. 2006 Nov 17;281(46):35176-85. PubMed.
11. Muresan Z, Muresan V. Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1. J Cell Biol. 2005 Nov 21;171(4):615-25. PubMed.
12. Baek SH, Ohgi KA, Rose DW, Koo EH, Glass CK, Rosenfeld MG. Exchange of N-CoR corepressor and Tip60 coactivator complexes links gene expression by NF-kappaB and beta-amyloid precursor protein. Cell. 2002 Jul 12;110(1):55-67. PubMed.
13. Collin RW, Martens GJ. The coding sequence of amyloid-beta precursor protein APP contains a neural-specific promoter element. Brain Res. 2006 May 4;1087(1):41-51. PubMed.
14. Cao X, Südhof TC. Dissection of amyloid-beta precursor protein-dependent transcriptional transactivation. J Biol Chem. 2004 Jun 4;279(23):24601-11. PubMed.
15. Kume H, Kametani F. Abeta 11-40/42 production without gamma-secretase epsilon-site cleavage. Biochem Biophys Res Commun. 2006 Nov 3;349(4):1356-60. PubMed.
16. Lu DC, Rabizadeh S, Chandra S, Shayya RF, Ellerby LM, Ye X, Salvesen GS, Koo EH, Bredesen DE. A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor. Nat Med. 2000 Apr;6(4):397-404. PubMed.
17. Passer B, Pellegrini L, Russo C, Siegel RM, Lenardo MJ, Schettini G, Bachmann M, Tabaton M, D'Adamio L. Generation of an apoptotic intracellular peptide by gamma-secretase cleavage of Alzheimer's amyloid beta protein precursor. J Alzheimers Dis. 2000 Nov;2(3-4):289-301. PubMed.
18. Lu DC, Shaked GM, Masliah E, Bredesen DE, Koo EH. Amyloid beta protein toxicity mediated by the formation of amyloid-beta protein precursor complexes. Ann Neurol. 2003 Dec;54(6):781-9. PubMed.
19. Shaked GM, Kummer MP, Lu DC, Galvan V, Bredesen DE, Koo EH. Abeta induces cell death by direct interaction with its cognate extracellular domain on APP (APP 597-624). FASEB J. 2006 Jun;20(8):1254-6. PubMed.
20. 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.
21. Cirrito JR, Yamada KA, Finn MB, Sloviter RS, Bales KR, May PC, Schoepp DD, Paul SM, Mennerick S, Holtzman DM. Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo. Neuron. 2005 Dec 22;48(6):913-22. PubMed.

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