Posted 31 August 2006

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Live discussion held 13 August 1999 and moderated by Chris Wheil.

Participants: Chris Wheil, Bruce Yankner, Ben Wolozin, Ken Kosik, Eddie Koo

Note: Transcript has been edited for clarity and accuracy.

Chris Wheil: Welcome Drs. Kosik and Wolozin. Thank you for joining us. We will try to start at 12pm EST.

Chris Wheil: We are waiting on Dr. Yankner and then we can begin.

Edward Koo: This is Eddie Koo, I'm here too.

Chris Wheil: Welcome Dr. Koo.

BWolozin: Hi Eddie!

Chris Wheil: Now we can begin. I want to welcome everyone to this discussion of the role of PS1 in beta catenin signalling. I thought that we might open the forum with a brief discussion about why papers have demonstrated different results in regard to PS1 function of beta-catenin. In particular what is the best measurement of beta-catenin function?

Bruce: I would say that the best measure is its biological activity in vivo.

Chris Wheil: Dr. Yankner suggested the best measure is in vivo function in animal models. He mentioned some preliminary data. Perhaps Bruce would elaborate.

Bruce: That remains to be determined

Edward Koo: I agree with Bruce to a large extent. However, the in vivo results have not all been consistent depending on the organism. So I think other evidence needs to be brought in to go along with an in vivo outcome.

Bruce: Our group and Rich Carthew's group have results which suggest that PS1 can potentiate the biological activity of beta catenin in Xenopus and Drosophila. Although these results are consistent with a stabilizing effect of PS1, the precise mechanism remains to be determined.

BWolozin: It looks like there is no response to the question about in vivo models, so perhaps we should try to figure out which are the best methods when using cell culture (acknowledging that in vivo is the gold standard).

Chris Wheil: Dr. Wolozin. Your data with the luciferase reporter seemed superior in gauging the function of beta-catenin compared with stability assays.

BWolozin: Perhaps we should focus on cell culture models, since there is more familiarity with that.

Kosik: Bruce, how did you do the experiment regarding the potentiation?

Bruce: We collaborated with Xi He to inject PS1 constructs into Xenopus and assayed axis duplication as an indicator of the Wnt/catenin activity

Chris Wheil: Bruce what were the results?

Bruce: Wild-type PS1 induces complete and partial axis duplication. FAD PS1 mutations show loss of function.

Chris Wheil: Dr. Koo would you please comment on this data as it is contrary to your theory of beta-catenin and mutant PS1.

Edward Koo: I'm trying to absorb Bruce's results. I would agree that our results did not have a functional read out, such as Bruce's xenopus injections. On the other hand, we've always looked at endogenous catenin levels, not co-expressed catenin. We now have some data using nuclear reporter assays that confirm our catenin data. So the differences to Takashima's papers may rest with cell type and method of expression, rather than different biologic effects per se.

BWolozin: Loss of function appears consistent with what many have observed - even the Aβ effects could be due to loss of function.

Edward Koo: Ben, what do you mean by loss of (presumably beta-catenin) function and Aβ effects?

zhenglab: I do not think the Aβ effect is a loss of function since in PS1+/- mice where the PS1 expression is reduced by half, there is no increase in Aβ.

Bruce: In all fairness to Eddie, our results are not completely at odds with his points about catenin pools because this assay required coexpression of catenin. However, Rich Carthew has selectively inhibited PS1 expression at different stages in Drosophila development and observes a partial Wingless (loss of catenin ) phenotype.

BWolozin: If PS1 is controlling degradation of proteins, then having half as much PS1 would still allow correct processing of Aβ and of catenin, hence no change.

Edward Koo: Rich Carthew's results surprise me. I asked Mark Fortini about his studies. So far, he tells me that none of his drosophila PS mutants look anything like Wnt/armadillo phenotype. They all look more like notch phenotypes.

zhenglab: But in any case, Aβ in PS1/FAD and PS1+/- is not the same.

BWolozin: Bruce, what is the role of catenin in axis duplication - is it due to adhesion effects? And........can you rule out effects on other signaling systems - like Notch?

Kosik: Regarding the expression of h-PS1 in other systems we have expressed h-PS1 in fly and see a lethal phenotype that is predominantly an adhesion defect.

Edward Koo: I think the adhesion versus signaling is very complicated. If you over-express cadherins, you can sop away the catenins from signaling, but it also increases catenins at cadherin sites. So it is not obviously an either/or situation.

Bruce: Eddie, Fortini knocks out PS1 expression from the start. Rich used a new method, double-stranded RNA interference, to selectively inhibit PS1 expression at specific developmental stages. In the Fortini experiments the Notch phenotype could have obscured the catenin phenotype - but I am certainly not an expert on Drosophila development. Regarding Ben's question, Wnt induces axis duplication through a primarily transcriptional mechanism. I don't know whether the effector phase involves adhesive interactions.

Zhuohua: Just curious about zhenglab's comment. PS1+/- does not really explain the loss of function. since a mutant ps1 expression may replace the wild-type ps1, as we all know PS1 expression is regulated by some unknown limited factor. It would be interesting to know what percentage of mutant PS1 is in the patient v.s. the wild type PS1 (I mean the protein).

Chris: What role does beta-catenin have in AD pathology?

Chris: Does increased or decreased Wnt signalling increase BAPP production?

Kosik: We have data that expressing Wnt-1 in PC12 cells will increase Aβ, but cannot assume that it is direclty related to the effect of Wnt or some downstream effect.

Bruce: Ken, do you know the relative effects of Wnt-1 on Aβ 40 and 42?

Kosik: Bruce, we found the effect on 40 an 42 remains indeterminate because its levels were below the threshold of the assay.

BWolozin: Ken, Do you think that the adhesion defect is mediated by GSK and APC (or could it be explained by another adhesion system)? So far there hasn't been much talk about the connection between APC and PS1.

Kosik: Ben, my first impression was that the adhesion defect was due to titration of beta-catenin from junctions by PS1.

Chris: Dr. Koo, did you find an interaction between APC and PS1?

Edward Koo: We only looked at this once but could not co-IP APC and PS1. But the catenin complex is getting larger (axin, catenin, APC, dsh, GSK, among others). Maybe we looked at the molecule in the incorrect phosphorylation state.

BWolozin: Ken - Chris brought up an interesting connection relating to APC. So far there has been little talk of connections with APC. Do you think that the PS1/APC/Aβ connection might be operative here? My sense is that the junctions represent APC/cat binding. Is this correct?

Edward Koo: I don't think anyone has shown how the stabilizing or destabilizing effects of PS1 on beta-catenin works. It could in fact be through GSK, APC, or other molecules. So the speculations are very reasonable.

Chris: Iva Greenwald found that SEL-12 associated with SEL-10, a slimb homologue involved in ubiquitin ligase. This could be a mechanism too. Dr. Yankner has done some work in this area.

Bruce: We do not yet know whether the Fbox protein beta Trcp is also present in the PS1 complex.

BWolozin: Since I use a lot of cell culture, I was wondering if people could give their opinions on the relative merits of various assays - cat levels, nuclear cat, nuclear translocation, PS1 binding, reporters, etc.

Chris: Dr. Wolozin this a key issue to understand why there are different results.

Bruce: Ben, none of them work.

BWolozin: Bruce - can you elaborate?

Bruce: Only joking - but I think at the end we have to understand the activity in vivo.

BWolozin: Agreed, but in the meantime....

Chris: Murayama's data came very close to functional data (use of reporter luciferase).

BWolozin: Chris - the odd thing about the luciferase data is that WT PS1 reduced Tcf activity. However, the loss of function in mutant PS1 relative to WT was consistent with what everyone is saying.

Chris: Perhaps this relates to transient overexpression?

Bruce: Regarding Ben's question, one important technical issue when comparing cell lines stably overexpressing PS1 constructs is to control for expression levels. This is not trivial in the case of the PS1 exon 9 deletion in which the protein is not metabolized to fragments.

BWolozin: That would be my bet. We see differences between transients and stables.

Chris: Ben- differences in what way?

BWolozin: For instance, looking at Elk (sorry to switch systems) we see large effects on reporters when the PS1 is expressed transiently, but less significant effects in stables.

Chris: Dr. Koo mentioned that there are problems with overexpression of PS1 and the increase in artifactual full length PS1? In addition, it appears that mutant PS1 fragments may be more stable as well. (Mike Lee's Nature Med paper). Dr. Koo please comment.

Edward Koo: We've noticed different results depending on transients or stables. In addition, in our inducible system, the length of time of induction and level of induction also provides varying results. Our suspicion is the full length and fragments. That's the obvious culprit.

chenel: Dr. Koo, do you see differences in the complex formations with PS1 depending on transient or stable transfection?

Edward Koo: As to stability of fragments, we haven't looked carefully. Mike Lee's paper used Bruce's favorite model, i.e. in vivo from transgenic mice. So most of the in vitro studies may not be comparable. The del x9 is obviously more stable than WT PS1.

Bruce: We found that the stabilization of catenin by wt PS1 was less in stables than transients, but the mutants still appeared to destabilize in our hands. We used inducible Heks from Sam's lab, and stable constitutive overexpressor's from Dennis Selkoe's lab.

Chris: Ben- do you think that PS1 affects several signalling systems?

BWolozin: No question of that. It affects many systems - the hard part is to figure out which effects are direct.

Edward Koo: In our hands, the complex are much more variable in transient transfections. In fact, they are very difficult to detect.

dekang: I would like to add to Eddie's comments. In our experimence, transient PS1 transfection does not augment the endogenous beta-catenin/PS1 complex.

Edward Koo: By complex, I mean PS1 with GSK and beta-catenin. We can't see APC.

chenel: Dr. Koo, which proteins appear/disappear in the stable vs transient transfections?

Edward Koo: In our hands, it's very difficutl to increase the levels of PS1 NTF and CTF in transients. The full-length protein obviously comes up readily, but not the stable fragments. On the other hand, in the stables or conditional stables (ecdysone inducible), the fragments come up much more, in parallel with the full-length protein.

Kosik: Bruce, we have tried several times unsuccessfully to see beta-catenin instability in Jie's KO animals, but beta-catenin always looks like the controls on western blots. Please comment.

Bruce: Ken, when we examined Jie's cells we saw no significant change in full-length beta catenin, but observed increased appearance of lower MW catenin fragments. However, in Bart's PS1-KO cells, we see both the fragments and reduced full-length catenin. Interestingly, Bart's cells also appear more severely affected by the KO phenotype in other assays as well, like Aβ production.

Edward Koo: I'm concerned about Bart's KO cells when differences come up since his KO is at a more downstream exon. I don't [think it has been excluded that an N-terminal PS1 fragments is produced.

BWolozin: One of the things that I am wondering is whether PS1 connects directly to the proteosome, since all of the effects of PS1 seem to ultimately funnel through the proteosome. Anyone have any data or thoughts on this?

Chris: Could the problem with transients relate to apoptosis and caspase cleavage of PS1 and disruption of beta-catenin binding?

Edward Koo: Chris, that is a possibility but I don't think PS1 WT should be killing cells normally, even if caspase results in loss of catenin binding, as reported by Rudy's lab.

Chris: Ed even if it is overexpressed?

Edward Koo: In our hands, overexpression of WT PS1 does not induce apoptosis. But admittedly, it's another of the in vitro type experiments. Let me ask a question for a change: Has anyone looked at PS2 and beta-catenin or GSK or any other part of the complex?

Kosik: Eddie we cannot find a PS2 d-cat or beta-catenin partnership.

BWolozin: It seems that PS2 has a stronger connection to things that directly affect cell death - like D'Adamio's Bcl-X results.

Bruce: Rudy has told me that his lab has not observed PS2: catenin co-recipitation.

Kosik: PS2 mutations are also not fully penetrant

Chris: Ben- Is there more neuronal loss in PS2 FAD families?

BWolozin: Not to my knowledge - even though there is a catenin connection, I still think the important thing is that PS1/2 affects Aβ.

Edward Koo: Ben, are you implying that the PS2 catenin connection is at the cell death level, a la your studies with Luciano?

Bruce: Eddie, we see no PS1 (or PS1 fragments) in Bart's cells using a very high titre antibody to the extreme N-terminus.

BWolozin: Eddie I think that there are two issues. How does PS1 cause AD and how does PS1 affect catenin. The latter question is exceptionally interesting, but unfortunately, probably not relevant to AD.

Chris: Ken-What role does PS1 play in the metabolism of other catenin?

Kosik: Chris--no data on that one yet.

Chris: Ken- do you think that PS1 role in delta-catenin will be more important that beta catenin, because delta is more neuronally expressed?

Kosik: Chris-many of the same doubts about the role of beta-catenin in AD apply to delta-catenin.

Chris grins evilly.

Chris: Does beta catenin relate to AD?

BWolozin: However...I'm not implying a PS2 catenin connection. PS2 probably affects cell death via BclX or JNK.

Bruce: Ben, what do you think about the absence of a phenotype in the PS2-KO given your point about the difference in PS1/PS2 functions regarding cell death?

BWolozin: Bruce - there are other regulators of Bcl-X. PS2 might regulate Bcl-X or JNK, but might not be the major regulator.

Chris: Dr. Koo-- does mutant PS1 affect GSK-3beta activity on beta catenin or tau?

Edward Koo: I think we tried some phosphorylation experiments a while ago and they weren't very clean. So it is possible that stability or destablity of PS1 on catenins is via GSK. Ben or his colleagues in Japan may have more on this than I do.

Bruce: Eddie, we have recently evaluated catenin phosphorylation using antibodies specific for catenin phosphorylated at the GSK3 sites in collaboration with Xi He, and find that PS1-/- cells have higher steady state levels of PO-catenin than controls.

BWolozin: Perhaps we should think of the term scaffold or complex - that includes GSK and catenin and PS1.

Edward Koo: Ben is absolutely correct. The signaling or stabilizing complex of catenin is getting larger. To think only about PS1-cat or PS1-GSK is almost certainly incorrect.

BWolozin: Okay, now for the messy question - to reiterate Chris' question. Can anyone come up with a cogent explanation for why the catenin connection would impact on AD?

Chris: Apoptosis? and beta catenin destabilization? Dr. Yankner?

chenel: Have there been any FAD cases that are related to mutations in catenin?

Edward Koo: I guess they (the catenin mutations) would die of cancer before getting AD.

BWolozin: Judah Folkman might allow us to address that once he cures cancer!!

Bruce: Ben, that would depend on whether the catenin mutations are gain of function, i.e the phosphorylation sites (the mutations causing colon cancer) or loss of function, i.e. C-terminal mutations.

Chris: Bruce what about FAD mutants and beta-catenin phosphorylation? I like that theory: An increase beta-catenin stabilty = cancer and decrease in beta-catenin stability = neurodegeneration? Please comment.

Bruce: Chris, we are just beginning to address that issue with the phosphorylation-dependent catenin antibodies. No definite results yet.

BWolozin: Good thought Bruce (as always).

BWolozin: I have to run off soon, but I would like people's input onto the merits of various catein assays. Any last thought - other than frogs and flies.

Edward Koo: Ben, my take is that the nuclear translocation is least informative since the complex does not have to signal in the nucleus. Levels of catenin need to be correlated with reporter assays (and biological effects, if possible) for the best in vitro readout at this time.

Chris: Well the hour is almost up. One last comment from our four participants regarding the role of PS1 in beta-catenin signalling. Drs. Koo, Yankner, Kosik and Wolozin....

BWolozin: Ciao to everyone.

Kosik: Re the request for a last comment, I would look to dysfunction within synaptic and adherens junctions for a possible role of these proteins in AD.

Bruce: Perhaps the most interesting biological question is how the same molecule, PS1, can affect the function or trafficking of a soluble cytoplasmic protein like beta catenin as well as proteins in the secretory pathway such as APP and Notch.

Edward Koo: I agree with Bruce. If PS1 is not gamma-secretase, then the most cogent explanation at this time is that PS1 functions as an escort protein of some kind.

Chris: Thank you again to all of our participants. We are still far away from any definitive answers. Maybe at our next discussion well have the problem of AD solved!