Posted 30 August 2006

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Participants: Chris Weihl, Gopal Thinakaran, Kudo, Kazunori Imaizumi, Nikolaos Robakis, Weiming Xia, June Kinoshita

Note: Transcript has been edited for clarity and accuracy.

Cweihl: Welcome to the panel discussion on PS1 and new theories of its functions. Let's begin by discussing the role of PS1 in the unfolded protein response (UPR).

kudo: Dr.Imaizumi and Dr.Thinakaran, firstly we should show what is the problem between our paper and Sato's paper (Nature Cell Biol, Dec 2000).

Thinakaran: Sure.

Cweihl: My first question is general. Do mutations in PS cause an increase sensitivity to ER stressors? Dr. Thinakaran?

Thinakaran: Good point. I haven't directly tested the sensitivity issue in my lab. However, a recent paper by Robert Siman casts serious doubts.

Cweihl: Please explain? Dr. Imaizumi and do you care to comment as well?

Imaizumi: We observed that FAD-PS1 mutation do sensitize cells to ER stressors.

Thinakaran: Siman and colleagues used neurons from their knock-in mice to address whether there is increased cell death, caspase cleavage, TUNNEL labeling etc with glutamate stress and also typical ER stress. Their paper just got published in J. Neuroscience. Also, he presented his data at the Neuroscience meeting.

Imaizumi: I also saw his data.

Cweihl: Dr. Imaizumi what are the key differences between your studies and the Thinakaran paper? Dr. Thinakaran, I'd like to ask you the same question.

Thinakaran: I will wait until Dr. Imaizumi has a chance to address.

Imaizumi: I think [they are] differences of treatment of stressors, dose and time course.

Cweihl: Please explain the differences.

Imaizumi: [Dr. Thinakaran's] data show that middle dose of tunicamycin affects sensitivity to ER stress, but higher or lower dose did not affect it. I personally thought that the doses were comparable and the time points the same as well.

Thinakaran: I am not quite sure what Dr. Imaizumi refers to as low/mid/high doses. We only used one dose and not a dose-response study. In fact, we tried hard to keep the dose and time of treatment as close to their Nature Cell Biology paper as possible.

Cweihl: Dr. Imaizumi. what happens to the UPR at high vs. low vs. mid dose?

Imaizumi: At high dose, the differences of ER stress response are difficult to detect. High dose causes cell death.

Thinakaran: But your Nature Cell Biology paper did report clear differences at the same dose we tried. This I can't explain. Dr. Imaizumi, if low dose shows the difference between wt and mutant PS1, and only high dose causes cell death (presumably more in mutant cells), how can we explain this difference in cell death based on "differential sensitivity"?

Imaizumi: At low dose, as cell death does not occur, the UPR is activated in normal cells.

kudo: I would like to add to Dr. Imaizumi's comment. Dr. Thinakaran checked relatively late time-points but we checked early points after adding stress.

Thinakaran: Dr. Kudo, not true. Our time points 5 h was shorter than the 6 h treatment reported in the Nature Cell Biology paper.

Cweihl: To me the differences between the papers were related to cell lines and experimental analysis. I thought that the differences between the two papers was reminiscent of the initial studies between cell lines looking at beta catenin stability vs. translocation of beta catenin to the nucleus. Please comment Dr. Thinakaran?

Thinakaran: Unlike catenin papers, both Imaizumi's study and our study (data from David Ron's lab and my own) examined "activation" of IRE. There are many ways of looking at activation. But, ultimately the function is the coordinate induction of UPR genes.

Imaizumi: I detected the disturbed activation of PERK and IRE1.

Cweihl: What is the best way to look at activation of IRE? Translocation, phosphorylation or ultimately mRNA levels?

Imaizumi: Phosphorylation.

Thinakaran: In my opinion, gene expression. It is the readout. Phosphorylation is like writing a check. Gene expression is cashing it.

Imaizumi: PS1 mutant affects the phosphorylation of IRE1 within 30 min. after treatment with tunicamycin.

Cweihl: I agree with Dr. Thinakaran. However you look at protein levels and mRNA levels, the protein levels are the sensor to activate the UPR. Is this a confounding problem in your experimental design?

Thinakaran: Again, I refer to Katayama et al. paper, where the difference was seen clearly at later time points. Is there a problem in reproducing that data?

Nikos enters

Imaizumi: It is reproducible data.

Nikos: Hi from Robakis lab. We had some problems logging in

Thinakaran: Dr. Imaizumi, then I fail to understand why you insist on the early time points.

Imaizumi: PERK and IRE1 are activated very early after ER stress.

guest10 enters

Imaizumi: I think this activation may be critical for induction of GRP78 mRNA.

Thinakaran: As far as I can tell (as an outsider to the ER stress field), no one has shown a functional role for "delay" of 10-15 min in activation of IRE or PERK. If I remember the data correctly, after 45 minutes of treatment, there is NO difference in the activation of either kinase.

Cweihl: Dr. Imaizumi, do your results agree with the Niwa paper in Cell?

guest10: This is Weiming Xia from Center for Neurologic Diseases. I forgot my password. Early onset?

Imaizumi: No, we could not detect any differences of UPR in PS1 knockout cells.

Cweihl: So there is agreement between your and Thinakaran's paper in regard to KO lines?

Imaizumi: Yes

Thinakaran: Has anyone talked with Dr. Walter about this?

Imaizumi: I have not talked with him.

Cweihl: So can we attribute the differences between the papers to experimental design? Dr. Thinakaran?

Thinakaran: I don't think experimental design is the problem. Our study followed Dr. Walter's experiment with regards to BiP Northern - concentration, time course.

Cweihl: Dr. Robakis or Dr. Xia do you care to comment on the differences between these two papers?

Nikos: (Robakis) Not at this point because we don't have experimental data to support one or another.

Imaizumi: We could not detect the cleaved fragments of IRE1.

Thinakaran: Oh oh...Someone really ought to sort this issue with Dr. Peter Walter. The AD field is citing his Cell paper whenever there is a chance to say PS1 regulates this cleavage, not detected by any other lab. Maybe other lab's haven't done their study as carefully as Walter's lab.

Cweihl: Let us shift discussion to the other topic.: PS1 and synaptic transmission. Do we have a consensus that PS1 mutations cause a perturbations in synaptic transmission?

Nikos: (Robakis) We think the action is at the cell surface including synaptic contacts where PS1 interacts with cadherins and participates in the cleavage of APP and Notch1.

Cweihl: Please explain, Dr. Robakis? Does PS1 exist at these synaptic contacts, Dr. Robakis?

Nikos: Yes it does. We have shown that in our paper in Molecular Cell. PS1 concentrates at synaptic and cell-cell contact sites.

Cweihl: Dr. Thinakaran can you comment on Dr. Robakis points?

Thinakaran: Dr. Robakis, has anyone addressed whether mutations in cadherins lead to altered APP or Notch metabolism?

Weiming Xia: I have not done any experiments to favor one side. My guess is that experimental methods used in two papers lead to the difference.

Nikos: PS1 regulates cell-cell adhesion and we believe that it also participates in synaptic structure. Dr Thinakaran, we have evidence that PS1 binds directly to cadherins and we have mapped the site (paper under review).

Thinakaran: What baffles me is that the levels of PS in cells are not nearly 1/10 of the levels of cadherins or other cell junction structural proteins.

Cweihl: Several lines of evidence suggest that the role of PS in synaptic output is at the level of the ER and calcium release or reuptake. Can this be the case as well as changes at the synapses?

Thinakaran: Synaptic transmission can be altered by proteins that are not localized in the synapse. There are several [lines of] evidence for this.

Nikos: We don't say that all of the PS1 molecules bind to cadherins. A portion of total PS1 is bound to cadherins and only at cell-cell contact sites.

Cweihl: Dr. Thinakaran, then could a trafficking issue be involved that alters the synaptic sites?

Thinakaran: Definitely.

Nikos: If calcium is not there and therefore cell-cell contacts are not forming, the cadherin-PS1 complex falls apart (see our paper in Mol. Cell).

Cweihl: Please explain and do you have any evidence for the trafficking synaptic proteins in particular? Dr. Thinakaran?

Nikos: In the absence of cell-cell contacts PS1 concentrates in the ER-Golgi (see the above paper).

Thinakaran: Is anyone studying second messenger cascade as a possible level of regulation by PS? Studies are under way in Sam Sisodia's and our lab to address the trafficking of proteins.

Nikos: We have already have shown that PS1 is found in vesicles and we have proposed in 1997 (Efthimiopoulos et al.) that PS1 trafficks to the cell-cell surface.

Cweihl: Dr. Robakis please expand on how PS1 association with cadherins alters synaptic transmission and in particular multiple tetanic stimulation paradigms that favor calcium levels?

Nikos: Again the data we have is that PS1 binds directly to E-cadherin and stabilizes the cadherin-catenin complex (paper under review). We don't have data that PS1 mutation affects synaptic transmission.

Thinakaran: The effect on synapse doesn't t necessarily happen in adult. It may be predetermined during development - for example a change in the number of spines and synaptic contacts during development.

Cweihl: Dr. Thinakaran, is there evidence for alterations developmentally in synaptic architecture in FAD patients or mice?

Thinakaran: The story on synaptic transmission is only starting to unfold. I am just throwing new ideas into the discussion.

June: Gopal touches on an issue that has interested me. Namely, how might effects of FAD PS mutations during development contribute to AD pathogenesis in carriers of those mutations?

Thinakaran: June, actually I am being the voice for my wife Dr. Angele Parent, who first published the LTP study on PS mice.

Cweihl: Is there evidence that changes in NOTCH can alter synaptic architecture?

Thinakaran: PS1 must be such a busy protein to be able to bind to cadherins, catenins and about 100 other proteins. Is there any cell-type selectivity of interactors?

Cweihl: Dr. Thinakaran, perhaps these bindings are all transient.

Nikos: (Robakis) Yes it seems that PS1 transiently binds to all these proteins.

Thinakaran: The lateral movement on the plane of the bilayer for a polytopic protein is unlikely to allow "transient" binding to a number of proteins localized throughout the cell - ER, cell surface etc.

Nikos: (Robakis) We have also evidence that PS1 expressing neurons are more resistant to degeneration than neurons with lower levels of PS1 (Giannakopoulos et al. American J. Pathology 1997).

Cweihl: Dr. Robakis does this mean that PS1 is protective and FAD mutations result in loss of function?

Nikos: (Robakis) Our data is on sporadic AD and suggests that PS1 somehow helps neuronal survival. June: Gopal, I'm wondering whether effects of mutant PS1 on Notch processing, for example, might alter neuronal populations or architecture in such a way as to reduce "cognitive reserve."

Thinakaran: June, I think the jury is still out on the issue whether PS mutations influence Notch processing. In our lab it has been inconsistent.

Cweihl: I would like to finally ask one broad question that can be commented upon by all present. Can we identify one unified function for PS1 and its mutations that address all of its multiple roles in synaptic transmission, ER stress, A-beta production, signaling etc.? And how might therapies that are targeted at PS1 disrupt its many putative roles?

Nikos: (Robakis) With regards to cadherin we have experimental evidence that the PS1-cadherin interaction is stronger than the PS1-catenin interaction with respect to detergent extraction

Weiming Xia: I would love to say it is the cleavage by PS1.

Thinakaran: Trafficking

Taisuke: Intra/juxtamembranous cleavage by presenilins.

Imaizumi: Trafficking and ER stress.

Cweihl: So would gamma secretase inhibitors have detrimental effects to synaptic transmission or notch cleavage or IRE1 cleavage?

Thinakaran: Perhaps we can change Presenilin to Polyfunctionalin PF1 and PF2.

Nikos: (Robakis) we cannot identify one unified function for PS1 yet that can can explain its role in Alzheimer's

Thinakaran: Chris, I think that treatments that target PS1 haven't been characterized well in terms of effect on other proteins.

Weiming Xia: DeStrooper and Wolfe showed that gamma secretase inhibitor blocked the Notch cleavage.

Cweihl: Is that a bad thing?

Thinakaran: Maybe the companies are looking at selective inhibitors. Most of the inhibitors are based on gamma-site of APP, so it surprises me that they all also block Notch.

Cweihl: Well it is nearing the hour and I would love final comments by Drs. Thinakaran , Imaizumi, Robakis and Xia.

Weiming Xia: It is not true that [gamma-secretase inhibitors] ALL ALSO block Notch. At least with different IC50.

Thinakaran: I think that cleavage and trafficking may be related functions of PS1. ER degradation in general is a likely area that PS may have a role - this will overlap with all the interactions and functions that we have been discussing.

Nikos: (Robakis) We believe that PS1 participates in the processing and presumably modulates the function of many type 1 receptors. We already know some, including APP, Notch1 and cadherins.

Thinakaran: Is cadherin "processed" by PS?

Nikos: (Robakis) We don't know yet. We are currently studying that.

Weiming Xia: It is interesting to identify additional "co-factors"/substrates involved in the high molecular weight complexes.

Cweihl: Thank you all for participating in this interesting discussion. Any other questions can be posted to the web site and will be directed to the investigator. Thank you again.

Gopal: I'd like to thank June and Chris for arranging this, and all the participants for an interesting discussion. Imaizumi: Thank you for interesting discussion.

Weiming Xia: Thank you, and have a wonderful holiday.

Nikos: Dr. Georgakopoulos represented the Robakis lab. Thank you. Weiming Xia: Gopal, watch out while you are driving....

June: Oh yes, I heard about the snow storm in Chicago. How are things there?

Cweihl: I'm at home.

June: Hurrah for the Internet!

Thinakaran: Me too.

Cweihl: ... and don't plan to leave.

June: By the way, Christian Haass wasn't able to participate today due to technical problems... Good bye for now!

Weiming Xia: My best regards to all of you. See you at the next live discussion.

END

Additional Comments
Kazunori Imaizumi: I would like to comment on differences of data between our and Dr.Thinakaran's labs. Previously, we provided the cells that we used in Nature Cell Biology paper (1,479-485,1999) to Dr.Thinakaran. Using these cells, they examined the induction of GRP78/BiP protein levels ( I think that they should examine the levels of BiP mRNA). They informed me that no significant differences of BiP induction were observed. Although the same cells were used in both labs, results were not consistent with each other. The causes may be due to the differences of experimental conditions (dose, cell types, medium change before stimulation) or sampling RNA or proteins. In these items, I think medium change before treatment with agents is most important because we could not obtain constant data if it was not performed. As we reported previously, the decrease of BiP mRNA induction in cells expressing FAD PS1 mutations is at 30 % compared with the controls. To detect the subtle defects in the UPR, the cells are carefully dealt with under the same conditions.