Live Discussion: New Theories of Presenilin Function
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Live discussion held on 12 December 2000 and moderated by Chris Weihl.
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).
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
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
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
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?
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?
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.
Imaizumi: I think this activation may be critical for induction of GRP78
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
Cweihl: So there is agreement between your and Thinakaran's paper in
regard to KO lines?
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,
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
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?
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
Cweihl: Dr. Thinakaran, perhaps these bindings are all transient.
Nikos: (Robakis) Yes it seems that PS1 transiently binds to all these
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.
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
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
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
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
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
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.