Posted 31 August 2006

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Participants: Benjamin Wolozin, Luciano D'Adamio, Ed Koo, Mark Smith, Donna McPhie, Soshi, Guest 3, June Kinoshita.

Note: Transcript has been edited for clarity and accuracy.

BWolozin: Okay, I believe that I am moderating this session, so let's Begin.

Eddie Koo: Good morning to everyone. It's still kind of early here.

BWolozin: Luciano, perhaps you could begin by stating your hypothesis.

Luciano: OK Ben. We are studying the role of the three FAD genes in the regulation of programmed cell death. Initially, we isolated a dominant negative form of ALG3 as an inhibitor of Fas-induced apoptosis. ALG3 is lso an inhibitor of the gamma secretase activity mediated by presenilins. The model we propose in this manuscript is that presenillins regulate the sensitivity of cells to apoptosis by controlling the release of the intracellular APP domain, that we have named AID. This biological role of presenilins and APP might underlie the pathogenic mechanisms of AD. In fact, conditions that cause increased APP processing cause AD but also sensitize cells to apoptosis by increasing the amount of AID that is produced.

MSmith: Hello everyone...is Perry on line?

June Kinoshita: No. He's in Italy... must be off having a bottle of Chianti. Just for clarification, is AID a precursor of C31, or is it the same thing called by two names?

Luciano: AID is the intracellular (c-terminal) APP fragment generated by gamma secretase (59 AA long). C31 represents the last 31 amino acids of AID.

Eddie Koo: Any APP C-terminal fragment is a potential precursor of C31. We never tested AID as the precursor, but certainly C99/C100 and C83 are precursors. No reason why AID cannot be but detecting the fragment of AID cleaved by caspase would be exceedingly difficult.

Eddie Koo: Luciano's hypothesis is consistent to what's been proposed by others. We know neurons die in AD but how and why is the $64[K] question.

June Kinoshita: There appears to be a growing family of APP-derived fragments that have toxic effects in vitro.

Eddie Koo: Luciano's results are quite interesting. Between the original results of Bruce Yankner and Rachael Neve, followed by all the other C99/C100 toxicity, our caspase results, and Luciano's AID all suggest that in vitro at least, something funky is going on in the C-terminus.

June Kinoshita: That is, there's more to it than just Aβ?

Eddie Koo: Well, June, that's also part of the equation. Is it just Aβ or more than Aβ. Although Luciano's results are different from ours, neither of our results implicates Aβ directly.

Luciano: Our studies indicate that the processing of APP, and in particular, the fragments released by gamma secretase have the biological role of modulating the threshold of cells to cell death.

Luciano: I agree with Eddie's remarks. Our results indicate that the last 31 C-terminal amino acids of APP are inert with regards to apoptosis. However, something is going with this C-terminal region of APP. The important concept is that gamma secretase can regulate programmed cell death.

MSmith: Luciano.....In vitro or in vivo?

Luciano: Mark, I think in vivo.

June Kinoshita: So this C31 (if I may call it that) fragment doesn't directly interact with an apoptotic pathway, but potentiates it?

Eddie Koo: Our original interpretation of the results is that C31, when formed, potentiates cell death. Luciano's experiments are the first to attempt at repeating our experiments and unfortunately is negative. But the conditions are not the same as ours.

Luciano: I agree with Eddie that the systems are different. We can't exclude that C31 has proapoptotic activity. In our hands it doesn't, while AID is highly toxic. We think that processing of AID by caspases represents a negative feedback mechanism that inhibits the toxicity of AID.

Eddie Koo: I don't understand Luciano's comment about negative feedback. If that's the case, then the AID mutant that cannnot be cleaved by caspase should be altered, which I believe is not.

Luciano: Eddie, the AID mutants (that can't be cleaved by caspases) are, in our hands, always more toxic. We are now attempting to quantitate this difference by using systems other than transient transfection.

June Kinoshita: Is there any evidence to support an in vivo role for C31?

Eddie Koo: We can only see the hallmark of APP caspase cleavage in AD brains.

Soshi: Do any of these toxic peptides occur in vivo at levels that can cause apoptosis? All in vitro experiments [involve] overexpression.

Eddie Koo: I agree with Soshi. The potential achilles heel of all the experiments is the overexpression. Where this fits in physiological levels is totally unknown.

Luciano: Soshi, this is an important question. The problem is that AID has a half-life of only ten minutes, so it is very difficult to quantitate. Besides the experimental differences, we agree with Eddie that the C-terminal APP region has important signalling properties that regulate apoptosis.

Eddie Koo: The one argument against AID in vivo is that in the PS knock in animals, Bob Siman cannot see any cell death. Thus, when more gamma 42 cleavage occurs, it doesn't seem to matter, at least to mouse neurons.

June Kinoshita: What differences might be critical between Eddie's experiments and Luciano's?

Soshi: I think the only toxic peptide known at high levels in brain is β amyloid. What does Dr. Koo think?

MSmith: Is amyloid toxic?

June Kinoshita: There you go again, Mark!

June Kinoshita smiles

Eddie Koo: I'm not really in the Aβ toxicity camp. I cannot believe for that amount of amyloid load in brain, the "toxicity" is really not that substantial in my mind.

Luciano: Yes, Eddie. I think the FAD mutations alter AID production to a measure that is not sufficient to directly kill neurons (otherwise these mutations would not be compatible with life). They might just make neurons more sensitive to apoptotic stimuli, i.e. you might need to trigger apoptosis to see any differences.

Eddie Koo: The key issue is that neurons are dying somehow, regardless of the artificial necrosis or apoptosis distinction. I think Aβ toxicity, if true, is only part of the answer.

Eddie Koo: Back to Bob Siman's animals: he reported that he was unable to kill the neurons with the insults he used. I cannot recall but probably excitotoxicity, ischemia, etc. But the neurons did not appear sensitized. Although Mark Mattson did report some sensitivity in his knock in animals, the changes were subtle.

Luciano: Our data do not exclude a role for Aβ;. They suggest that another APP-derived peptide (AID), which is stoichiometrically produced along with Aβ, is toxic and might participate in the neurotoxicity in AD.

Eddie Koo: I would say Luciano's statement is a good summary and a very reasonable interpretation. The $64K again is which of the several in vitro models are physiologically relevant. And this, I don't think anyone can say yet.

MSmith: Sorry guys..this computer is driving me crazy....would love to stay and discuss the nuances of amyloid toxicity (sic) and rampant apoptotic cell death in AD (double sic!) but cannot see what you are all saying...toodle pip!

Luciano:Eddie: Did Bob Siman's animals show plaques or neurological symptoms?

Eddie Koo: Bob Siman's animals were quite normal. But he recently crossed them to APP knock-in swedish animals and plaques formed early. I would be curious to see what they show when the analysis is completed.

Luciano: Yes. It would also be interesting to see if they now show more sensitivity to apoptosis.

June Kinoshita: Are you looking in human AD brain tissue for these fragments, or are they too transient to be seen postmortem?

Luciano: We reported in our JAD paper the isolation of AID-like peptides from the brains of normal and sporadic AD patients. Postmortem analysis is always tricky. It is difficult to distinguish between cause and effect, that is, pathogenetic mechanisms versus protective responses of the organism

Soshi: If C-terminal fragments are toxic, then why is the Swedish family with a ten-fold increase in gamma secretase ok until there are amyloid deposits? Data seem to support amyloid deposits.

Eddie Koo: But in the swedish mutation, 40 and 42 ratio is the same. So I assume Luciano can argue that there is no preferential increase in the 42 AID form.

Luciano: Soshi, they don't show symptoms till enough neurons are eliminated.

Eddie Koo: Others would argue that it is synapse loss, which precedes neurons loss, that is the early and more important substrate for clinical symptoms.

Soshi: Dr. D'Adamio, where in the cell is GFP AID fragment found?

Luciano: We saw AID everywhere---in all cell compartments.

Ben: Luciano - how might you relate the AID to early synaptic loss?

Luciano: Early synaptic loss could be mediated by a local activation of apoptotic mechanisms

Eddie Koo: With regard to postmortem brain, Andrea LeBlanc reported in D.C. that she sees more caspase activation in AD (I think caspase 6), as we showed with caspase 9. But again, there's smoke but no gun.

Ben: I guess the critical question is developing the right model system.

Luciano: Yes, Ben, you are right.

Eddie Koo: Ben, got any ideas which is the right model?

Luciano: The right model? That's a tricky question. AD is defined by the presence of plaques. Therefore, if you generate the symptoms of AD but not the plaques: would that be considered a relevant AD model ? (I think it should).

Ben: Eddie or Luciano, any thoughts about the relevence of that NGF deprivation model? There certainly would be apoptosis there.

Luciano: Yes, and as we showed together, Ben, it requires presenilins and APP.

Eddie Koo: Gene Johnson has certainly developed that model elegantly. But I don't know whether you can extend what happens in the sympathetic ganglia to the CNS. I don't know how much has been examined in the basal forebrain.

Ben: I guess the thing that strikes me in favor of Aβ is that in disease after disease, aggregation is the major problem.

Luciano: Are you referring to the Danish and British dementia ?

Ben: BDI, Huntington's, Parkinson's, ALS, etc. So MANY diseases have aggregates.

Luciano: Yes, but in which of these diseases have the aggregates been shown to be the cause of the disease ?

Ben: In Huntington's, the aggregate does not cause the disease, but the micro-aggregate (oligomer) seems to bind caspases. In PD, the aggregate is associated with death of dopaminergic neurons.

June Kinoshita: I thought the evidence in Huntington's is pretty strong, at least based on the reversal of symptoms in the mouse models when the expression of the mutant huntingtin is turned off and the inclusions go away.

Soshi: World Alzheimer Congress meeting data suggest that Aβ vaccine makes improvement in memory testing but no effect on Aβ levels - just Aβ aggregation. Please comment.

Ben: The only way to PROVE causation is to induce and reverse the aggregate. Soshi's comment is very appropriate.

Eddie Koo: In this regard, Lennart Mucke's recent paper comparing the APP and APP 717 mutant is quite informative. His group showed that synaptophysin loss can occur without plaques. It's how much 42 that's around. One can also interpret this in favor of Lucian's AID. Back to Ben's earlier statement, there is at least this instance where plaque aggregates are not so abundant.

Luciano: Soshi: In my opinion, there are several possibilities: for example, the polyclonal antibody response may recognize membrane bound APP and affect APP signalling through its cytoplasmic tail.

Ben: Luciano - the membrane bound APP would be intracellular.

June Kinoshita: {public msg} There is also the work from Dean Hartley and Bill Klein suggesting that non-aggregated forms of Aβ may be important.

Soshi: Is Mucke mouse a good model? Mouse already a stupid animal

Eddie Koo: At least in that paper, the stupidity of mice was not an issue.

Ben:Perhaps it was the stupidity of the reviewers!

June Kinoshita laughs hysterically

Luciano: Ours and Eddie's data show toxicity of the c-terminal region of APP. They also show that gamma secretase generates toxic intracellular peptides

Luciano: It is difficult to assume these peptides have no role in AD development.

Luciano: Ben: There is some APP at the cell surface as well.

Ben: Yes, but not the majority. OK Luciano - here is the question. What would constitute proof for AID in your mind?

Luciano: Ben: I think the development of an animal model in which you recapitulate symptoms, intracellular paired helical filaments and neuronal loss.

Ben: Don't the symptoms include plaques? Guest 3, will you identify yourself?

Eddie Koo: Guest 3 must be deep throat.

June Kinoshita: A spy from the NIH?

Ben: Must be Harold Varmus...........Hi Harold!!

Luciano: Behavioral symptoms. Plaques are histopathology. Harold has followed me to New York!!

Ben: But many things mimic the behavioral symptoms. A hippocampal lesion will give that. Even in Harold in NYC.

June Kinoshita: Poor Harold!

June Kinoshita: What about subtler aspects of neuronal dysfunction that precede neuronal death?

Luciano: June, neuronal dysfunction may be the consequence of activation of cell death in peripheral extensions of neurons. Also, it might be the consequence of attempts of cells to block apoptosis.

Eddie Koo: Didn't Dennis Dickson and the Mayo group report the NFT (tau mutant) crossed with the APP mice develop both tangles and palques? I seem to recall something about his saying APP increases the tau pathology but I can't remember precisely. Too much booze at the WAC

Ben: Hmm.....don't remember, but sounds believable. My sense is that many groups are seeing associated memory deficits.

Eddie Koo: Luciano, do you have any idea as to how AID works? What does AID activate? Your bcl-2 effects are similar to ours with C31.

Luciano: We have found several AID interacting proteins. Some of those are good candidates as mediators of AID induced cell death.

Eddie Koo: Luciano, are these interacting proteins different to what's been reported to bind the APP C-terminus? There are a bunch of them, some already hypothesized to be associated with increased cell death, like the G proteins.

Luciano:Yes. We have some new interesting candidates.

Eddie Koo: I need a deep throat in Luciano's lab.

Eddie Koo grins evilly

Luciano: It's the guy who is typing !

mcphie enters

Eddie Koo: Dr. McPhie, do you have any thoughts about this issue.? Rachael [Neve] obviously has worked with C100 for sometime.

June Kinoshita: Donna might want to comment on C-terminus-binding proteins as well.

mcphie leaves

June Kinoshita: Woops, looks like Donna is having problems with her system too.

Luciano: I think another important issue is: how is Aβ and AID generation (i.e. gamma secretase activity) regulated?

Ben: Yes, separate from whether Aβ or AID are the cause of AD, abnormal regulation must be somehow connected to the aging process.

June Kinoshita: So what enzyme cleaves the AID (C59) fragment to make C31?

Luciano: Caspases

Eddie Koo: The assumption from our data is caspase 8 and caspase 9 are required. We don't know which is the actual caspase.

Luciano: This is an important distinction because AID toxicity would imply gamma secretase as a regulator of programmed cell death. Caspases that generate C31 are already known to play a pivotal role in apoptosis.

Ben: Luciano, since PS2 is more closely connected to apoptosis - perhaps PS2 is more important for generating AID during proapoptotic conditions, and PS1 is more important under basal conditions. What do you think?

Luciano: Ben: This is an interesting possibility.

Ben: You should look at AID generation in cells lacking PS1 or cells lacking PS2.

Eddie Koo: So Luciano, have you done this experiment: express C99 in the setting of gamma-secretase inhibitor?

Luciano: We have been unable to obtain gamma secretase inhibitors from Merck. I am sure that Michael Wolfe will be kind enough to give us the reagents!

Eddie Koo: Ironically, in most if not all the studies with toxicity associated with APP C-terminus, none of the results are mutually exclusive. They could all be operating to some degree.

June Kinoshita: I wish Donna were here. I believe she and Rachael have found in their C100 cell cultures that gamma secretase inhibitors led to increased cell death.

Eddie Koo: I own no Merck stocks so you'll have to ask Mike and Dennis [Selkoe]. I am under the impression that Mike's inhibitors are also somewhat toxic.

Luciano: The important thing is that, initially, we cloned a C-terminal fragment of PS2 (ALG-3) as an inhibitor of fas-induced cell death. Now, we know that ALG-3 blocks gamma secretase activity and that's why it inihibits cell death

Ben: I have to run off to another meeting. Ciao!!

Ben bows gracefully

June Kinoshita: Thanks so much, Ben!

June Kinoshita: Any closing remarks?

Eddie Koo: It will be fun to see how the C-terminal toxicity plays out.

Luciano: Yes, June, I think the field should give more space to alternative hypotheses and possibilities. It is difficult to sell at any level data that does not directly supports the Aβ hypothesis and that is unfortunate.

Ben: Hmm.....No great wisdom. I really think the challenge, as always, is to examine the issues in a way that can exclude possibilities (e.g., Aβ toxicity or AID toxicity).

Luciano: I think we have to understand the mechanisms by which AID induces cell death. Second, we need to understand the mechanisms that regulate C99 processing and gamma secretase activity.

June Kinoshita: Thank you for participating today.

Eddie Koo: Gotta run, thanks for organizing this, June and Ben. And to Luciano for an interesting paper.

Ben: Eddie - I agree. Initially I was very enamored with apoptosis, but now I am more enamored with the idea of rust (e.g., cars die because of rust, etc). Ciao!

Luciano: Thanks to everyone for an interesting discussion !!

Ben: Thank you June and thanks Luciano and Eddie and Guest 3...........by Harold!

Luciano: Ben, the rust is due to oxidation.