Posted 5 September 1999

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Live discussion held on Tuesday, 3 August 1999, 12-1 p.m. EST, and moderated by June Kinoshita

Participants: June Kinoshita, Peter Davies, Kun Ping Lu, Mark Smith, Dmitry Goldgaber, Ben Wolozin, Luc Buee.

Note: Transcript has been edited for clarity and accuracy.

June Kinoshita: Hello. Just checking this out.

Peter Davies: Hi June, just checking in.

MSmith: Everyone seems early. Am I in the wrong room?

Peter Davies: This is not the amyloid chat, if that's what you are looking for, Mark.

MSmith: A sick humor...I like that.

Luc Buee: Hi everybody.

June Kinoshita: Luc, why aren't you on vacation like everyone else in France?

Luc Buee: I am the youngest tenure position in the lab. I have to be at the bench.

June Kinoshita: Guest1, identify yourself!

guest1: Alex Osmand.

MSmith: Hi, Alex.

Luc Buee: Shall we start?

June Kinoshita: Where is Ping?

MSmith: Lets get ready to rumble....

Peter Davies: You sound in an aggressive mood today, Mark. Maybe we should send you to the amyloid chat.

June Kinoshita: Well, I think we can begin now. Welcome all! Pete Nelson wanted to join us today, but he couldn't make it. So I'll present the questions from his discussion. First, let's talk about tau's "normal" role in neuronal mitosis. Ping, would you like to respond?

Ccalz: I will try.

June Kinoshita: Take your time. Others are welcome to respond too, as long as we stick to this topic.

Peter Davies: Dmitry and I talked about this just a few hours ago. We don't know where tau is in mitosis, in any cell type

Ccalz: Neuronal mitosis likely occurs in fetal brains and stem cells in adult brain.

Luc Buee: Are we focusing on neuronal mitosis or is it possible to talk about cell mitosis?

June Kinoshita: We can broaden it to other cells. Where IS tau in mitosis?

MSmith: Also, specifically tau or tau phosphorylation?

Luc Buee: I think both aspects are important.

Ccalz: Tau is present in mitotic spindle in normal mitotic cells

Peter Davies: Can anyone cite a paper which shows immunofluorescence of tau in mitosis? (With a really specific antibody: most phosphoantibodies see other MAP's too.)

MSmith: I forget details, but Pope did a lot. Brion also, then there is vague recollection of a European Journal of Cell Science paper on this.

Peter Davies: I'm not convinced that tau has been visualized in mitotic cells at all.

Ccalz: Peter, do you means that your TG3 and other also recognize other MAPs?

Peter Davies: TG3 sees nucleolin as well as tau in mitotic cells. Dmitry showed this very convincingly

Ccalz: I think there are several papers looking at mitotic tau in tau overexpressing cells.

Luc Buee: Tau-1 also labels nucleole.

MSmith.: Do they have any alterations in proliferation? The tau overexpressors, that is.

Peter Davies: I have never seen a convincing double labeled mitotic cell I challenge anyone to show a picture of tau in a dividing cell.

June Kinoshita: We should have a picture contest!

Peter Davies: I agree with June.

MSmith: The challenge is there....let's post on site!

June Kinoshita: OK!

Ccalz: Mark, yes, I believe that they found that tau transient associates with MT.

June Kinoshita: Is the problem that tau antibodies are not specific enough?

Ccalz: How can we solve the problems about tau antibodies?

Peter Davies: There has been a real problem making stably transfected cells expressing decent amounts of tau. It seems they don't divide. We have plenty of good antibodies.

MSmith: Has anyone looked at expression of tau during mitosis?

Peter Davies: It is impossible to do without immunofluorescence

Luc Buee: Yes, during Xenopus oocyte maturation

MSmith: In synchronized cells????

Peter Davies: Where are the pictures, Mark?

MSmith: I am fast, but not that fast....I do not have on hand.

Peter Davies: Poor Dmitry must be going nuts.

dgoldgaber: I can see last entries. Dmitry.

Peter Davies: Welcome!

Ccalz: I remember I did see some papers looking tau-MT interaction in cultured cells.

Peter Davies: It is a critical question. Do tau and Pin1 see each other in a normal cell?

Ccalz: Peter, we have not looked at tau and Pin1 in mitotic cells yet, but will do so.

Peter Davies: I hope you are right, Ping

Ccalz: But I have to add that Pin1 is everywhere in mitotic cells, as expected from its binding a lot of mitotic proteins

June Kinoshita: What types of cells?

Ccalz: June, most of our studies are on HeLa cells and normal fibroblasts

June Kinoshita: Well, now that we've established the dimensions of our ignorance about tau and Pin1 in normal mitosis, let's expound on what these proteins are doing in mature neurons!

Peter Davies: We badly need to know where Pin1 is in normal neurons: our data, Ping, suggests it is nuclear.

Ccalz: My sense is that Pin1 is important for normal neurons to divide at fetal stage, but will be important for keeping postmitotic neurons from entering mitotic stage, which is lethal.

MSmith: What is Pin1 doing in diseased neurons?

Peter Davies: Yes, Mark, although it seems to be in tangles, but we don't know how early it gets there.

Ccalz: I agree with Peter.

June Kinoshita: Which goes awry first in AD: Pin1 or tau?

Peter Davies: I would love to think it's Pin1, but don't have the data

MSmith: Pin seems like some markers that we have been looking at related to the cell cycle, in that normally it's in nucleus but in AD it's in cytoplasm.

Luc Buee: Does Pin-1 always bind its substrates through one unique site?

Peter Davies: We need good icc antibodies for Pin1.

Ccalz: I feel that genetic alterations or other stimuli will somehow trigger neurons to enter cell cycle. This may be first.

MSmith: Maybe I missed it but is it just tangles or other neurons, and also, what about controls (aged)?

Ccalz: Luc, I think that Pin1 will bind and release.

Peter Davies: Only tangles in the cases we looked at, but there's lot's more to do.

Ccalz: Mark, Pin1 is in the nucleus in normal neurons.

June Kinoshita: To follow up on Ping's previous remark, what are the stimuli that are triggering neurons to enter the cell cycle?

Peter Davies: We need to do double labeling with Pin1 and the antibodies that Mark mentioned: cdc2, Cdk4, MC1 etc.

MSmith: June comes in with the easy questions!

Peter Davies: If anyone knows the answer to June's question, please share it.

June Kinoshita: I welcome speculations, the wilder the better!

MSmith: If only NFT, why would you think this is early? Come to think of it, why would you think this is bad?

Luc Buee: Elevated neuronal cdc2 like kinase activity in AD. An article just came out in Neuroscience Research

Peter Davies: What causes cells of the liver, or muscle, or lung, to re-enter the cell cycle? It is usually injury of some sort. But it can be genetic, or environmental, or smoking (Mark?)

dgoldgaber: A recent paper suggests that cyclin D is degraded by proteasome in neuronal cells undergoing differentiation. One can speculate that in AD proteasome is not functioning properly, cyclin D is not degraded, and neurons therefore may be pushed into the cell cycle by cyclin D and other proteins that are important in the initiation of mitosis.

MSmith: This may be stimulus?

MSmith: We are trying to address stimulus by following signaling pathways back to cell surface receptors...its painful

MSmith: Could AD be a cancer?

Peter Davies: If it looks like a duck, quacks like a duck

Ccalz: Ad is arrested cancer

MSmith: See cancer literature.

June Kinoshita: Wasn't this cancer idea proposed decades ago? Why hasn't it taken hold?

Ccalz: Cancer idea may be difficult to sell in most people's mind

Peter Davies: Too much attention on amyloid

MSmith: No!

Ccalz: As more cell cycle markers found in AD, cancer idea may come back.

Luc Buee: Is tau phosphorylation really a major issue in AD?

Peter Davies: To follow Luc's provocative line, the FTDP cases would argue that fooling with tau, as Pin1 seems to be doing, may not be good.

MSmith: Like fooling with amyloid?

Luc Buee: What is abnormal phosphorylation on tau? AT100 or PHF27-ir and phosphorylated Ser422. Is it related to cell cycle?

Ccalz: In normal cells, there are wide ranges of stimuli that can trigger cells to enter cell cycle.

MSmith: Does anyone think the increases in growth factors in AD brain may be responsible? Iqbal had a recent paper showing FGF causes increased tau phosphorylation.

Peter Davies: The Mandelkows show that phosphorylation of serine 214 dissociates tau from microtubules. This site is phosphorylated at only very low levels in the normal human or animal (J. Neurosci, in press).

Luc Buee: FGF binds HSPG, activates MAPK pathway and cell cycle... Why not??

MSmith: Luc, I like it. Activated MAPK is sky high in these neurons.

June Kinoshita: Can you update me on how growth factors are altered in AD?

MSmith: e.g., NGF increased...but, therapeutic trail still initiated I think.

Ccalz: There are many pathways leading to activation of MAP. How about stress?

Luc Buee: SAPK pathway (JNK, p38...)

MSmith: Ping, stress, oxidative stress, that was our interpretation in addition to cell cycle.

Ccalz: Mark, I think both stress and cell cycle can come together.

MSmith: What were results of NGF trial in AD?

Peter Davies: The NGF trial was a disaster in the first patients. No hard evidence of deficits, but it is complicated by the fact that activated glia produce lots of growth factors, and in AD....

MSmith: Perhaps it stimulated more cells into mitosis?

Ccalz: Mark, I agree.

Peter Davies: Whatever NGF did, was not good for the patients.

June Kinoshita: Very interesting. What happened to the patients?

Peter Davies: They were much worse, but only for the duration of treatment. It wore off.

Ccalz: Mark, do you know how stress works?

MSmith: Getting back to the paper, since Pin1 restores MT / tau-P....why MT alteration in AD?

Ccalz: Mark, at early stage, Pin1 can restore the function of tau, but not when it is depleted.

dgoldgaber: Prevention of amyloid depositions by immunization with amyloid peptide and activation of the immune system would also argue for "cancer-like" conditions in AD.

MSmith: That a trial I am NOT looking forward to.

Peter Davies: It may not be a good idea to try to suppress the immune response in AD.

June Kinoshita: Hi Ben! Glad you could make it.

Luc Buee: I do not understand how Ig can cross the BBB in these mice.

BWolozin: Hi June and Mark and Peter!

Peter Davies: Hi Ben.

June Kinoshita: Peter, why do you say it may not be a good idea to suppress immune response in AD?

Peter Davies: Because the immune system might be doing it's best to get rid of the junk accumulating in the AD brain, June

MSmith: But creating havoc in the process?

Ccalz: Does anybody think antibodies to tangles will have any benefit?

Peter Davies: The immune system may respond to more than just amyloid.

Luc Buee: The extracellular junk may be better than the intracellular. Thus inflammation process may not be so good.

dgoldgaber: The immune system probably works very well in people who will have AD (like those who have APP or PS1,2 mutations) but before they have the disease. But with age, like in cancer, it may fail to counteract the effects of the mutations and this may be the start of the disease.

Peter Davies: Dmitry and I think the same way.

MSmith: Did we get to the MT question?

Peter Davies: No: but again, we don't know whether changes in tau produced by Pin1 are good or bad: they may make tau more likely to form tangles.

Ccalz: Mark, Pin1 can restore tau function only at early stage, but would not have any effects, when it is depleted.

BWolozin: Can I ask some questions about pin1?

June Kinoshita: Yes!!

BWolozin: Since pin1 accumulates in tangles, wouldn't that reduce apoptosis?

MSmith smiles

MSmith: AHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGH

BWolozin: Hmm, what does a smile mean?

Peter Davies: Is there apoptosis or mitosis in the AD brain?

Ccalz: Depletion of Pin1 into tangle may in fact induce apoptosis.

Luc Buee: Does Pin1 overexpression induce changes in tau expression?

Peter Davies: Luc: we don't know yet.

Ccalz: I do not know the effect of Pin1 overexpression on tau expression.

June Kinoshita: Mark, you're the apoptosis maven. What's your response to all of the above?

MSmith: Sounds like deja vu all over again......BTW, the noose broke!

BWolozin: OK, fine, forget apoptosis, but if there were mitosis, might you not see dividing stem cells (just to be provocative).

MSmith: You do.

BWolozin: Significantly?

MSmith: Neurons, not just stem cells , can in fact divide (Brewer showed this). He finds 70% in 3 year old rat neurons.

Peter Davies: Lots of stimulation of glial elements, remember.

BWolozin: True, lot's of glial stimulation.

dgoldgaber: Is it known to what molecules Pin1 binds in AD brains?

Peter Davies: Aside from tau, no.

Peter Davies: We don't know what Pin1 is doing in a non-dividing cell.

Ccalz: Pin1 likely binds other phosphoproteins in AD.

Peter Davies: I hope you are right, Ping

Luc Buee: Do we know if Pin1 binds to other phosphoproteins that are also glycosylated (O-GlcNac type)?

Ccalz: We do not know that

Ccalz: Peter, at least we are trying to identify other Pin1-binding proteins in AD.

Peter Davies: Sorry, I have to go. Thanks, everyone. It was fun!

BWolozin: Is there a pin1 knockout?

MSmith: So, Ping, what's next?

Ccalz: Yes there is Pin1 knockout

BWolozin: What is the phenotype in the brain?

Ccalz: We are examining the phenotype of Pin1 knockout in brain.

MSmith: Let's mate it with APP/PS1.

Luc Buee: OK, I am also leaving, Bye everybody, see you in Miami.

Ccalz: Mark, we are trying to determine whether Pin1 levels can alter NT phenotype.

June Kinoshita: The animals are viable?

MSmith: Pin overexpressor transgenic?

Ccalz: Mark, we are making Pin1 overexpressing transgenic.

MSmith: Would love to work on this when available.

Ccalz: Mark, I will let you know.

BWolozin: If pin1 turned on mitosis in neurons, wouldn't you see excess neuronal proliferation in the k/o?

Ccalz: Probably not.

BWolozin: Why?

Ccalz: There are other Pin1-like molecules.

BWolozin: Any idea how other pin1 like molecules respond in the AD brain?

Ccalz: We do not know yet.

June Kinoshita: We've run over the hour, so I would like to thank you all very much for attending.

Ccalz: Thank you, bye.

MSmith: I'll fix the noose for the next round of "Apoptosis and AD."

June Kinoshita: Stop in next week, Friday (13th) for our next live event!

dgoldgaber: Thank you everybody and good bye!

June Kinoshita: Thanks, Dmitry. Ciao!

MSmith : See yer.

June Kinoshita: Ben is a featured panelist next week. Beta catenin and beyond!!

BWolozin: Absolutely - to infinity and beyond. Ciao!