Posted 6 September 2006

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Participants: Fred van Leeuwen, Elly Hol, Ahmad Salehi, Paul Coleman (moderator), Howard Federoff, Reinko Roelofs, Ruth Perez, Rachel Aronoff, Chris Weihl, Marc Paradis, Jill Weimer, J. Shioi, Keith Crutcher, Kieran Breen.

Note: Transcript has been edited for clarity and accuracy.

Asalehi: Welcome to Ichat about frameshift mutations of beta amyloid precursor protein and ubiquitin-B in AD and DS

Howard Federoff: Good morning (US EST) from Howard Federoff.

Fred van Leeuwen: Hello Howard, How are you?

Asalehi: Greetings from Amsterdam. Is 14.40 here in Amsterdam.

Howard Federoff: I am well thanks Fred, how are you?

Fred van Leeuwen: I am fine, thank you, the traffic is quite slow.

Elly Hol: Good afternoon Paul and Howard. Hi Reinko.

Asalehi: Greetings from Amsterdam Paul.

Howard Federoff: Good day to all.

Fred van Leeuwen: Hello Paul, welcome to the site and thanks for chairing the session.

Fred van Leeuwen: Howard, do you already have a burning question?

Howard Federoff: Fred: I am wondering whether the dinucleotide deleting may be a general phenomena?

Fred van Leeuwen: Yes, we think. As indicated in the Science paper ref.30 we have found similar events outside the brain in a vasopressin transgene. We hope to submit this manuscript very soon.

Paul Coleman: Fred, I am wondering if the +1 immunoreactivity in IHC is limited to cells that are Alz 50 or Bodian or MAC-1 positive.

Fred van Leeuwen: We found colocalization of +1 proteins as a sub population of Alz-50, Bodian and MC1 positive neurons.

Howard Federoff: Is there evidence in other AD-derived material for other mRNA modifications?

Elly Hol: Howard, we are currently checking other AD tissues if +1 proteins are present. Data are in progress.

Howard Federoff: My line of inquiry is more general: Is there evidence that a systematic alteration in mRNA biogenesis exists in AD?

Fred van Leeuwen: We presently do not know. At the moment we are checking whether it is present in more transcripts, indicating that we are dealing with a general mechanism.

Paul Coleman: How do you relate the limited IR of +1 proteins, particularly the ubiquitin, to the more widespread IR for lysosomal enzymes shown by Randy Nixon's group?

Fred van Leeuwen: Good morning Rachel.

Rachel: Hello!

Howard Federoff: Rachel- how are you?

Paul Coleman: Rachel - good to have this chance to chat.

Rachel: Hi, again. It's an early morning, and El Nino isn't over yet.

Rachel: I'm sorry to come in the middle.

Paul Coleman: Actually, you are on time. We just jumped the gun.

Fred van Leeuwen: Ubiquitin is as you know a different protein degradation pathway. How they are exactly related is not yet clear to our knowledge.But can you rephrase the question?

Paul Coleman: Hello Keith.

Howard Federoff: On the subject of a general mechanism: Do you have thoughts as to whether some pressure exerted at the cellular level may account for the propensity of vasopressinergic and AD neurons to undergo nucleotide deletion?

Fred van Leeuwen: Yes we know it is the transcriptional activity as is clear from the Brattleboro rat but also the Down syndrome patients.

Paul Coleman: Hi June.

June Kinoshita: Sorry I'm late.. Hi all!

Paul Coleman: Fred, to try to rephrase the question, I am thinking about where the frameshift editing you describe may fit into the pathological cascade of AD.

Fred van Leeuwen: Whether it is cause or consequence is still under investigation. However it must be a very early event as in Down syndrome patients it precedes neuropathology. More data will be presented at the SFN meeting in LA.

Asalehi: In DS, There is very clear overexpression of APP. which we think is related to the frequency of occurrence of +1.

Fred van Leeuwen: You can fire the next question.

Paul Coleman: I wonder whether it could be an early event in view of the IR being limited to a subset of Alz-50, Bodian, Mc1 positive neurons.

Fred van Leeuwen: Paul, from the cover of Science you can see that more neurons are stained in Vibratome sections. Paraffin is not ideal. But we feel that it starts in a few neurons as seen in non-demented controls.

Paul Coleman: Hello Jill.

Rachel: In the Brattleboro rat, and for the wt rats, frameshifted RNAs appear in the neurons. Is this dependent primarily on age? i.e. have stronger promoter constructs been tested in transgenic animals?

Fred van Leeuwen: Rachel, in rats it is dependent on age. That relation was not found in humans for vasopressin, but the sample might have been too small.

Howard Federoff: Are there any data from transgenic as of yet?

Elly Hol: We have a few transgenic lines and we are currently analyzing them. Chrisw: I was interested if the +1 proteins led to altered distribution of the WT proteins in transfected cells (maybe aggregates).

Elly Hol: About the transfected cells: we do not have any indications that +1 proteins lead to an altered distribution of the WT proteins.

Howard Federoff: Have other post-mitotic tissues from AD patients been examined for deletional editing?

Elly Hol: No, we didn't check other post-mitotic tissues from AD patients.

Rachel: But how highly expressed is vasopressin in rats relative to humans?

Fred van Leeuwen: We do not know, Rachel.

Paul Coleman: I have looked at your web site. Actually, the few neurons that are positive in non-demented controls raises another question in my mind, which is, what constitutes a control in AD studies. We have found that a significant proportion of our so-called control cases (based on clinical studies) are actually Braak Stage I-III on neuropath exam. In the latest consensus statement on the neuropathological diagnosis of AD, the statement is that any evidence of NFT or SP in normals is not normal. So, I guess I am raising the question of whether the +1 IR you see in controls represents the same kind of co-localization you see in AD brain.

Asalehi: Paul, it could be the case. But all the brains here diagnosed for AD are stage IV of Braak.

Fred van Leeuwen: Yes Paul. If the first tangles appear there is +1 immunoreactivity as well. Once again this work was based upon paraffin sections. Using Vibratome sections it might even be seen earlier.

Paul Coleman: Ahmad - that's good. But really my question relates to the presence of NFT in control brains, and whether from a neuropath point of view, these control brains are really early AD, with some cells, the ones with NFT, representing more advanced disease.

Rperez: This is Ruth Perez in Pittsburgh. Hello to all. Do you have an idea why the +1 APP was found in only 50% of the AD patients? Were they sporadic cases or FAD?

Elly Hol: Hi Ruth, All the cases we studied were sporadic cases. The 50% is not correct, if you accumulate all brain areas we looked at is 70%. This is based on immunostainings on paraffin sections. We now prefer to study Vibratome sections, and we are convinced that we will find APP+1 immunoreactivity in almost all AD cases. The Vibratome method is more sensitive.

Howard Federoff: Federoff will return to the mundane. Thanks to all.

Paul Coleman: Fred, I think you have a very important point about Vibratome sections, in that I believe it would be of great interest to the issue of whether the +1 IR is an early event in the pathology of single cells or a later event.

Asalehi: Indeed the controls with some IR +1, were the same with higher numbers of NFTs or plaques, However, they did not reach to that point to be diagnosed as AD.

Chrisw: Is there any evidence that in addition to the increase in +1 proteins that there is an increase in undegraded and perhaps untranslated aberrant mRNA in AD patients suggesting the phenomenon is related aberrant transcription (and the accumulation of junk RNA) and not the +1 proteins?

Elly Hol: Chris, you might be quite right that there is an accumulation of junk RNA or mutated RNA. One of the possibilities is that a system like the 'RNA surveillance' system in yeast is not functioning in AD patients. If this is true than mutated RNA is not degraded anymore and can be translated in aberrant proteins: such as the +1 proteins.

Rperez: Could the accumulation of the +1 proteins be toxic to neurons? Or could the loss of the trophic APP secretion products diminish neuronal survival?

Fred van Leeuwen: +1 proteins could indeed be toxic but it is possibly not acute. See our discussion about for ubiquitin(slow accumulation of aberrant proteins). AD lasts 5-10 years.

Rachel: How does the presenilin story fit with +1 products?

Fred van Leeuwen: The presenilin story will be checked quite soon in brain material from different presenilin families.

Paul Coleman: Fred et al, the reason I am pursuing the line of inquiry that I am following relates to the data from Brad Hyman's lab, as well as data we have, suggesting that the formation of NFT is not necessary to the loss of neurons in AD. If your +1 IR is largely in NFT or PHF + cells, then it might not be a primary factor in neuron loss. It could, of course have other great significance.

Asalehi: Paul, you are right that NFTs may not induce cell death, However, only NFTs and not NPs correlate with the severity of dementia.

Fred van Leeuwen: Paul, Heiko Braak has reported that tangles correlate only with dementia.

Marc_paradis: Hello all - my question concerns the specificity of your finding. Why should a brain (i.e. a large assemblage of anatomically distinct neurons and/or astrocytes) suddenly start producing post-transcriptional +1 mutations only in APP and ubiquitin B? Especially if a similar post-transcriptional mechanism is postulated for ALS?

Elly Hol: Marc, I believe that most certainly not only APP and ubiquitin-B are affected. But we started out with these two.

Chrisw: Do you think that the phenomenon of +1 proteins is a generalized pathology of several neurodegenerative diseases. Have you looked at animal models of AD (APP mice)?

Marc_paradis: Precisely, Chrisw, if +1 proteins are a general phenomenon for neurodegeneration, some preceding disease-specific insult must also exist (as with people who argue a central role for apoptosis). What might this AD-specific +1 insult be?

Chrisw: Marc that is why I was curious about transgenic models have the same phenomenon of +1 IR.

Fred van Leeuwen: Marc, it is probably the common denominator but we don't know what that may be.

Marc_paradis: Speaking of apoptosis, is there evidence that apoptotic cells generate +1 or any other kind of post-transcriptional mutations?

Paul Coleman: Ahmad, I think the bulk of evidence does support that NFT correlate with dementia. However, see a fairly recent paper by Carl Cotman and B. Cummings that indicates that NFT and plaque burden correlate equally well with dementia, depending on the IHC procedures used.

Asalehi: Paul, that is fine, but still it does not rule out that the process of neurofibrillary degeneration plays a major role in AD.

Paul Coleman: Ahmad, I agree. I see synapse loss as really the underlying best correlate presently known, and we have evidence that NFT neurons contribute significantly to synapse loss, not non-NFT neurons. The proportion of synapse loss due to still live NFT neurons and to death of neurons is undoubtedly different in different projection systems, but remains to be determined for any system.

Asalehi: Paul, I agree. But you can add decreased neuronal metabolism as another hallmark of AD.

Msmith: Sorry, I'm late...damn traffic.

{JUNE KINOSHITA} Rachel tells Fred van Leeuwen I am sorry not to be saying much, but this is interesting!

Elly Hol: About the transgenic lines. We tested several transgenic lines (from Karen Hsiao, Staufenbiel, Fred van Leeuwen, Bruce Lamb) with different mutations in the APP molecule and wt APP. None of them detectably produced APP+1.

Chrisw: Elly Hol, that seems contrary since you speculate that high APP levels result in +1 APP reactivity.

Elly Hol: Chris, these mice are healthy and may be able to cope with aberrant RNA. Thus if mutations do occur the RNA, this RNA is efficiently degraded.

Marc_paradis: But then for each of those lines, strong arguments could be made that they are poor models of AD.

Rperez: Since the transgenics did not generate +1 APP, does this suggest that aging plays a role?

Chrisw: or perhaps environmental factors (in regard to the transgenic data)?

Fred van Leeuwen: The APP transgenic mice may bear incomplete constructs (without introns, except those of Bruce Lamb). Thus the environment of the transgene may be important.

Rachel: Did these mutants affect the dinucleotide repeat regions?

Fred van Leeuwen: Rachel, the APP transgenic mice do not have a dinucleotide deletion.

Kieran_breen: Have you looked for +1 APP in any of the PS mutant mice?

Fred van Leeuwen: Kieran, no we did not look in the PS transgenic mice.

Paul Coleman: Marc wants to know what role apoptosis may play in generating +1 or any other post-transcriptional modifications.

Asalehi: Paul, I don't know about the role of apoptosis in generating +1. However, there are not much data supporting the notion that apoptosis may play a major role in AD.

Fred van Leeuwen: Marc's question is a good one. It may be that an inefficiently working ubiquitin system may somehow induce apoptosis. But cell death in AD is only clear in the hippocampus(CA1 and subiculum) , locus coeruleus and the entorhinal cortex. It is not a clear phenomenon for every brain area in AD.

Paul Coleman: Breen asks whether there is anything known about the role of free radicals in generating post-transcriptional modifications.

Fred van Leeuwen: Not known as yet. SOD models may interesting.

Rachel: I wonder if there is an AD transgenic model that everyone agrees on. Does it make APP+1??

Rachel: Also, isn't the hippocampus problem the most important in terms of memory and Alzheimer's?

Paul Coleman: Rachel, I regard your question as one open to all. My opinion is that at present transgenics model very well selected aspects of AD, but I know of no tg that models all currently known aspects, or even all the major pathologies.

Elly Hol: Rachel, the transgenics we tested did not make APP+1. Maybe crossbreeding of several lines, can lead to an improved AD transgenic model. I think Ubiquitin +1 overexpression can lead to problems in the transgenic lines, as it probably will interfere with the proteasomal protein breakdown. Although it remains to be seen if AD-like neuropathology will appear in these mice (plaques and tangles).

Marc_paradis: The other half of the apoptosis question is necrosis, including sick cells which do not commit to the apoptotic pathway. What role might such necrotic cells play in generating post-transcriptional mutations?

Fred van Leeuwen: We have addressed that question in multiple sclerosis patients and did not find any +1 immunoreactivity.

Chrisw: Is there any evidence that mutant transcriptional machinery might be involved and resulting in +1 proteins. Have you looked at RNA polymerase etc? Maybe these proteins are susceptible to environmental factors (free radicals)?

Elly Hol: Chrisw, no we did not look at RNA polymerases. It might be well possible that the transcriptional machinery is involved.

Paul Coleman: Your Science paper refers to data from PD. Have you examined other dementing disorders?

Fred van Leeuwen: Yes we are currently studying Pick's disease, ALS. Multiple system atrophy and Lewy Body Disease. No definite results as yet.

Rperez: Have the Athena PD-APP mice been analyzed?

Fred van Leeuwen: We have asked Karen Chen for these mice but so far we did not receive them.

Msmith: Obvious Q but here goes anyway...any effect of ApoE? Or, have you looked at the ApoE Tg animals yet?

Fred van Leeuwen: As yet we did not look to ApoE Tg animals.

Asalehi: No clear effect of ApoE was found on IR of +1.

Chrisw: Do you speculate a causal link between environmental factors resulting in + IR?

Elly Hol: Everything that increases the speed of aging and the transcriptional activity might result in +1 IR.

Msmith: Tissues aside from brain?

Fred van Leeuwen: We did not yet study tissues outside the brain but see our ref.30 in the Science paper. What about progeria?

Elly Hol: I am sorry...Progeria?????

Msmith: I forget the other names.....disease of premature aging...12 year old kids look like grandfathers/mothers!

June Kinoshita: We've now gone past one hour. Let's let the authors wrap up the remaining questions. Any further questions can be submitted to the news group. Rperez: Thanks to all.

Elly Hol: Oh progeria, Might be very interesting to study. Do you know somebody who can provide us with material of these patients?

Paul Coleman: Sorry I have to leave. This has been most enjoyable. Thank you June for organizing this, and thanks to Fred, Ahmad, Elly and all participants.

Fred van Leeuwen: Thanks for all the interesting questions and feel free to send e-mails.

June Kinoshita: Thank you very much Paul!

Elly Hol: Thanks to you too Paul.

Asalehi: Thanks very much Paul.

Chrisw: Thank you for an excellent discussion.

June Kinoshita: Thank you for the excellent discussion text, Chris! Thank you Fred, Elly and Ahmad. I have to leave now. See you in Amsterdam.

Msmith: Toodle-pip.

Chrisw: Thank you again.

Asalehi: Thank you June. Hope to see you here in Amsterdam.