Mon Sep 29 1997:

June_kinoshita says, "My clock says it's noon. I suggest we wait just a few more minutes for late arrivals. In the mean time, those of us who are logged in are free to introduce ourselves."

Dennis_selkoe says, "May I ask who Skeptic is?"

Therman says, "bye skeptic."

Dennis_selkoe smiles.

Steveyounkin says, "Golde speculates that skeptic may possibly have been Alan Roses."

Nigel_clarke says, "I'm Nigel Clarke, from the Mayo Clinic

Rochester. I work with Steve Younkin. I work on the mass spectrometry of abetas"

Nigel_clarke bows gracefully.

Kieran_breen says, "Hi, I'm Kieran Breen from Dundee and I will be moderating the session."

Keith_crutcher says, "Keith Crutcher here. I was bumped out and had to come up with another password so I was skeptic for a brief period. I still confess to being a bit skeptical regarding amyloid toxicity."

Steveyounkin says, "Golde is wrong one more time!"

Steveyounkin says, "Hello Allison [Goate]"

Steveyounkin says, "Hello Gopal [Thinakaran]"

Thinak says, "Hi Steve"

June_kinoshita says, "Why don't we begin. First, thank you all for coming here. Kieran Breen will be moderating the discussion."

June_kinoshita applauds Kieran_breen fervently.

Kieran_breen says, "Thanks June. Well, I presume that everybody has had a chance to read the presentations. Perhaps I could strart. If both extra- and intracellular A-beta play a role in AD, which do you think occurs first?"

Steveyounkin says, "I would suggest that nucleus formation likely occurs intracellularly because the concentration is most likely higher there. In addition the environment may be more favorable for fibril formation."

Kieran_breen says, "Is there any evidence that an increase in intracellular A-beta results in a secondary build up of the peptide extracellularly?"

Steveyounkin says, "There is no evidence for this, but the experiment has been quite hard to do because of the difficulty of measuring intracellular Aß quantitatively."

Kieran_breen says, "Another possibility is that the extracellular A-beta, especially the amyloid fibrils, may be due to neuronal degeneration - waste from the cells as it were. What do you think of this?"

Steveyounkin says, "I should add that it is possible to get marked increases in intracellular Aß with mutant forms of APP that are retained in the ER without increasing secreted Aß. That, of course, is a very special situation, however."

Kieran_breen says, "That is interesting - do you think that this may be how the presenilin mutants may be acting?"

Dennis_selkoe says, "I've been offline. Where are we?"

Steveyounkin says, "Probably not, because presenilin mutations clearly increase secretion of Aß42"

Steveyounkin says, "Hello George"

Kieran_breen says, "Would anybody else like to join in at this juncture?"

Thinak says, "Steve/Dennis, surely you have examined intracellular abeta 42 in PS cells"

Rneve says, "What about the experiments in which, e.g., intracellular Abeta has been overexpressed in transgenic mice and no phenotype has been detected? How would you interpret these?"

Dennis_selkoe says, "Steve: I think intracellular abeta may well aggregate , but since we see no intracellular aggregates so far and since we do see extracellular aggregates very early in DS and in the mice, long before evidence of neuronal injury, it seems that extracellular accumulation is necessary. What do you think?"

Thinak says, "I agree with Dennis"

Mdparadi says, "Extracellular accumulation may be necessary, but that accumulation may require intracellular aggregation, if I understand Steve's argument"

Steveyounkin says, "I think it may be very difficult to see intracellular aggregates, and that we should be cautious about the apparently negative data obtained to date."

Friedhoff says, "The problem is, intracellular may not be enough. One has to distinguish cytosolic aggregation from aggregation in compartments."

Rneve says, "So you suggest that in the cases in which Abeta has been overexpressed intracellularly, it is released by the cell rather than accumulating in the cell?"

Dennis_selkoe says, "I agree [with Steve], but very sensitive antibodies that readily detect extracellular deposits have not yet seen intracellular material. Could it be that small amounts of intracellular abeta oligomers are released into the extracellular space where they act as a seed for aggregation of the much more dilute extracellular pool?"

Steveyounkin says, "In response to Rachael, I believe it may be that intracellular aggregates are responsible for the behavioral changes seen in Tg mice where there is no overt pathology. Most important, this is a testable possibility."

Kieran_breen says, "It may be interesting to try microinjection and to look for aggregation or even the induction of apoptosis - what do you think?"

Steveyounkin says, "It is entirely possible that seeds form intracellularly are secreted and form the nidus for plaque formation which then proceeds largely in the extracellular space."

Dennis_selkoe says, "I concur with this model. The transgenic mice that only show intracellular abeta production may not have sufficient release from cells to initiate extracellular plaques."

Swbarger says, "Yes, Dr. Younkin, I was initially curious about how you thought the transgenic models make your hypothesis testable? What would be your approach to distinguish intra- and extracellular effects in the mice?"

Mdparadi says, "Why should it even be necessary for the cells to actively secrete oligomers? Perhaps when neurons are damaged (by stroke, anoxia, trauma, etc.) they release these tiny, dangerous oligomers."

Dennis_selkoe says, "Can the moderator ask Steve or me to answer each question in order and no new questions be entered until an old question has been answered?"

Thinak says, "In tg mice with amyloid deposition, there is no evidence for neuronal damage/degeneration; equally conceivable is that abeta 42 acts as a nucleator when bound to ECM molecules like HSPG"

Steveyounkin says, "I would propose to look in young mice where there is no pathology. Then look for changes in cellular Aß defining cellular Aß as Aß that requires detergent for solubilization."

Kieran_breen says, "Yes - it is getting a bit confused. After each question, please let the two speakers answer in turn before a new question is put."

Dennis_selkoe says, "Steve, I agree with your last point about looking for intracellular abeta in young transgenics. We already know that ICC cannot pick up any such aggregates in young mice prior to the first appearance of plaques."

Kieran_breen says, "I have had a suggestion that all questions should be sent privately to me - I will then ask them in order."

Kieran_breen says, "Keith Crutcher asks: doesn't the absence of neuronal death in the transgenic mouse suggest that aggregated amyloid kills neither from inside or outside the cell?"

Steveyounkin says, "Why don't you answer first, Dennis."

Dennis_selkoe says, "Although death has not yet been seen, there is clear evidence of neuritic dystrophy and brisk gliosis. I am not terribly concerned about the current lack of actual neuronal cell body loss. I think there is good evidence that neurons are compromised in these mice, e.g., Karen Hsaio's behavioural changes."

Keith_crutcher nods at Kieran_breen.

Keith_crutcher nods at Dennis_selkoe.

Steveyounkin says, "No. Intracellular Aß could destroy neurites, but have little effect on the number of perikarya. If one can get robust Aß deposition by destruction of a small % of neurites, one would also see very little in terms of loss of synapses initially."

Kieran_breen says, "Cummings asks DS: what kind of neuronal dystrophy?"

Dennis_selkoe says, "Both the PD-APP and the Hsaio mice show abnormal neuritic profiles around abeta plaques with markers such as MAP2 and synaptophysin." vKieran_breen says, "Apart from neuronal cells, do you think that microglial activation may also contribute to the extracellular A-beta?"

Steveyounkin says, "Sounds interesting lets talk."

Dennis_selkoe says, "Shall I answer?"

Steveyounkin says, "Yes"

Dennis_selkoe says, "I think the initial extracellular abeta aggregates (perhaps coming from even earlier intracellular oligomers ) are responsible for activing local microglial cells. These may then initiate an inflammatory and neurotoxic cascade. Perhaps more abeta is then released from the activated microglia. But I don't think activated microglia are there before the first extracellular abeta aggregates."

Cummings says, "With regard to human abeta deposits, activated microglia are certainly present at the earliest stages."

Dennis_selkoe says, "Are they there before the first abeta deposits are detectable, for example, in DS?"

Cummings says, "I'm not certain how to "prove" that activated microglia are responding to a newly, or as yet unformed plaque in DS. But, I do see reactive microglia surrounding complement positive neurons in DS and AD."

Swbarger says, "Yes, Dr. Selkoe, microglia overexpress IL-1 in fetal Down's brains!"

Dennis_selkoe says, "So do you think that IL-1 positive microglia precede any abeta deposits in DS? If so, does this make DS a less than ideal model for AD, since we assume that no such preceding microglial activation occurs in AD subjects?"

Swbarger says, "I don't think we have to assume that. Head injuries precede AD and certainly involve microglial activation, as an example."

Cummings says, "Perhaps activated microglia don't "Precede" abeta, but rather go hand in hand with its release. Afterall, a percentage of diffuse plaques (25%) don't have activated microglia in them."

Dennis_selkoe says, "In PS mutant forms of AD, I assume increased AB42 production, internal oligomerization, release into the extracellular space and plaque formation is what is occurring. Thus, there may be a role for both intra- and extracellular abeta."

Cummings nods

Kieran_breen says, "Rachel Neve asks: have the appropriate controls been carried out for the PD-APP mousew i.e. overexpression of wold type APP in ther same transcriptional unit?"

Dennis_selkoe says, "I don't believe so. What's your concern?"

Rneve says, "I asked Dale Schenk if he saw increased C100 in the neurons of these mice, and he said yes, but he couldn't really interpret it because they didn't have the proper control, which would be overexpression of wild type."

Swbarger says, "Fetal Down's microglial activation was mentioned in Griffin et al PNAS 86: 7611. Subsequently, the actual data were presented at a Neurosci. meeting."

Dennis_selkoe says, "True, but I feel that intracellular accumulation of abeta without plaques is not a close pathological model of AD. So, I am more interested in looking for small amounts of intracellular in the current mice with extracellular deposits."

Steveyounkin says, "Apart from the rapidity of development, I don't see any reason to think that the FUNDAMENTAL mechanisms in PS AD are any different from those in sporadic AD. The central questions for me are (1) is there considerable intracellular Aß that is hard to detect conventionally and (2) can modest amounts of intracellular Aß aggregates impair normal neuronal function, and (3) do intracellular aggregates large enough to see grow rapidly in the intracellular compartment rapidly causing neuritic degeneration so that there are few intracellular aggregates to see."

Rneve says, "I also think it's important to determine whether the mutation in APP or simple overexpression of APP, with or without the mutation, can cause the phenotype seen in the PD-APP mice."

Cummings agrees with Younkin.

Steveyounkin says, "I agree with Rachael, but note that simple overexpression of APP models Down's syndrome."

Rneve says, "Yes, that is what I had in mind."

Dennis_selkoe says, "I agree with both Steve and Rachael, but the central question is can one get typical AD pathology without extracellular abeta deposits? I don't think this has been shown so far."

Dennis_selkoe smiles.

Rneve says, "How do you define "typical AD pathology"?"

Cummings says, "Nice tautology Dennis."

Keith_crutcher says, "I thought typical AD pathology assumes extracellular abeta deposits?"

Keith_crutcher says, "Oh, I get it."

Cummings laughs

Keith_crutcher smiles at Rneve.

Dennis_selkoe says, "The classical Khachaturian criteria: abundant extracellular abeta plaques neuronal loss and tangles. Tangles have not yet been seen in mice , of course, but abnormal tau-reactive neurons have."

Keith_crutcher says, "So the mice have one of the features."

Dennis_selkoe says, "One and a half."

Rneve says, "And not others."

Steveyounkin says, "What I find intriguing is the POSSIBILITY that much of the typical AD pathology is not causing disease. This pathology MAY be an associated rather weak effect with intracellular Aß causing functional impairment independent of the classic pathology."

Rneve says, "I agree with Steve."

Dennis_selkoe says, "Then all those early and abundant extracellular deposits are a sidebar?"

Kieran_breen says, "Cummings asks: Do you all agree with the classical Kachaturian criteria? He doesn't!"

Rneve says, "It's POSSIBLE :-)."

Steveyounkin says, "Probably not, but who knows for sure?"

Dennis_selkoe says, "I agree that they are by no means perfect!"

Cummings says, "Could we agree that the actual pathological criteria for AD ought to be reconsidered and re-evaluated by active scientists?"

Dennis_selkoe says, "Yes!"

Rneve says, "I think that it would be a useful endeavor"

Dennis_selkoe says, "They just have been by Trojanowski's committee."

Rneve says, "Will their criteria be published?"

June_kinoshita says, "You can read their recommendations on the Alzhiemer Research Forum website! (Sorry about getting that plug in.)"

Dennis_selkoe says, "They are in press in.......Neurobiology of Aging with many comments."

Cummings says, "They may have been, but "modern" staining/quantification protocols are still not mandatory."

Dennis_selkoe says, "I agree. This is a shame."

Kieran_breen says, "I think that we are getting into cause and effect here. If the AD pathology as it stands is not a primary event, what are people's views on the key pathological events?"

Cummings agrees with Kieran's point.

Rneve says, "I like the work of Terry and Masliah and their collaborators, in which they emphasize the axonal/synaptic pathology."

Dennis_selkoe says, "I disagree that they are not a primary event. I think abeta accumulation is. The work of Terry relates to downstream events."

Kieran_breen smiles at Cummings.

Cummings agrees vigorously with Dennis.

Rneve says, "Can you expand on that, Dennis? Why do you think those are downstream events?"

Dennis_selkoe grins evilly.

Steveyounkin says, "The classic AD pathology probably is main cause of dementia. It's just that we should be clear that close association does not prove causality. Maybe, intracellular Aß is causing functional problems that have not been appreciated and that we are being pointed to by the behavioral deficits observed in mice with no overt pathology."

Dennis_selkoe says, "Because genetic causes of AD all seem to directly increase abeta deposition. So I assume that neuritic and glial changes follow abeta deposition in at least the known genetic forms of AD."

Cummings says, "As Kieran and others were stating, the REAL issue is what perturbs neuronal function. The neuron doesn't have to be dead to be dysfunctional. Plaques and nasty tangles assume that the only bad thing is a clearly dead thing. Let us modernize!"

Dennis_selkoe says, "Agree. As it is now 1:13 I will be leaving in the next couple of minutes."

Kieran_breen says, "Are there any final questions then, before we wrap up?"

Cummings agrees with Steve's point about behavioral deficits preceding detecting all pathology.

Rneve says, "Dennis, we have a paper coming out in which we show that all FAD APP mutations cause increases in accumulation of intracellular C100 in neurons. In addition, C100-expressing transgenic mice show synaptic/axonal pathology. Just wanted to place those data out there."

Steveyounkin says, "So long Dennis, I too must go soon."

June_kinoshita says, "Thank you so much for participating. A round of applause to Dennis and Steve!"

June_kinoshita applauds fervently.

Dennis_selkoe says, "Rachel: that's very interesting, can you send me a preprint?"

Cummings says, "Thanks to everyone for participating."

Cummings claps

Rneve says, "Sure."

Dennis_selkoe bows gracefully.

Kieran_breen says, "I would like to thank Dennis and Steve for participating today, and to all the audience for a very interesting discussion."

Kieran_breen applauds Dennis_selkoe fervently.

June_kinoshita says, "Thank you Kieran for moderating."

June_kinoshita applauds Kieran_breen fervently.

Kieran_breen says, "Just to remind people of the next discussion tomorrow at 1500 EST - Rachael will discuss the role of the C-terminal of APP"