Tuesday, September 30, 1997

June_kinoshita says, "I suggest we wait a few minutes for others to arrive and have a chance to look at Dr. Neve's web page."

Rneve says, "Hello Mark, I'm glad to see you; thanks for "attending"."

June_kinoshita says, "She's put together a very impressive website. We very much appreciate the work you put into it, Rachael!"

June_kinoshita applauds fervently.

Rneve says, "Thanks, June, and of course TVisions was responsible for the beauty of the presentation."

June_kinoshita says, "But content is king (or queen), as they say."

Rneve says, "I see that anonymous left. Who could that have been?"

June_kinoshita says, "Who, indeed? Rather like having a masked stranger appear at the ball..."

Rneve says, "I have a question I could throw out to those who are here. Suppose you do a silver stain of mouse brain and you see what looks like tangles. How do you prove that they are tangles? Are ultrastructural studies necessary?"

Shapirop says, "Not my expertise precisely... but couldn't you also look for phospho-tau epitopes."

Rneve says, "Yes, I was thinking about that, but the hard core pathologists in the field might feel that that is not enough."

Shapirop says, "....but the hardcore NPathologists may not be in the room !"

June_kinoshita says, "But they're out there reviewing for the journals!"

Rneve says, "Yes; well, maybe some will show up and I can have a private chat with them at the end."

June_kinoshita says, "Hi Kieran. Glad you could make it back. I was suggesting that we begin without Mark. His system probably crashed too."

Rneve says, "June is right; you can't leave any loose ends for those pathologist."

Kieran_breen says, "Anyway, perhaps we could start, and our scintillating discussion may attract the pathologists!"

Kieran_breen grins evilly.

Shapirop says, "Dr. Neve; Do you know what the binding site for N-PAK is on C-104 ?"

Rneve says, "I just wanted to say that Dr. Donna McPhie, who did the studies showing that C100 is increased in neurons expressing FAD mutations of APP, is with us, and may want to field questions related to that."

Rneve says, "Right; I'm reasonably sure that it is within the Abeta region. That is based on some solid-phase pull-downs I did with GST fused to Abeta or to C60. I saw binding with the former construct only."

Kieran_breen says, "Rachael/Donna - does full length APP bind to N-PAK as well as C100?"

Rneve says, "Full-length APP binds to N-Pak. In fact, full-length APP seems to bind better to N-Pak than does C100."

Kieran_breen says, "Is there any difference in their ability to activate it?"

Shapirop says, "Fascinating that Abeta is the binding site for N-pak."

Shapirop says, "Does Abeta activate. 40 vs 42 any difference ?"

Rneve says, "Actually, Donna has done the preliminary experiments on that. Remember that these are very preliminary. But we have done two different types of experiments that indicate that C100 inhibits activation of N-Pak."

Rneve says, "The 40 vs. 42 experiment is a good idea. Right now I have only 42, and we haven't tested that for activation, only binding. But I like that experiment and will do that."

Shapirop says, "What sort of kinase is N-Pak ?"

Rneve says, "I'd like to put out on the table that I suspect Abeta, whether 42 or 40, will not have an effect on N-Pak ."

Rneve says, "It's a serine kinase. It's activated by the p21 proteins rac and cdc42."

Shapirop says, "Any evidence for substrates yet ?"

Rneve says, "The binding site on N-Pak for rac and cdc42 is immediately adjacent to the binding site for APP."

Kieran_breen says, "Last night, we had a presentation from Steve Barger who suggested that an APP/PS complex may activate a G-protein-linked cascade. Do you think that the PS proteins play a role in the C100 system?"

Rneve says, "No; everybody who works with PAKs is trying to find their substrates; no clues yet."

Shapirop says, "Any reason to think that Go modulation by APP cytodomain is involved in N-Pak modulation ?"

Rneve says, "I like Steve's emerging story. I think that we definitely need to find a point of convergence with him, and he is probably looking at the same signalling pathway that we are."

Shapirop says, "I guess it is actually part cytodomain and part TM domain that has been reported to modulate Go"

Rneve says, "Go modulation by APP -- yes, I think so. Let me write down why -- just wanted to let you know I'm writing it."

Rneve says, "I think that Nishimoto is correct that APP modulates Go in some way, although I'm not sure that it is mediated through a direct interaction of APP with Go. My working hypothesis is that the interaction of APP with N-Pak modulates the activity state of Go. That's very hard to show, of course -- any ideas?"

Rneve says, "The problem we're facing is that the interaction of APP with N-Pak is probably dependent on ligand binding, and no one knows what the ligand for plasma membrane APP is."

Kieran_breen says, "Is there any evidence for the phosphorylation of Go by N-Pak?"

Rneve says, "Nishimoto mimicked that using the 11C22 antibody is a ligand mimetic, but I would like to identify the ligand itself. We're working on that."

Rneve says, "Are there other situations in which G proteins are phosphorylated? Am I exposing my vast ignorance by asking that?"

Kieran_breen says, "I don't know - I'm not a G-protein person!"

Kieran_breen says, "One possible ligand could be heparin"

Rneve says, "Well, usually they're activated by activating their GTPase activity."

Rneve says, "Yes; and you know, we should try that. It seems that it would be fairly straightforward."

Markmattson says, "One issue that needs to be addressed is the impact of overexpressing the carboxy terminal fragment of APP on the processing of endogenous full-length APP. What is known about this point?"

Rneve says, "Welcome back, Mark. Donna, do we know about processing APP and how it's affected by,e.g., expressing C100 in neurons?"

Dmcphie says, "No since we have not as yet co-infected with C100 and APP"

Rneve says, "My recollection from the fractionation experiments is that excess C100 does not seem to alter the amount of endogenous p10 fragment."

Shapirop says, "Oops. fell off the internet"

Kieran_breen says, "Have you any species-specific antibodies that you could use to examine this?"

Rneve says, "We do know that all known FAD mutations of APP, when expressed in neurons, cause incrreases in intracellular C100."

Shapirop says, "Does N-Pak co-precipitated with APP phosphorylate APP cytodomain?"

Rneve says, "Kieran, that's a good point. We do. 10D5 is pretty good for that."

Rneve says, "That's a difficult question to answer. We don't get robust co-precipitation of N-Pak with APP, although the two proteins clearly interact in the yeast 2-hybrid system. What we can do is look at the phosphorylation state of APP from neuronal cultures overexpressing N-Pak. A person at New England Biolabs is trying to make APP phospho-specific antibodies for this."

Shapirop says, "Are there any inhibitors of N-pak kinase activity - might be a therapeutic target !"

Kieran_breen says, "Has anybody looked at the subcellular distribution of N-Pak - does it interact with APP at the plasma membrane or elsewhere within the cell?"

Rneve says, "Well, our preliminary data suggest that C100 actually inhibits N-Pak kinase activity (although I don't want to be held to that one year from now), so it might be better to think in terms of tying up C100."

Shapirop says, "Good point... you might want to find an activator or ??"

Rneve says, "Donna's done fractionation studies that show strong co-localization of N-Pak with APP in synaptosome fractions. N-Pak is also present in the cytosol."

Rneve says, "We're presently doing immunofluorescence/confocal microscopy looking at subcellular localization of N-Pak and APP in neuronal cultures."

Kieran_breen says, "Do you know if N-Pak translocates, like PKC?"

Rneve says, "One problem I ran into while hypothesizing that plasma membrane APP is ligand-activated is the problem that many people don't see it on the plasma membrane. However, there was a neat paper in J. Neurosci. Res. that showed that acutely dissociated bra"

Rneve says, "Kieran, you're amazing. Donna is working on that. Donna can you tell him your preliminary results?"

Shapirop says, "Have you looked at N-Pak modulation of Ca channel or other Go-modulated effect ?"

Rneve says, "I see that my last comment got cut off: acutely dissociated brain cells expressed APP on their surface; cells from other tissues did not."

Rneve says, "No, we haven't looked at N-Pak modulation of Ca channel or other Go-modulated effect, but that will be important for interfacing with Mark and Steve Barger."

Dmcphie says, "In the perlim results it seems that when we express equal amount of pak with or with out App the membrane fractions that have both App and Pak expressed have more Pak there versus the same fractions with Pak alone expressed"

Rneve says, "Mark, I am aware that you're looking at potassium channels -- we need to look at those, too."

Kieran_breen says, "As it happens, we have also noticed a great disparity of surface APP levels between different cells."

Rneve says, "Did you get the same results vis a vis brain vs. other tissues?"

Kieran_breen says, "Generally, yes, although the higest surface expression appears to be on our friendly AtT20 cells!"

Rneve says, "Really!?! So I should use them for more than our GAP-43 studies...."

Rneve says, "By the way, how did you show surface expression -- by FACS?"

Kieran_breen says, "Frankly, they're more trouble than they are worth!! By immunofluorscence microscopy using non-permeabilised cells."

Rneve says, "You make me really look forward to the experiments we have planned for the AtT20 cells :-)."

Rneve says, "So did you all get what Donna said -- that overexpression of APP in neurons expressing N-Pak seems to cause an increase of membrane-associated N-Pak."

Shapirop says, "... the experiments we have planned for the AtT20 cells :-). Do tell !!!"

Rneve says, "Maybe that is the mechanism by which APP modulates the activation of Go."

Rneve says, "Oh, that's not APP. That's GAP-43. The smile is because I have discovered that everybody who works with AtT20 cells hates them."

Kieran_breen says, "Yes - that sounds very interesting - it would appear that APP activation causes N-Pak binding with parallel translocation."

Rneve says, "Donna should look at Go activation in those same cultures. We need to find out how to do that."

Rneve says, "I'm still waiting for a hard-core neuropathologist to come in."

Shapirop says, "so N-Pak recruitment by APP may activate Go by a Gap-43 dependent mechanism ???"

Rneve says, "Well, that would actually be fantastic. For years I've been looking for a way to bridge the APP and GAP-43 projects!"

Dmcphie says, "We have not looked at Pak and Gap 43 together"

Dmcphie says, "The Gap 43 experiments are a separate project"

Rneve says, "Yes, Donna, let's do it next week!"

Dmcphie says, "OK"

Shapirop says, "but then how does one reconcile Nishimoto's 40-mer peptide and Go activation ?"

Rneve says, "It is true that both GAP and APP, and also N-Pak, are all at the presynaptic terminal."

Shapirop says, "is N-Pak a phosphoprotein ?"

Dmcphie says, "It can undergo autophosphorylation"

Shapirop says, "How do you know it is autophosphorylation ?"

Rneve says, "Well, the 40mer peptide can activate Go without actually binding to it. That's the only point on which I take issue with Ikuo. In other words, the 40mer peptide may interact with N-Pak (I can't remember -- is it in the Abeta domain?) and through that interaction activate Go."

Rneve says, "Donna, you want to answer the autophos questions?"

Dmcphie says, "A number of papers have come on its autophosphorylation as I recall one group even found the key amino acids"

Shapirop says, "proving autophosphorylation is not entirely trivial"

Rneve says, "And you yourself have immunoprecipitated it and activated its autophosphorylation, have you not?"

Dmcphie says, "I see your point and will have to go over the papers again"

Dmcphie says, "yes"

Rneve says, "How _does_ one prove autophosphorylation?"

Dmcphie says, "based on a mobility shift in a protein gel"

Rneve says, "You mean on an immunoblot."

Shapirop says, "mobility shift in a protein gel demonstrates phosphorylation not AUTOphosphorylation"

Shapirop says, "demonstrates POSSIBLE phosphorylation actually"

Kieran_breen says, "Presumably incubation of purified N-Pak with ATP, followed by a gel shift would be pretty good evidence, would it not?"

Shapirop says, "bacteria are kinase poor. incubation of a bacterially-expressed N-pak (with and without active site incapacitated) could serve as proof of AUTOphos"

Rneve says, "That is a good idea."

Rneve says, "The only problem is that N-Pak kills bacteria!"

Shapirop says, "the problem with purified kinases is how do you know there is not a catalytic N-Pak kinase impurity that is undetectable ?"

Rneve says, "That's right; how _do_ you know?"

Shapirop says, "The problem with purified kinases FROM EUKARYOTIC CELLS is how do you know there is not a catalytic N-Pak kinase impurity that is undetectable"

Rneve says, "OK; so you simply would avoid doing the experiment in eukaryotic cells."

Kieran_breen says, "How about a baculovirus system?"

Rneve says, "You're still in eukaryotes, and I think those insect cells have been shown to have lots of kinase activity."

Shapirop says, "yes, baculovirus-infected insect cells have some kinases"

Shapirop says, "I wonder if active-site disabled N-pak kills bacteria ???"

Rneve says, "I believe the autophosphorylation experiments have been done as blot overlays. The N-Pak band lights up when the blot is incubated with P32-ATP and an active p21. What do you think of that?"

Shapirop says, " Very Good :-) Re:The N-Pak band lights up when the blot is incubated with P32-ATP and an active p21. What do you think of that?"

Rneve says, "We were having a lot of trouble expressing N-Pak in yeast, and then Donna made a dominant negative point mutant and had no trouble expressing that in yeast."

Shapirop says, "what makes it dominant negative ?"

Rneve says, "Donna, I don't remember the mutation. Was it at the ATP binding site?"

Dmcphie says, "I believe so"

Kieran_breen says, "Mark, have you any comments at this stage?"

Rneve says, "Is Mark back?"

June_kinoshita says, "I just called Mark. His computer crashed again. I think he may not come back, given the time. Sorry!"

Shapirop says, "Is there any idea of what the concensus phosphorylation site is for N-Pak ?"

Rneve says, "Donna would know that."

Dmcphie says, "Yes but I would need to look it up"

June_kinoshita says, "Gosh, it's already 4. We're supposed to have a closing "party," but this is more fun. Should we "party" here?"

Kieran_breen says, "Yes, why not!"

Rneve says, "Where's the party supposed to be?"

Shapirop says, "is the concensus site in the literature. i know little about these kinases."

Rneve says, "Maybe there will be a hard-core pathologist at the party."

Rneve says, "Yes, the consensus site is in the literature."

June_kinoshita says, "The party is supposed to be in the virtual lounge, but I don't see anyone there. So we might as well stay here."

Rneve says, "The Pak field is moving EXTREMELY rapidly, and we have all we can do to keep up."

Dmcphie says, "Yes the consensus site is published I could e-mail you the reference later"

Rneve says, "Maybe we should post a sign in the lounge asking neuropathologists to come to the lecture hall :-)."

Shapirop says, "I could e-mail you later . thanks. shapirop@ohsu.edu"

Rneve says, "My brother is at ohsu; do you know Kim?"

June_kinoshita says, "Well, I just tried to get someone over at MGH, but he's in clinic."

June_kinoshita says, "A neuropathologist."

Rneve says, "Yes, it would be good to pull in Brad."

Rneve says, "Let's page him."

June_kinoshita says, "Hah! good luck."

Shapirop says, "well. i do know Kim by face and all. don't know him well."

Rneve says, "He's a dopamine pharmacologist, probably not your field :-)."

Shapirop says, "True. dopamine is not my bag... and i have to stay focussed !!"

Rneve says, "I am curious, mjcwrl. Who are you?"

Mjcwrl says, "I am Woan-Ru Lin in UMIST, Manchester, UK"

Rneve says, "Thanks for identifying yourself! It's always nice to know who I'm talking with."

Mjcwrl says, "Sorry I am not in the amyloid side, so I cannot ask many questions"

Rneve says, "Are you on the tangle side? (Rachael says with hope)"

Mjcwrl says, "Nope. I am doing aetiology- the role of virus and AD"

June_kinoshita says, "HSV1 and AD!"

Mjcwrl says, "But we are involved in some studies about amyloid."

Rneve says, "That's very interesting! What have you found in that area? HSV1! Haven't you published on that? I work with HSV1 as a gene delivery vector...."

Mjcwrl says, "Yeah in the Lancet this February. We found that HSV1 is a risk for AD"

Rneve says, "I'll check it out. Really! For sporadic AD?"

Mjcwrl says, "Yeah. We found that the combination of ApoE4 and HSV1 in the brain is the risk for late onset AD"

Rneve says, "Do you think it could be due to chronic inflammation during HSV flare-ups?"

Mjcwrl says, "It is a possibility."

June_kinoshita says, "See poster 8.2 when you have a chance."

Shapirop says, "Thank you for the exciting/stimulating conversation. shapirop@ohsu.edu will check out now. (need to work on my typing ;-))."

Rneve says, "When you say HSV1 in the brain, do you mean in the PNS? Thanks, June; I'll check it out."

Mjcwrl says, "thanks june"

Mjcwrl says, "It is both actually. IN PNS, apoE4 are likely to suffer from cold sores. In CNS, apoE4 carrier are likely to suffer from AD"

Rneve says, "That's _extremely_ interesting. I will visit your poster. Thanks for telling me about it."

Mjcwrl says, "Thanks for your interests"

Kieran_breen says, "Before everybody suddenly leaves, I would like to thank Rachael and Donna for taking part this evening."

Kieran_breen applauds fervently.

Dmcphie says, "Thanks to everyone for many new , interesting ideas"

Rneve says, "It was a pleasure. Donna is going to take speed typing :-)."

June_kinoshita applauds fervently.

Mjcwrl says, "Thanks June for this interesting conference"

June_kinoshita says, "It was Kieran's idea originally."

Dmcphie says, "yes thank-you June!"

Rneve says, "Yes, June, you and all the presenters clearly put an enormous amount of work into it."

Kieran_breen says, "Also, before we finish, could we have some feedback on the conference. What are people's views"

Dmcphie says, "I thought it was a great way to discusss topics with people in a number of locations in real time"

Rneve says, "I enjoyed it a lot because I love conferences but hate traveling. But it seems that a lot of people need the traveling."

June_kinoshita says, "Clearly, the technology was a stumbling block for many."

Mjcwrl says, "I think that it is a very good idea to have this kind of conference. Maybe someone can maintain a web site like this."

June_kinoshita says, "We're already doing it!"

June_kinoshita smiles.

Rneve says, "I personally would love to see a trend towards internet conferences. End the Society for Neuroscience zoo!"

June_kinoshita says, "Yes! Although people do like to get together for drinks..."

Kieran_breen says, "Funny that you should mention that! I used to be involved in an amyloid discussion group, but audiences fell away. Do you think that these should be tried again?"

June_kinoshita says, "I'd like to see them revived."

Mjcwrl says, "The only inconvinence is that we are in the different time schedule. So we missed quite a few discussions""

Rneve says, "Oops, a person I'm to meet with has arrived. I shall have to sign off. But on the amyloid discussion group question: I was disappointed that most people would not reveal unpublished data. I can just go to the library."

June_kinoshita says, "I think it's crucial to have exciting topics, high-profile speakers and lots of publicity."

Kieran_breen says, "June, maybe you and I could liaise on this aspect over the next few months."

June_kinoshita says, "Yes. I want to do a thorough post mortem so we can keep improving."

Kieran_breen says, "Unfortunately, I won't be at neuroscience, so we'll have to continue to meet virtually!"

June_kinoshita says, "Among other things, we should send out a questionnaire."

Mjcwrl says, "When will be the next internet conference"

June_kinoshita says, "How did this meeting compare with the one you organized previously?"

Kieran_breen says, "Actually, we should have a breakfast in Amstrdam next year, like we did at Osaka, to get people to discuss the format of 1999"

June_kinoshita says, "The idea was to have the next one in 1999. They would alternate with the International Conference."

Mjcwrl says, "I will be there."

June_kinoshita says, "A breakfast in Amsterdam is a good idea. We'll have time to plan this time."

Kieran_breen says, "With regards to the previous meeting, which was on Glycobiology, it compared well. The ichat was a good idea."

June_kinoshita says, "Thanks for your support, Dr. Lin."

Mjcwrl says, "Thanks I like this conference very much."

June_kinoshita says, "Now that we have ichat, we can build up a community of researchers who can use it. I'd like to incorporate it into the on-line journal clubs and other on-line forums."

Mjcwrl says, "maybe you can try internet phone next time."

June_kinoshita says, "Just to keep people on their toes!"

Mjcwrl says, "Then we can hear the speaker talking"

Kieran_breen says, "Yes, if we organise regular (?monthly) sessions, by the time 1999 comes around, everybody will have had plenty of time to come to terms with it."

June_kinoshita says, "Dr. Lin suggests audio. It would be nice, but it's yet more technology for people to cope with."

Mjcwrl says, "Yeah the connection is not perfect yet."

June_kinoshita says, "You are more than welcome to hold the Amyloid discussion group meetings at the Alzheimer Research Forum."

Kieran_breen says, "Yes, I have found that the fear of new technology is a barrier for a lot of people - its difficult to believe that we are all high-tech scientists!"

Kieran_breen laughs hysterically.

June_kinoshita sobs.

June_kinoshita says, "I tried to help Nick Robakis set up ichat over the phone. It was really something!"

Mjcwrl says, "Congratulations to you again. I got to go now. See you sometime in the future."

June_kinoshita says, "He has Windows, which as a Mac devotee I know nothing about. Talk about the blind leading the blind."

June_kinoshita says, "Thanks for coming! See you in some future chatroom!"

Kieran_breen says, "Yes, I have to go as well. Thanks for everything June. I'll be in touch over the next few days to discuss the future."

June_kinoshita says, "OK. Talk to you later. Thanks for everything, Kieran."

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