Friday, March 20, 1998

Participants: Jinhe Li, Huntington Potter, Marc Paradis, June Kinoshita, Oksana Berezovska, Chris Weihl, MQ Xia 

Marcparadis says, "Hello Jli and June"

June_kinoshita says, "Hi Marc!"

Jli says, "hi mark!"

June_kinoshita says, "Jinhe, where are you these days?"

Jli says, "Hi June! I'm with Pharmacia & Upjohn since Jan."

Marcparadis says, "Hello Hunt"

Marcparadis waves at everyone.

June_kinoshita says, "Hi Hunt!"

Hpotter says, "Hi folks. Jinhe and I are on the phone together, so I may do the answering for both of us"

June_kinoshita says, "That's real multimedia!"

June_kinoshita chuckles in amusement.

Marcparadis laughs hysterically.

June_kinoshita says, "Hello Chris and Oksana."

Cweihl says, "hi June am I late"

June_kinoshita says, "No. We haven't begun yet."

Hpotter nods solemnly.

June_kinoshita says, "No need to be solemn, Hunt. A good time should be had by all."

Marcparadis says, "Nice Monty Python reference June ;-)"

Marcparadis grins evilly.

June_kinoshita bows gracefully.

Marcparadis says, "I am still getting used to these emotions here"

Oksana says, "Hi, June. This is Oksana Berezovska from Brad Hyman's lab. I am still trying to figure out how to use this ichat"

Marcparadis says, " I wouldn't say that I am grinning evilly"

June_kinoshita says, "Glad to "see" you Oksana. Take your time with iChat. It has some quirks, like those adverbs for emotions."

June_kinoshita says, "Why don't we begin. Anyone want to lob the first question?"

Jli says, "I'm ready."

Marcparadis says, "I'm ready as well"

Cweihl says, "Me too"

Jli says, "Ready to take the bullets."

Marcparadis says, "I would like to start by thanking June for giving me the opportunity to review and also to thank Hunt and Jinhe for writing an excellent paper that was fun to read and think about."

Hpotter says, "You did a great job"

June_kinoshita says, "I would like to thank Marc for the wonderful job he did."

Marcparadis says, "I would just start with a question from my review if that is alright?"

Hpotter says, "Sure"

Marcparadis says, "One of the most interesting questions, I thought, concerned your mechanisms for non-disjunction in post-mitotic neurons and how this might cause AD. I would like to hear more of your thoughts on this"

Hpotter says, "We don't actually know how many neurons are totally post-mitotic. As we mentioned in our formal response, there is a fairly large number of new neurons generated in the dentate. However, if we accept the premise that neurons are normally post-mitotic, we have to explain why there are so many mitotic markers in AD neurons."

Jli says, "So, 1-neurons have the potential to divide, 2-there are non-neuronal cells undergoing cell division, 3-the effect may occur during early development in all cells including those in brain."

Cweihl says, "Dr. Potter, I was interested if there is any precedent for a multi-transmembrane protein associating with chromosomes? And serve a role in mitosis?"

Marcparadis says, "Perhaps a very simple question, but I have never heard of this neuronal replacement before. Are there supposedly stem cells that generate the new neurons or do neurons actually make a decision to perform one division at some point in life?"

Hpotter says, "I don't think that is known. Apparently the new neurons just appear in the midst of the old ones. It is not as though there were a region where all the stems cells are and then they migrate as in other regions."

June_kinoshita says, "I believe there are significant numbers of stem cells in adult brain."

Marcparadis says, "If a structure like the dentate can turn over every 6 years, but is only allowed one turnover (because each cell only divides once) what happens after one complete turnover?"

Hpotter says, "I did not mean to imply that each cell would turn over only once. It could turn over many times during the course of a life. But for the sake of the calculation I assumed one turnover per cell. If some cells turned over more than once in a given time and others not at all, then it might take more than 6 years for the entire dentate to turn over."

June_kinoshita says, "Have you been able to look at the number of trisomic neurons in the dentate across different age groups?"

Hpotter says, "I have a question for discussion. What do you folks think of the idea that all of the effects of the APP and presenilin mutations on Abeta production are mediated through their effects on apoptosis?"

Marcparadis says, "I would say that it can't simply be apoptosis, because many stimuli and insults result in apoptosis, but only APP and PS mutations seem to cause AD (ignoring sporadic issues for the moment)"

Hpotter says, "What I mean is that since apoptosis induced by normal means causes increases in Abeta, then maybe it is the apoptosis that the APP and PS mutants induce that leads to the increase in Abeta1-42."

June_kinoshita tells Oksana "I'm curious what you might be observing with respect to presenilin localization..."

Hpotter says, "Marc has a good point"

Marcparadis nods solemnly.

Jli says, "To Cweihl's Q: after looking at so many stainings for a long time, I think PSs are associated with some kind of cytoskeleton protein. And centrosome is the center of the organization of cytoskeleton protein. So they seem to have a chance to get together. But I don't know how, especially for a 7-tm protein."

Oksana says, "In mouse and human tissue we occasionally see the PS1 in nuclei, but it is also localized in the cytoplasm in cell body and processes"

Marcparadis says, "The real test would be other neurologic diseases which cause apoptosis but do not cause AD or AD like symptoms"

Hpotter says, "June: we have tried to use fluorescence in situ hybridization to look at brain sections for trisomy 21. The problem is that the lipofuscin is so strongly fluorescent in aged brain that it obscures the hybridization spots. We will have to try other techniques."

Marcparadis says, "I agree that Cweihl's question is a good one although not impossible given ER and Golgi localization."

Marcparadis says, "Sorry, maybe also nuclear membrane localization too!"

Cweihl says, "In your experiments involving interactions with other proteins. (2 hybrid) what regions of PS are you interested in?"

Hpotter says, "To cweihl: Yes we see cytoplasmic staining in sections of brain tissue too. We discuss this a bit in the formal response. Probably different cells use the presenilins for different things. That might make a neuron that hopes not to divide use the PS proteins for some other membrane-cytoskeleton interactive function such as vesicle transport."

Jli says, "Cweihl'Q--n-term and the loop."

Hpotter says, "Good point about other apoptosis diseases. One possibility is that they do cause some Abeta deposition, as does head trauma, but that if the apoptosis is transient, so will be the Abeta deposits."

Cweihl says, "To me the most compelling evidence of PS and apoptosis is the anti apoptotic nature of ALG-3."

June_kinoshita says, "It's been speculated that apoptosis is involved in all kinds of neurological diseases: ALS, Huntingtons, etc. How convincing is the evidence? I guess I should ask that about Alzheimer's too."

Hpotter says, "Cweihl: Absolutely. Also PS mutants cause apoptosis as do APP mutants"

Jli says, "Maybe apoptosis is not a process specific for any disease, but rather one of the final steps in many diseases."

Marcparadis says, "I would tend to agree with Jli, simply by Occam's razor type arguments"

Cweihl says, "As far as PS. It appears to play a real role in apoptosis with caspase cleavage etc. Whereas APP mutants may just add to the overall sickness of the neurons"

Hpotter says, "June: Apoptosis is hard to be sure about in post-mortem brain because of all the insults that have occurred before you get the tissue. Also some of the techniques for measuring apoptosis are so sensitive that you can easily get false positive results."

Oksana says, "Hi, this is MQ Xia from Brad Hyman's lab, I just jump in the middle of this discussion. We have observed both nuclear staining and cytoplasmic staining of PS-1 in mouse and in human brains using polyclonal antibodies against the N-terminal of PS-1. In human brain, a lot of nuclear staining was observed, particularly granule cells of dentate gyrus. The nuclear staining appears to be related to postmortem interval. This paper is coming out very soon in Journal of Neurological Sciences"

June_kinoshita says, "Very interesting, MQ!"

Marcparadis says, "So perhaps PS1 is "moved" to the nucleus with apoptosis/cell death/post-mortem interval"

Cweihl says, "I am confused. What is post mortem interval?"

Hpotter says, "Has anyone else used commercial antibodies (as we have, with an antibody from SANTA CRUZ ) We found the same nuclear membrane staining in the fibroblasts"

Marcparadis says, "Such movement would be analogous to localization of steroid receptors to the nucleus on binding to steroids. This was actually a possibility that came to mind while reading Jli and

Hpotter's paper, but that I didn't have the space to get into in my review. BTW I would also be interested to hear about commercial antibodies"

Hpotter says, "PMI is the time from death to the time the autopsy is complete and you can get the brain. You can imagine what your tissue culture cells would look like if you left them with no oxygen or glucose for a few hours--lots of death"

Oksana says, "Postmortem interval is the time after death and before the brain was fixed. The same phenomenon was also observed in mouse brains of different postmortem interval. In human, after 12 hrs, PS-1 nuclear staining gets really weak -- sometimes cannot be seen at all."

Marcparadis says, "Throwing out ideas here, perhaps the nuclear localization is related to the caspase cleavage and thereby to cell death/apoptosis?!"

Cweihl says, "I have used Chemicon's rat monoclonal on western blots and Immunohistochem, and it works well."

June_kinoshita says, "Oh, so PS1 staining gets WEAKER with increased PS1."

Marcparadis says, "Sorry, looks like I may have the sequence backwards here, agree with June?"

Jli says, "I'm really happy to hear that from MQ Xia ."

Oksana says, "Sorry , that I forgot to mention that our PS-1 antibody is a rabbit polyclonal to N-terminal 81 amino acid, similar to Dr. Potter's"

Hpotter says, "Actually the antibody was"

Oksana says, "MQ Xia: usually in human brain, after 12 hr PMI, nuclear staining gets weaker or invisible. This was most obviously observed in granule cells of dentate gyrus"

Marcparadis says, "So I did have it backwards."

June_kinoshita says, "Are you suggesting others may have missed seeing the nuclear staining because the PMI was too long?"

Oksana says, "MQXia. Very likely, and the antibody we used was by far the strongest. We seem to prefer polyclonal antibody against longer peptide."

Cweihl says, "Nuclear staining is far different from kinetechore localization during mitosis."

Hpotter says, "That is fascinating. We were happy to see the association with notch and PS expression during development since that would fit with a joint function in cell cycle regulation. Indeed several components of the notch signaling pathway have been shown be involved in cell cycle and in chromosome segregation."

Marcparadis says, "The rapid disappearance post-mortem could be explained by rapid cleavage and processing of the PS, which is consistent with a lot of previous work"

June_kinoshita says, "For Hunt and Jinhe: Did Bruce McEwen's paper indicate whether granule cells were undergoing division more than other types?"

Hpotter says, "J: He only could see it there for technical reasons"

June_kinoshita says, "So is it still an open question whether there is neurogenesis going on in adult animals in other regions of the brain?"

Oksana says, "About PS1/notch colocalization. We have data that they do colocalize both temporally and, anatomically (in the same brain areas) and in the same neurons. It was double fluorescence."

Hpotter says, "The cleavage of PS proteins does seem to be related to some mistreatment of the cells. When we harvest, we put the cells directly into lysis/sample buffer with SDS and see full length protein. Others may trypsinize or harvest by centrifugation that would stress the cells. Maybe that is why so many see only the cleaved forms."

Jli says, "to Xia: Very interesting. Is it coming out soon?"

Hpotter says, "Also Dewji and Singer said that full cleaved PS was only present when the cells were harvested carelessly: PNAS a few months ago"

Cweihl says, "Hunt, is the full length that you see from cell overexpressed or endogenous PS"

Hpotter says, "both"

Oksana says, "Xia: yes, it is in press, if not this month then next month will be likely."

Hpotter says, "What journal?"

Oksana says, "xia: J. neurological sciences."

Cweihl says, "So Hunt do you believe in the cleavage of PS1?"

Hpotter says, "Actually. we think it possible that the cleavage is a part of the normal function of the presenilins. If the proteins function as kinetochore receptors, they need to let go every time the cell enters enaphase. What better way than to be cleaved and stop functioning."

Jli says, "PS1/2 may be cleaved, but not necessarily completely for their function."

Marcparadis says, "That's great stuff Hunt! What kind of cells are you looking at?"

Hpotter says, "Full length in fibroblasts and transfected cos cells, and also fragments"

{PRIVATE} Marcparadis tells Hpotter "Hi Hunt, I would like to talk with you more about how you harvest your cells and look at the PS's. Maybe sometime after this JClub? I could e-mail or call whichever you like."

Cweihl says, "Do you see the cleavage products at all or just full length?"

Jli says, "using both anti-flag and anti-ps abs"

June_kinoshita says, "If you speculate that AD pathology is seen early in hippocampus because of cell division there, how do you account for the spread to other brain regions? Do you think there is cell division also going on at a slower rate, or is there some other mechanism by which dysfunction and death are spreading?"

{PRIVATE} June_kinoshita tells Marcparadis "So you started out pretty skeptical. What do you think of this idea now?"

Hpotter says, "Actually, we are much more inclined to though think that the regional specificity reflects an astrocyte-microglial cell difference coupled to a difference in neuronal cell vulnerability. Both astrocytes and microglia divide throughout adulthood, and we know they are weird in Down syndrome. They could be the most affected by PS mutations causing problems with the cell cycle."

Jli says, "to June: different brain regions or cell types may have different extent of vulnerability to the abeta toxicity. That’s why only some of the regions are affected in AD."

June_kinoshita says, "In what ways are astrocytes and microglia weird in Down's?"

{PRIVATE} Marcparadis tells June_kinoshita "I am less skeptical, but still not convinced. I need to read this neuronal division stuff and pay a bit more attention to Hunt and Jinhe's responses. But overall, he's done a good job of replying and defending"

Hpotter says, "One thing is clear. The cerebellum has amorphous amyloid but not mature amyloid. It also has no expression of the amyloid promoting factor ACT and almost no expression of the ACT and inflammation-inducing cytokine IL-1."

Mqxia enters.

Marcparadis says, "Hello again, Mqxia"

June_kinoshita says, "Welcome back MQ. You're not in drag anymore!"

Hpotter says, "Astrocytes and microglia are spontaneously activated even in fetal brain There are 30 times as many IL-1-positive microglia in DS brain of all ages (Griffin et al , 1989)"

Mqxia says, "Hi, nice to be back here."

June_kinoshita says, "Didn't know that. So inflammation may be important in Down's too?"

Marcparadis says, "Inflammation, I think, plays a role analogous to apoptosis, a final common pathway, not necessarily specific to AD. Of course, this doesn't negate the importance of studying or designing treatments based on inflammation or apoptosis. It just means that you probably won't cure AD that way, just slow it down"

Hpotter says, "Yes, inflammation (which we define in the brain as activated microglia and astrocytes and induced expression of inflammation proteins such as ACT and IL-1) is apparently constitutively activated in DS brain. I would expect that anti-inflammatory drugs would help them [Down syndrome individuals] develop better."

June_kinoshita says, "Interesting. I wonder if anyone is studying that possibility."

Jli says, "Very good point."

Marcparadis says, "Hunt, you should start epidemiological studies of DS mothers with arthritis or some other condition for which they chronically take anti-inflammatories. Their DS children would be expected to have better outcomes... By DS mothers, I mean mothers of children with DS"

Hpotter says, "The only thing is that the inflammation is clearly responsible for expressing ACT and apoE, which appear to be necessary to get Abeta to polymerize efficiently. We have shown this in vitro, but Steve Paul's group at Lilly has shown it for ApoE in vivo."

Hpotter says, "I agree. That would be a good experimnt. No one is trying I don't think."

Marcparadis says, "Well, I'd be happy to help get that experiment going in any way that I can."

Hpotter says, "Lets talk. Maybe a drug company would help."

Jli says, "Let's try it."

June_kinoshita says, "It's a few minutes past 3PM, so I would like to release Hunt and Jinhe of their obligation to stay on line. Of course, everyone is free to continue chatting here. I want to thank Hunt and Jinhe for being here, and Marc for the wonderful discussion text. Our next live journal club is next Thursday, 4-5PM EST, with Rachael Neve, Nick Robakis and Dennis Selkoe. Should be another lively event!"

{PRIVATE} Marcparadis tells Hpotter "I'd love to talk more. One place to start would probably be with the Massachusetts Association for Retarded Citizens"

Jli says, "Thanks to June and Marc."

Marcparadis says, "I would like to say my thanks as well to Hunt, Jinhe and June. As well as to everyone who participated today. I learned quite a bit."

June_kinoshita says, "So did I."

June_kinoshita applauds Marcparadis fervently.

Marcparadis applauds fervently.

Mqxia says, "Thank you very much"

Marcparadis bows gracefully.

June_kinoshita applauds Hpotter fervently.

Hpotter says, "Great. See you all next week at the next roast."

June_kinoshita applauds Jli fervently.

Jli says, "And I am looking forward to your paper, Xia."

Mqxia says, "Applauds fervently"

Hpotter says, "Me too"

Mqxia says, "I can send you a copy"

Marcparadis says, "And general applause, and standing ovation to all!!!! ;-)"

Hpotter says, "Yea"

Hpotter says, "Bye for now."

June_kinoshita says, "I'll say farewell for now. Off to lunch (I'm in California). Thanks again!"

Marcparadis says, "Bye to all, as well"

Jli says, "See you all next time. so long!"

June_kinoshita bows gracefully.

Mqxia says, "Bye bye."