Posted 29 August 2006

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Live discussion held 9 July 2002, 12 noon-1 p.m EST with Kiminobu Sugaya.

Participants: Kiminobu Sugaya, George C., Peter Soba, June Kinoshita, J. Wesson Ashford

Note: The transcript has been edited for clarity and accuracy.

Kiminobu Sugaya: Well, I guess we can start. Recent progress in stem cell technologies suggests the probability of using neuroreplacement strategies as an AD therapy, but we may have to clear several hurdles. For example,

• Can we replace long-projecting cholinergic neurons?
• Does AD pathology affect stem cell biology? If so, how can we control it?
• Is it a good tactic to make cholinergic cells in vitro then transplant them into the brain just throw these few ideas out to get us started. Anyone got any thoughts.

George C.: It seems that a key question is whether the neuronal loss/damage is due to extracellular factors, which would be expected to damage the stem cell derived-neurons or whether intracellular metabolic effects are responsible for the dementia.

Kiminobu Sugaya: George, I do not think A toxicity is the only factor. We found a direct effect of AbPP on stem cell migration and differentiation.

George C.: How was this demonstrated?

Kiminobu Sugaya: When we apply sAbPP to the stem cells, they differentiate more and, interestingly, more differentiate into glia.

George C.: Is there an effect of Ab on stem cell differentiation in culture? In vivo?

Kiminobu Sugaya: We also found AbPP transfection to the cell causes glial differentiation rather than neural differentiation. In vivo, we are conducting transplantation into AbPP transgenic mice, and will present data in this coming Neuroscience. We have also transplanted the stem cell to AbPP-knockout mice. In this case, they did not migrate well. We have not done any particular study with Ab , though I do not expect cell toxicity.

Peter Soba: But still the question remains, even if neuroreplacement works in the normal brain, does it also work in Alzheimer's brains?

June Kinoshita: How about nontoxic effects, e.g., on migration or differentiation?

Kiminobu Sugaya: Ab may have some effect, which we have to look for in a future study.

June Kinoshita: I saw a poster--I think it was at the Neuroscience meeting last year--by Barbara Tate, showing that stem cells migrated to the sites of Ab injection. It wasn't clear whether this was a response to Ab-induced injury, or whether Ab was acting as a signaling molecule. Could you comment on that?

Kiminobu Sugaya: Yes, it even may be because of the injury caused by the injection.

June Kinoshita: I don't think this effect was seen in the controls, which received sham injections.

Kiminobu Sugaya: Any brain injury increases AbPP expression in the brain, though. Anyway, we are thinking AbPP, not Ab , is the signal molecule for migration of stem cells.

June Kinoshita: Yes, so an interesting question is whether AbPP or an AbPP derivative is part of the brain's normal injury response, and whether this response includes the recruitment of stem cells to the injury site.

Kiminobu Sugaya: I think AbPP is the molecule to initiate migration or differentiation of stem cells.

June Kinoshita: Kiminobu, could you summarize for us the strongest evidence you have to date to support your hypothesis?

Kiminobu Sugaya: Okay.

• sAPP treatment increases differentiation of stem cells in vitro.
• 22C11 suppresses this effect
• High doses of sAPP increase glial differentiation
• AbPP transgene increases glial differentiation
• Stem cells transplanted to AbPP-knockout mice did not migrate or differentiate.

George C.: I believe Mark Mattson and coworkers have evidence in AbPP-transgenic mice that endogenous stem cell activity is depressed. Any evidence for this with transplanted stem cells?

Peter Soba: But still you see an increase in neuron numbers in AbPP-Tg mice.

June Kinoshita: On the surface, the Mattson results and yours would seem to contradict each other. What was the difference between his experiment and yours?

Kiminobu Sugaya: Stem cell activity depressed in AbPP transgenic mice could be caused by the over differentiation to glial cells. Actually, in AbPP transgenic mice, the stem cell population may be decreased by the glial differentiation, and this could happen in the AD brain, too.

June Kinoshita: Just trying to make sure I understand: From what you are saying, AbPP overexpression doesn't decrease stem cell activity per se, but it pushes stem cells towards differentiating into glia rather than neurons. Is that right?

Kiminobu Sugaya: AbPP overexpression just pushes glial differentiation of stem cells, which results in decreased stem cell population. I am not sure if Mattson counted neural differentiation of transplanted stem cells in control and AbPP-transgenic mice, though.

George C.: I don't think Mattson assessed differentiation.

Kiminobu Sugaya: Some Japanese groups reported increased proliferation of stem cells by sAbPP.

George C.: Any ideas on a mechanism for the effect of AbPP on stem cell migration and differentiation? What about endogenous stem cell activity in AbPP null mice?

Kiminobu Sugaya: We have not tested endogenous stem cell migration or differentiation in AbPP-knockout mice yet. It may be interesting to do.

Kiminobu Sugaya: I think brain injury increases AbPP expression. AbPP expression then induces stem cell migration and differentiation to the site. First, the cells would differentiate into glia, such glial cells may induce neuronal differentiation later, or support the neuronally differentiating stem cells.

Peter Soba: But the proliferative effect is only seen when growth factors are present, e.g., EGF, FGF.

Kiminobu Sugaya: Peter, you are right. In our case, we do not use any of the factors in vitro.

Peter Soba: So the effect of sAbPP might depend on which factors are present, and these might work cooperatively.

June Kinoshita: Are there any obvious abnormalities in the brains of AbPP-knockout mice? I was under the impression that there aren't.

Kiminobu Sugaya: There is no obvious phenotype in AbPP-knockout mice. AbPP may be more important for adult neurogenesis rather than corticogenesis.

June Kinoshita: So you would argue for AbPP not playing an important role in differentiation of brain cells during development, or are other proteins, e.g., AbPP homologues, able to step into that role? For example, if one knocked out AbPP and its homologues, how does that affect stem cell differentiation in development? And is that relevant to understanding neurogenesis in an adult brain?

Kiminobu Sugaya: During development AbPP may also work, because we can see increased expression of AbPP at some point. But APLP might be able to compensate for this effect in AbPP knockouts.

Kiminobu Sugaya: In our case, we saw some migration of stem cells to the hippocampus. This may be because APLP is rich in this area. As a matter of fact, if we knock out both AbPP and APLP, we don't get offspring.

June Kinoshita: Kiminobu, in what system did you observe this?

Kiminobu Sugaya: After AbPP-knockout mice transplantation.

J. Wesson Ashford: It seems that some of this might be like saying that the frontal lobes are not important since we can't measure their functions. I would think that we need to determine the function of AbPP carefully. One recent thought is that the two pathways-- - versus -secretase--may be important, but there would still be some balance, though probably less adaptive, without AbPP.

George C.: In general, how reproducible is stem cell migration and differentiation, and how difficult is this to quantify?

Kiminobu Sugaya: Good question, since the stem cell forms spheres, we cannot count the cells inside of the sphere.

June Kinoshita: Is that in vitro or in vivo?

Kiminobu Sugaya: Sorry, this is in vitro.

Kiminobu Sugaya: To overcome this problem, we have to use rather small spheres.

George C.: So you are transplanting whole neurospheres rather than dissociated cells?

Kiminobu Sugaya: Yes, we are transplanting whole spheres.

J. Wesson Ashford: So, Kiminobu, is your inference that AbPP and APLP can cross-cover for each other? It seems that ApoE-knockouts may be relatively normal. Also, there is a related case where the absence of the protein is not clearly bad, probably because others can cross-cover.

Kiminobu Sugaya: Wes, thank you for your input. I think they can cross-cover each other.

June Kinoshita: We need a conditional double or triple APP/APLP1 and 2-knockout, perhaps.

Kiminobu Sugaya: That is a good idea. Anybody have one?

June Kinoshita: Don't know. I'll keep an eye out for one!

Kiminobu Sugaya: I think such a compensation of knockout genes is always a problem with knockout mice models.

George C.: If quantification is difficult, how many mice of each genotype need to be transplanted to demonstrate a difference between null and wildtype mice, for example?

Kiminobu Sugaya: In vivo quantification is rather easy, because we do not see any migration of stem cells to the cortex. We can see the cells in the needle track, though.

George C.: Very interesting.

Kiminobu Sugaya: For the hippocampus, we may be able to count the cells reaching the pyramidal cell line. Lacking migration of stem cells in AbPP knockout mice may also be related to the Ab immunization problem.

J. Wesson Ashford: Kiminobu, I am still not completely happy with the cross-coverage issue. There must be some basic function that the AbPP provides, without which there is some deficit. Otherwise, all we would have to do to solve Alzheimer's disease would be to turn off the AbPP altogether.

George C.: Good point, and tet-on tet-off or cre-LoxP mice should be able to answer this question.

Kiminobu Sugaya: If we turn off AbPP, the stem cell could not migrate. Meaning that there would be no adult neurogenesis.

J. Wesson Ashford: And migration is an active process, probably related to dendrites and axons searching for new connections--neuroplasticity.

June Kinoshita: Sorry, Wes, I'm missing a step here. If AbPP is involved in neural stem cell differentiation, how would shutting it down cure Alzheimer's?

Kiminobu Sugaya: Again, shutting down AbPP causes no neuroplasticity. It should not cure the disease. I guess that is why Ab immunization failed.

June Kinoshita: I think that had more to do with an inflammatory response. There aren't any public data to help us understand what happened there with the Elan trials.

J. Wesson Ashford: My point, June, was that if AbPP-knockout mice were normal, and APLP cross-covered for it adequately to produce an animal without deficits, then it really would not be needed. If shutting down AbPP led to a lack of neuroplasticity, that would be bad for memory but good for AD. But the issue is more complex, I am sure, as Kiminobu is suggesting, probably underlying the reasons that the immunizations failed--about which we need to learn much more.

Peter Soba: What I heard is that there was also loss of synaptic plasticity, June.

June Kinoshita: Wes, that seems paradoxical. Alzheimer's involves a loss of plasticity, so how would even less neuroplasticity help?

Kiminobu Sugaya: June, maybe so, but in the long run, suppressing stem cells could be a big problem.

J. Wesson Ashford: I need to run. Sorry I was late. Very interesting and just getting better. The neuroplasticity question is a longer answer; check our papers and I will talk with you about that answer later. Bye.

Kiminobu Sugaya: Wes's point would be, even if we knock out AbPP, APLP may help to migrate the stem cells. So, I guess it's about the time to close this.

June Kinoshita: Well, there's clearly a lot of exciting work to be done to sort this all out. Let me know if you have any afterthoughts to add to the chat.

June Kinoshita: Kiminobu, thank you very much. Thanks to the audience.

Kiminobu Sugaya: Thank you, everybody. Talk to you later.