Posted 26 August 2006

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Berislav Zlokovic, University of Rochester, led this live discussion on 23 June 2003.

Participants: Kim Green; Ladislav Volicer, Boston University Medical School; Berislav Zlokovic, University of Rochester Medical Center, New York; Martha E, Stokely University of North Texas Health Sciences Center; Dave Holtzman, Washington University, St. Louis, Missouri; Alexei Koudinov, Peoples Friendship University of Russia, Moscow; Anne Fagan, Washington University in St. Louis; Yasuji Matsuoka, New York University School of Medicine; Jorge Busciglio, University of Connecticut Health Center; Rolf W, Warzok University of Greifswald, Bonn, Germany; Tadafumi Hashimoto, University of Tokyo; Andrea, Case Western Reserve University; Leigh Holcomb, Texas A&M Neuropsychiatry Research Program; Gaku Sakaguchi, Nathan Kline Institute; Dietmar Thal, University of Bonn Medical Center, Germany; Atul Deshpande, University of Connecticut Health Center; Michal Schnaider, Beeri Psychiatry Department at Mount Sinai School of Medicine.

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

Berislav Zlokovic
In principle, Ab-binding agents could be, and sRAGE is part of that group. So the answer is yes.

Dave Holtzman
Berislav, is it known where sRAGE is metabolized systemically after it is injected?

Berislav Zlokovic
The pharmacokinetic studies are in progress; it is likely to be excreted by the kidney and perhaps liver.

Yasuji Matsuoka
Are there any compounds with an affinity to the physiological form of Ab that could be sequesterers?

Berislav Zlokovic
The higher the affinity, the better.

Yasuji Matsuoka
Berislav, any Ab deposition in peripheral organs after treatment?

Berislav Zlokovic
Not observed, but we did not look hard enough into it.

Gabrielle
Perhaps we could address the first question in the backgrounder: What should the next experiments be to advance this potential therapeutic approach?

Berislav Zlokovic
Perhaps to look into high affinity Ab peripheral binding agents that could also reduce inflammation and improve cerebral blood flow (CBF). Possibly further chemical design of the sRAGE molecule to optimize its binding to Ab, and producing smaller molecules, such as V-domains.

Kim Green
As RAGE is transporting Ab into the brain, would this suggest that Ab is being produced in the periphery?

Berislav Zlokovic
Ab can be produced in the periphery, but also during processes that dissolve amyloid plaques from the brain, so a bulk may come from the CNS.

Kim Green
So you would surmise that increased cerebral AbP leads to decreased CBF?

Berislav Zlokovic
Yes, increased Ab decreased CBF. After treatment with sRAGE, the levels go up from normal 50-100 pmol/L to about 1.5 nmol/L, about a 20-fold increase.

Kim Green
Interesting, as I would guess that a decrease in CBF would cause an increase of AbP!

Berislav Zlokovic
Kim, what exactly is AbP?

Kim Green
Amyloid b peptide production.

Berislav Zlokovic
We did not look into it; I was referring to Ab levels.

Alexei Koudinov
Berislav, add (with regard to the possibility of the systemic production of Ab) that we showed some years ago that Ab is produced in HepG2 hepatocytes (Cell Biol Inter, 1997).

Dave Holtzman
How far along are experiments to determine whether RAGE KO x APP transgenic mice exhibit changes in Ab deposition/Ab metabolism?

Berislav Zlokovic
Some of it has been completed, but only in the earlier age group, five to six months, I believe. They do much better on behavioral tasks and develop less Ab. More conclusive studies are in progress with older age groups.

Yasuji Matsuoka
Berislav, what APP mice did you cross with?

Berislav Zlokovic
With RAGE KO. This work has been done with Shid Du Yan and Mark Kindy. The APP was PD-hAPP.

Gabrielle
Berislav, did you do your sRAGE treatments in different mouse models that varied in terms of how much cerebral amyloid angiopathy (CAA) they had? I wonder what your data reveal about CAA.

Berislav Zlokovic
Detailed studies are in progress. In terms of CAA, we showed that sRAGE treatment increases blood flow.

Yasuji Matsuoka
Berislav, any notable amount of peripheral Ab in PDAPP?

Jorge Busciglio
Berislav, I have a basic question: Is RAGE a receptor for the monomeric, oligomeric or fibrillar form of Ab?

Berislav Zlokovic
According to literature, it can bind both fibrillar and monomeric forms.

Dave Holtzman
Berislav, while it seems likely from your paper that the effect on Ab deposition of chronic sRAGE treatment of APP mice over three months is due to a peripheral effect, can you rule out a central effect of the small percentage of sRAGE that crosses into the CNS?

Gabrielle
While Berislav is answering some questions put to him, I was wondering if Yasuji could tell us about ongoing work with small molecules that bind Ab in the periphery. Anything you could tell following your paper on gelsolin?

Yasuji Matsuoka
We are testing other classes of Ab binding agents because chemical modification of gelsolin and GM1 is not realistic. I found a few Ab binding agents that could alter both brain and plasma Ab.

Gabrielle
All: Do receptor-mediated transport mechanisms respond in the same way to peripheral sequestration as passive flow would, i.e., will CNS levels go down if Ab is removed from the peripheral pool?

Berislav Zlokovic
I think that capturing Ab in the periphery will lower the total amount of Ab from the peripheral pool available for exchange with its central pool; this might move Ab from brain to periphery.

Alexei Koudinov
Dr. Zlokovic should be an expert in this subject with his pioneering earlier papers on receptor-mediated Ab transport. I mean receptors related to lipoprotein (LP) transport.

Berislav Zlokovic
Alexei, we believe that LRP-1 is important in elimination from brain.

Tadafumi Hashimoto
Berislav, is LRP-1 the only one?

Alexei Koudinov
...and this Q points to a pre-discussion background point on AD as a systemic disease. If so, the change in the systemic pool of Ab should affect the CNS levels.

Berislav Zlokovic
We found that LRP-1 clusters II and IV bind Ab and its mutants with high affinity. It is probably a major efflux mechanism at the BBB. There may be some other mechanisms, depending on which form Ab is in.

Dietmar Thal
Dr. Zlokovic, what do you think is the role of astrocytes in regulating Ab homoeostasis in the neuropil, especially in regard to their function in the blood brain barrier?

Berislav Zlokovic
Dietmar, I am not sure that the regulatory role of astrocytes in Ab BBB transport is well-understood at this time. Very interesting possibility. Perhaps Dave can make some comments regarding GFAP-ApoE mice.

Dave Holtzman
I think it is likely that ApoE regulates BBB transport of Ab. ApoE is made in astrocytes. Whether astrocytes play a role outside of ApoE and also ApoJ production, I don't think anyone knows.

Gabrielle
Rolf Warzok has worked on the p-glycoprotein (known as the transporter that pumps drugs out of tumor cells), suggesting it may transport Ab out of endothelial cells, as well. Do we know anything about its role vs. LRP? Berislav, all?

Rolf W. Warzok
In our studies we found an inverse correlation between P-glycoprotein and Ab load. Subjects with ApoEE4 had lowest PGP levels.

Gaku Sakaguchi
Does LRP-1 have a sensor machinery for decreasing amyloid in the periphery?

Berislav Zlokovic
Hi, Gaku; yes, it has high affinity to bind Ab in periphery as well, and soluble fragments may act as binding agents.

Dave Holtzman
In regard to Ab in the blood, if it is bound to a large molecule, that should prevent it from reentering the brain unless the large molecule is actively transported into brain. If there are transport systems that "sense" Ab levels in the periphery and somehow respond to that in some way, it is not known, I believe.

Berislav Zlokovic
I agree with Dave.

Gabrielle
Again, while Berislav is busy replying, can I ask Dave and Yasuji one of the questions in the background text? Is serum Ab truly out of the picture once it is bound? Could transport systems "sense" that bound serum Ab is increasing, and reduce Ab efflux in response? If so, peripheral sequestration could lead to an unintended increase of CNS Ab. Have animal models ruled this out? Or is there an error in the thought?

Yasuji Matsuoka
Plasma Ab levels returned to the baseline quickly after treatment with simple Ab binding agents, such as gelsolin, GM1 and new testing compounds. Ab disappeared from the blood in the case of simple Ab binding agents. We are working out the pathway.

Atul Deshpande
Recently there was an article suggesting that astrocytes internalize Ab. Would that be of any significance in decreasing the Ab load in the brain and transporting it across the BBB into the blood? (See ARF related news story).

Dave Holtzman
It would seem that astrocytes have the potential to play a major role in Ab metabolism, since they express many receptors for molecules that bind Ab.

Alexei Koudinov
Dave, that's what I meant by saying earlier that Ab transport regulation is a systemic event, the sensing machinery of transport.

Dave Holtzman
Alexei, I agree with you that it could be a "systemic" event.

Berislav Zlokovic
If astrocytes can resolubilize amyloid and release free Ab, in the presence of a stable efflux system at the BBB, this can be a possibility. I am not aware, however, of any study that has tested this possibility in an animal model.

Gabrielle
Another question for all from the background text: Are there ways of stimulating peripheral degradation of Ab in the liver and the kidney? That would take it out of the equation faster, too.

Berislav Zlokovic
Yes, I totally agree—same as dialysis.

Dietmar Thal
What is the role of ApoE in regard to the drainage of Ab from the brain to the vessels? Do you have an explanation for our finding that capillary Ab deposition is strongly associated to the ApoE4 allele?

Alexei Koudinov
Dietmar, what is your explanation for this event?

Berislav Zlokovic
We are studying details now. In our earlier work (J Neurochem, 1997), we showed that ApoE2 and 3 prevent Ab transport across the BBB, while ApoE4-bound Ab can get into the brain from periphery.

Dietmar Thal
Alexei, our attempt to explain this finding is that Ab-ApoE complexes are less soluble when E4 is present.

Dave Holtzman
It is very interesting that one of the major effects of ApoE on Ab is on CAA (even more than on parenchymal plaques). Perhaps drainage of Ab from brain and/or via the BBB is influenced by the transport/drainage of ApoE out of brain via receptors/heparin sulfate proteoglycans (HSPGs) and others.

Berislav Zlokovic
Dave, I agree; this is an interesting possibility. It could also be related, though, to the level of LRP-1 expression at the BBB.

Leigh Holcomb
Could you explain how HSPG would be involved?

Dave Holtzman
HSPGs are concentrated near vessels, and ApoE binds strongly to HSPG. I agree LRP-1 may also help co-localize ApoE at the BBB.

Berislav Zlokovic
This is also a possibility, as ApoE binds to clusters II and IV as well as Ab.

Gabrielle
Berislav, for the non-expert: What are clusters II and IV?

Berislav Zlokovic
These are soluble forms of LRP-1 receptors part II and IV. (There is VI + cytoplasmic tail.)

Alexei Koudinov
Dietmar, I remember that we discussed while referring to and discussing papers by Berislav that there could be a competition for Ab and ApoE (E2-, 3-, 4-specific) for binding to LPR.

Dietmar Thal
Are there any other pathological alterations in blood vessels, besides CAA, that could be responsible for lowering Ab resorption?

Berislav Zlokovic
Yes, this includes senescence of the vascular system (replicative or stress-induced) and aberrant angiogenesis in response to VEGF and bFGF.

Gabrielle
Berislav, Yasuji, Dave, others, are you trying to find and develop lead compounds on your own, or have companies licensed the drug development piece? I am asking because Alzforum is thinking about what sorts of information resources we could help establish for academic scientists who are pursuing their treatment hypothesis themselves, at least part of the way, to create more validated drug leads. What sorts of information can be hard to find that we could help assemble? Contacts and names of contract research organizations (CROs) who do toxicology and pharmacokinetic studies? Places to purchase compound libraries? Any needs we should think about?

Yasuji Matsuoka
Gabrielle, yes, I am trying.

Rolf W. Warzok
Dietmar, vessels with high Ab have low PGP and vice versa. We never registered a coexpression of Ab and PGP in the same vessels. In other words, only in vessels with no PGP was Ab observed in double staining immunohistochemistry.

Gabrielle
Interesting; so you suspect PGP to be a major exporter? How about LRP? Did you assess that?

Dave Holtzman
If PGP is a major exporter, wouldn't you expect PGP KO mice to have higher levels of Ab in the CNS? Has this been done?

Rolf W. Warzok
So far we haven't looked at LRP. We are just now looking at this in mice.

Berislav Zlokovic
Rolf, we were not able to observe so reproducibly that ABC transporters are associated with CAA using microarray analysis, but in about 50-60 percent of AD cases we also found downregulation of ABA-1 associated with Tangier's disease, and MRP-1 associated protein. We also reported that reduced expression of LRP-1 is associated with increased levels of vascular Ab. The CAA here may also be secondary to senescence, and Ab accumulation secondary to LRP-1 downregulation that is down in senescent endothelium.

Atul Deshpande
In AD, due to inflammation and activation, the BBB is probably compromised; that could also play a role in the entry of Ab into the brain and vice versa.... Just a thought.

Gabrielle
Also, Berislav, do you know the effect of RAGE deficiency on the steady-state endogenous Ab levels in the brain and plasma? Someone asked that in a comment.

Berislav Zlokovic
It's in works currently with Shi Du Yan from Columbia.

Gabrielle
We are nearing the end of the hour. Let me thank Berislav and everyone very much for coming. This looks like a promising line of investigation and we sure hope something comes of it.