Summary

Ashley Bush led this live discussion on 15 January 2004. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Transcript:

Ashley Bush led this live discussion on 15 January 2004.

Participants: Ashley Bush, Massachusetts General Hospital, Charlestown, Massachusetts; Gabrielle Strobel, ARF; Anatol Kontush, Hôpital de la Pitié; Diane Lacombe, Neurochem Inc., Montreal; Alexei Koudinov, Neurobiology of Lipids; Ed Pores, Long Island Alzheimer's Foundation; Jeanne Loring, Burnham Institute, La Jolla, California; Marc Paradis, MIT; Roger Streit, West Orange, New Jersey; Edward Zamrini, Birmingham ADRC.

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

Gabrielle Strobel
I am Gabrielle Strobel, managing editor of the Alzforum, and glad to moderate today.

Jeanne Loring
Jeanne Loring from the Burnham Institute in La Jolla. Expecting 70 F today!

Roger Streit
Snowed in—West Orange, New Jersey.

Diane Lacombe
Diane Lacombe from Neurochem Inc., Montreal, expecting −23 C.

Marc Paradis
Hi, Ash!

Ashley Bush
Hi, Marc, hi, everyone…I feel like this is my bar mitzvah!

Marc Paradis
Nobody's going to give you any checks, though!

Ashley Bush
No, just pens.

Marc Paradis
I might be convinced to do the Hava Negila, though.

Alexei Koudinov
Hello to everybody. I first thought I was late… because did not make earlier calculation of across-Atlantic time difference. Fortunately, I was early.

Ashley Bush
Must be late there…you're in Moscow?

Alexei Koudinov
In Israel.

Anatol Kontush
Yes, I am here to talk to Dr. Bush.

Alexei Koudinov
Thanks, Anatol, for coming. And thanks to Ashley for quoting Dr. Kontush in the background text.

Ashley Bush
Yes, Alexei; in fact, one of our future interests is the metals in HDL, referring to your work. I am particularly interested in the SOD activity of HDLs.

Alexei Koudinov
Everything gets in one complex—an even more interesting/puzzling picture.

Ashley Bush
Exactly—a lot of people do not realize that SOD activities exist outside of SOD1, but in HDLs you see SOD, ApoE, APP, Aβ, cholesterol….

Alexei Koudinov
Yes, lipoproteins (LPs) seem to do the most work in buffering oxidative vulnerability.

Ashley Bush
I agree. That's probably why HDLs are good for you….

Alexei Koudinov
…and possibly oxidative stress modulation when there is an urgent need for it.

Ashley Bush
So, Anatol showed how Aβ can form favorable complexes with Cu in CSF. In CSF there is only HDL as LP.

Alexei Koudinov
Similarly, our explanation of LPs serving a reservoir for immediate access for cholesterol during synaptic activity-dependent rearrangements.

Anatol Kontush
HDL is really a multifunctional particle which carries such different proteins as apolipoprotein A-I, paraoxonase, PAF-AH, LCAT, and at least three of them are present not only in plasma, but also in brain, and all can protect against oxidation.

Alexei Koudinov
I would mention—a totally different story—the work I was involved in a decade ago at NYUMC, when, with Prof Nussenzweig, we showed that HDL helps to kill Trypanosome brucei (African sleeping sickness). As for CSF, you are right; HDL seems to be the only particle normally present there, though there is a particle size/chemical heterogeneity.

Ed Pores
Dr. Bush, what are the side effects from clioquinol? Also, when and where will phase III drug trials take place?

Ashley Bush
Ed, in the Archives of Neurology paper we reported that the side effects were not remarkable. Previously, clioquinol (CQ) was allegedly associated with a neurological syndrome that resembled B12 deficiency. As an antibiotic, CQ was generally well-tolerated. As we pointed out at the end of the CQ paper in Archives, 10 patients (more now) have been on the drug for over 18 months or longer with no side effects.

Gabrielle Strobel
Ashley, have you gotten any signals from the FDA regarding clioquinols' approvability? For example, thalidomide eventually was reapproved with restrictions, after a hard struggle and for conditions that have no good treatment.

Ashley Bush
The FDA is being conservative, but encouraging. They have given us good guidance and feedback. The phase 2B trial has been designed considering FDA input.

Gabrielle Strobel
Ashley, could vitamin B12 be formulated into a clioquinol pill to lessen the need for monitoring?

Ashley Bush
B12 can indeed simply be put into the capsule.

Anatol Kontush
I have another question related to CQ and its side effects. What happens to Aβ after it is extracted from the plaques by CQ? Where does it go and could it cause side effects?

Roger Streit
I usually look to the Life Extension Foundation for information on health issues. Their website (LEF.org) mentions a long list of possible alternative treatments for Alzheimer's: phosphatidylcholine (lecithin), pantothenic acid, vitamin E, ginkgo biloba, vitamin C, acetyl-L-carnitine, coenzyme Q10, N-acetyl-cysteine, flavonoids, curcumin, essential fatty acids, DHA, EPA, GLA, vitamin B12, folate, SAMe, phosphatidylserine, inositol, vitamin K, idebenone, melatonin, music therapy, tryptophan, DHEA, vitamins B1 and B6, carnosine. Have you communicated with the Life Extension Foundation? Any comments on their list?

Ashley Bush
Generally, I can see merit in considering many of these approaches. However, we need clinical trial data.

Gabrielle Strobel
Roger, I believe many of these drugs, while perhaps useful, don't target the underlying pathogenic process.

Ashley Bush
Idebenone was recently reported to be ineffective (marginal).

[Editor's Note: See Thal et al., 2003]

Roger Streit
Who is going to pay for clinical trial data on supplements where no one has a patent?

Ashley Bush
Good question. Sometimes the NIH will fund a trial, as, for example, for vitamins E and C.

Gabrielle Strobel
Roger, NIA, at least, funds some preventive trials on over-the-counter NSAIDs.

Roger Streit
Gabrielle, that's a start.

Gabrielle Strobel
Ashley, you've encountered many misconceptions about your approach. Do you want to take this opportunity to clarify the one or two you find the most serious?

Alexei Koudinov
Good suggestion, Gabrielle, especially after having WSJ article describing Ashley's story.

Ashley Bush
Misconceptions: that exposure to zinc and copper is the problem, that chelation therapy is the intervention we study. To summarize, the brain has plenty of its own zinc and copper. Zn is released during neurotransmission. Somehow, this zinc gets harnessed by soluble Aβ leading to its precipitation. The Aβ, we believe, is traveling in the extracellular spaces carrying copper ions. That may be its function.

Anatol Kontush
Does anybody know whether Zn is released in a free or bound form?

Ashley Bush
Zn is released in an ionic "free" form.

Gabrielle Strobel
Being a copper transporter may be its physiological function?

Ashley Bush
Well, APP knockouts have elevated Cu in the brain, and APP transgenic animals have lowered Cu in the brain.

Roger Streit
Ashley, regarding chelation, I had heard that NIH was studying chelation (not necessarily for Alzheimer's). Anything going on there?

Ashley Bush
Roger, I think NIH is looking at chelation for heart disease.

Anatol Kontush
Yes, it is ethylenediamine tetra acetic acid (EDTA) for cardiovascular diseases.

Ashley Bush
What we are trying to do is pass the blood-brain barrier.

Gabrielle Strobel
Ashley, for the record, can you explain to everyone the difference between chelation therapy, and why you are not proposing that, and the specific version you are pursuing?

Ashley Bush
We use specific small molecules that are targeting only the metals on Aβ (in principle).

Alexei Koudinov
I do not remember the explanation of the zinc-based staining for the mossy fibers in the hippocampus, the pathway we found to be especially sensitive to the cholesterol (lipoprotein or cyclodextrine dynamics increase) mediated neurodegeneration (tau phosphorylation/neurofilament fragmentation).

Ashley Bush
Mossy fibers are the richest "zincaminergic" pathway. They represent the zinc that is stored for passage into the bouton. This is fluorescent and represents 15-30 percent of brain zinc.

Alexei Koudinov
Great story to think about and explore (APP transgenic/knockouts and Cu). Nice link, again, on the mossy fiber pathway specificity, cholesterol (and presumably lipoprotein) and Zn dependency.

Ashley Bush
There will be interactions with cholesterol for certain.

Gabrielle Strobel
Were the trial results strong enough that Prana is continuing to develop clioquinol for FDA approval? Their website mentions a second-generation compound. I wonder whether that is where the energy and funds are going. Can you tell us? (see ARF related news story on the clioquinol trial).

Ashley Bush
The academic community is developing clioquinol. Prana is developing new, safer, more selective drugs. However, like Cognex (remember the precursor to Aricept) clioquinol shows promise, and Sam Gandy and Barry Rovner have applied for a phase 2B trial based at Jefferson in Pennsylvania.

Gabrielle Strobel
Good to hear it. How about Duke?

Ashley Bush
It will be multicenter, but I am not certain about Duke.

Gabrielle Strobel
How do you explain the different effects of clioquinol on CSF Aβ42 levels and ADAS scores? Why did the drug seem to slow cognitive decline in severe AD but had no effect on plasma Aβ?

Ashley Bush
Gabrielle, firstly, it is a small study, so the ADAS-cog scores are too noisy to draw firm conclusions unless the difference is very conspicuous. Keep in mind that we had 18 AD patients and 18 controls (N=18 + 18). For something like Aricept, the similar phase 2 trial used N=200 + 200. Therefore, we need to see what the ADAS will do on a larger scale, hopefully the phase 2B. Regarding plasma Aβ, no one has done a longitudinal study on a monthly basis yet, like ours. We know that from CSF data Aβ levels go up and then down (profoundly down) probably in the late phase of AD. The plaques suck up the available soluble Aβ.

Gabrielle Strobel
I see. Pretty often though, phase 2 results prove to have been outlier effects when retested in a phase 3 with a larger, more diverse group of patients. The effects here were only visible upon subgroup analysis to begin with, which weakens statistical power. Is it misplaced skepticism to be wary of that?

Ashley Bush
Gabrielle, I agree we have to be conservative. The phase 2B will have larger N (40 + 40), and PET imaging. Of course, we did subgroup analysis, since this was the first study of its kind, and we were also doing dose finding. The trial was a promising first step, and indicates that we should keep going, in my opinion.

Alexei Koudinov
Good luck to you and Prana on this project. Ashley, can you give some specific values: From-to? For CSF, I mean roughly.

Ashley Bush
Alexei, one thing we learnt was that the individual variation in Aβ levels is great, and you can reduce variance by referring each individual to their own baseline reading.

Alexei Koudinov
You are right. I was just finishing a review and referenced the article where this point was highlighted—an individual patient variability.

Anatol Kontush
For me, lowering of plasma Aβ by CQ was the most questionable point, since low plasma Aβ is actually used to ensure the presence of AD, together with elevated tau. Are you sure that your patients were at the early stage when Aβ is (still) high?

Ashley Bush
Back to plasma Aβ: Colin Masters makes the point that late in AD, perhaps the plasma Aβ levels drop.

Gabrielle Strobel
Oh, and since you did spinal taps anyhow, could the 2B trial measure clioquinol's effect on oxidative markers?

Anatol Kontush
Should this initial increase in Aβ be regarded as a response to something like oxidative stress? Which is followed by peptide accumulation and drop in its plasma levels?

Ashley Bush
Anatol, I think so. I like this model.

Anatol Kontush
Ashley, it is logical, but direct data which support it are lacking.

Ashley Bush
Indirect evidence is there. APP translation is regulated by iron (Rogers et al., 2002).

Alexei Koudinov
Anatol, what is the basis of your concept of Aβ/oxidative stress causation in AD? I thought others should be interested to integrate it with Ashley's concept?

Ashley Bush
Regarding our plasma Aβ data—at least we have on record now data from nine months' sequential readings in two severities of AD.

Edward Zamrini
Did you measure oxidative markers?

Ashley Bush
No oxidative markers…on my wish list.

Gabrielle Strobel
What happens to the Aβ after clioquinol pries the metals away from it? Do you have data showing how it gets cleared? For example, data about the proportion that is locally degraded by enzymes, like IDE, and the portion that's transported into plasma and degraded in liver/kidney?

Ashley Bush
Aβ looks like it's being degraded. Not certain yet how it gets degraded…but presumably passes into the blood to the liver. Also, local degradation is possible.

Gabrielle Strobel
Mostly in brain, by IDE and neprilysin, or mostly in periphery? I am confused by that. Is this an important open question?

Anatol Kontush
Aβ can be expected to immediately bind to something like HDL due to its hydrophobic nature. And HDL's apolipoprotein A-I (AI) can cross the blood-brain barrier (BBB), so it could end up in the liver.

Ashley Bush
Note that IDE is a Zn-dependent protease.

Gabrielle Strobel
Does this imply clioquinol would inhibit Aβ degradation?

Ashley Bush
Much of the APP proteolytic pathway is regulated by zinc proteases. So yes, CQ could potentially inhibit degradation. It would be a dose-dependent pleiotropic effect. At [the Society for] Neuroscience [meeting], an Israeli company, D-Pharm, reported that CQ and similar molecules inhibited TNF-α generation, for example. So one must be careful with the α-secretase cut, as well.

Gabrielle Strobel
That would be good, right? Elevated TNF α is a risk factor for AD, if a minor one.

Ashley Bush
 Correct.

Alexei Koudinov
I remember this presentation by DPharm. If I am not mistaken, it was authored together with some Korean company.

Ashley Bush
DPharm's work was in collaboration with Jae Koh at the University of Ulsan College of Medicine in Seoul.

Gabrielle Strobel
You can find a summary of some of Koh's work in the Alzforum news section.

Alexei Koudinov
In the CSF AD patients, we noticed a change in Aβ distribution across HDL subspecies with more Aβ in HDL2 and HDL1 particles (contrasting with the normal case where it is mostly in HDL3 and VHDL). We explained it in terms of cholesterol changes, a need to manage affected cholesterol dynamics, and greater cholesterol removal, with Aβ as a functional part of it (as we showed earlier, it affects cholesterol esterification). But in light of today's discussion, HDL could manage Aβ clearance.

Anatol Kontush
Wouldn't inhibition of Aβ degradation by CQ be inconsistent with a drop in its plasma level?

Ashley Bush
True. We think that we got the dose correct, and the net effect is clearance and degradation.

Gabrielle Strobel
Why did copper levels not change in the treated people? Did you expect them to? I thought clioquinol binds copper as well as zinc.

Ashley Bush
Brain levels of Cu are about 10 percent those of zinc. We have found that the ZnT3 system that loads zinc into synaptic vesicles adds about 10-20 percent of the zinc in plasma.

Ashley Bush
We think the zinc gets trapped in the amyloid mass in the brain. When the amyloid mass breaks up, the zinc is restored to circulation.

Anatol Kontush
Why did Cu not get trapped?

Ashley Bush
Copper levels are lower in the brain, and brain copper cannot be appreciated in plasma in the same way as zinc.

Gabrielle Strobel
I understand about the copper measurement. Thanks.

Alexei Koudinov
Regarding the regulation of APP processing by the pathway favored by Ashley and Anatol, I would suggest that the APP/secretases processing is dependent on cholesterol. It seems that we are returning to the starting point of today's chat: The complexity that may well have functional importance, and duplication of safeguards to protect neuronal function when either cholesterol or oxidative stress goes wrong.

Gabrielle Strobel
On breaking up the amyloid mass, Ashley: A criticism of your work is that dissolving Aβ deposits will "release" soluble Aβ and make more of it available to form toxic oligomers? I know you see this differently and think this may be an old view. Do you want to correct it here?

Ashley Bush
I am one of the workers proposing that soluble Aβ forms mediate toxicity, but CQ neutralizes soluble Aβ toxicity by shutting down hydrogen peroxide production mediated by copper.

Gabrielle Strobel
Yes, but breaking up the amyloid mass would release more oligomers. Don't you think that metals are needed even for the oligomers to form?

Ashley Bush
Oligomers form by a number of means, e.g., ADDLs, e.g., SDS labile oligomers, etc.

Anatol Kontush
Does it mean that soluble Aβ oligos do not contain Cu but rather other metals (Zn)? Can you imagine Aβ aggregation in the absence of metals?

Ashley Bush
The data so far suggest that Aβ will form oxidatively modified soluble oligomers induced by copper.

Alexei Koudinov
Ashley, Anatol, does your model allow plaque reversibility? I ask because it was shown/suggested to be the case with the reversal of amyloid in animals fed a cholesterol diet, and then returned to a normal diet (the change that should bring brain cholesterol to "normal" after a prolonged period of time.

Ashley Bush
Cholesterol is a substrate for peroxide formation by Aβ:Cu complexes (Opazo, 2002). Aβ bound to copper forms a nasty catalytic complex that converts cholesterol into hydrogen peroxide. In terms of activity, Aβ42 is stronger than Aβ40, and human Aβ is more potent than rat.

Gabrielle Strobel
Regarding copper, can I ask about contradictory evidence out there? Cappai, Sparks, other labs have shown it is harmful, and you have worked on it extensively. But how about the Phinney/Westaway and Bayer/Multhaup PNAS papers of last month? They say increasing brain copper levels protects against amyloid toxicity and prolongs life in transgenic mice. Can you explain? Or do you think this is a diversion, irrelevant to your main point? Sorry, I am confused.

Ashley Bush
Regarding the Phinney and Bayer papers, we have data that agrees with them. Also, CQ raises brain Cu and Zn levels (Cherny, 2001). In transgenic mice, CQ takes from the rich (e.g., plaques) and gives to the poor (e.g., neurons). It seems that when the cell senses a high intracellular-to-extracellular copper ratio, it decreases Aβ generation.

Alexei Koudinov
Gabrielle, regarding Aβ release to form oligomers, I should mention the issue raised earlier by Anatol, and elsewhere by us: that Aβ-to-LP association is a critical matter that oligomer studies should address. In a proper close-to-physiological condition, Aβ may well have preference to bind to LPs than to self-aggregate.

Gabrielle Strobel
I see, Alexei. Ashley, we need you to sort out the chemistry behind this and design a diagram or graphic flow chart so others can get their minds around this. I'd love to post something like that on Alzforum!

Ashley Bush
Gabrielle, sure thing—it is a complex model. It is the extracellular copper that is the problem for Aβ. But AD is a complex disease. Look how complex heart disease is, for example.

Gabrielle Strobel
I know. I think some people get lost because there's a lot of chemistry behind your hypothesis. But the chemistry is of key importance, and I'd be pleased to use the Web to help display it.

Ashley Bush
Absolutely. The key word is pleiotropy. Copper and Aβ are functionally related and dysfunctionally related. One hypothesis is that Aβ is geared to bind one atom of copper. However, if it binds another atom at a low affinity site, it [Aβ] gets oxidized, and leaves its correct cellular compartment, i.e., the membrane. Most of the Aβ in the normal brain is in the membrane fraction. So how come it gets elevated as soluble Aβ in AD? Kevin Barnham published a nice paper recently showing that Cu causes methionine 35 oxidation and that causes release of the membrane-tethered Aβ (see Barnham et al., 2003). I suspect that that is step one. The soluble, oxidized Aβ oligomerizes (??ADDLs) and drifts away extracellularly.

Gabrielle Strobel
I fear in my exuberance I have silenced some here who may have much better questions and thoughts. Let me invite everyone expressly to ask their favorite questions, and I'll stay mum for a while.

Anatol Kontush
I do not think we can talk about "free" Aβ. It is so hydrophobic that it aggregates immediately or binds to lipids.

Ashley Bush
Or enters the synapse and is precipitated by zinc. Guys—remember, you can add a chelator to brain homogenate and dissolve the amyloid.

Anatol Kontush
 Exactly!

Ashley Bush
It doesn't behave like a hydrophobic aggregate in postmortem brain. It is reversible with chelation. Even in vitro.

Anatol Kontush
Sure, but it is never "free."

Ashley Bush
I agree, not really normally free. That's where we started—it is found close to lipids. Plenty of access to cholesterol.

Anatol Kontush
A component of HDL that is functionally critical, as is HDL.

Ashley Bush
Good hypothesis.

Alexei Koudinov
I agree on membrane binding of Aβ to membranes; see our SFN 03 abstract.

Anatol Kontush
We should finally accept that it is an apolipoprotein whose function is to bind metals, for example.

Ashley Bush
Also interesting.

Anatol Kontush
Alexei proposed this about a decade ago.

Alexei Koudinov
We later added to this proposition by showing that Aβ may also have a function in lipid (particularly cholesterol) metabolism, as other apolipoproteins (by definition) do; see ARF hypothesis page.

Anatol Kontush
Or maybe it functions as SOD, as you proposed, Ashley?

Ashley Bush
Note that ApoE is a Zn/Cu metalloprotein, as well. In the HDL there is clear SOD activity, implying plenty of Cu. We published the SOD structure of Aβ in JBC (see Curtain et al., 2001). But we see function, too (unpublished).

Anatol Kontush
Has somebody ever measured HDL-bound Cu? I mean in vivo, because in vitro it binds a lot—we checked it.

Ashley Bush
Cu in HDL has not been measured, to my knowledge. But SOD activity implies Cu presence. By the way, I see the HDL as normally clearing Aβ to the liver.

Anatol Kontush
Ashley, it is very possible, but HDL has many other functions which are consistent with permanent presence of Aβ in the particle.

Ashley Bush
It is a doable study.

Alexei Koudinov
Adding to the interrelation of oxidative stress and cholesterol: As was shown by Sparks a decade ago, cholesterol feeding changes SOD activity in rabbit brain (see ARF related news story on Cu and induction of AD in rabbits).

Ashley Bush
I agree; I think in AD, the HDL "drops" it[s] Aβ into extracellular fluids. But not in healthy brains. There was a fascinating paper in the American Journal of Pathology recently that showed that APP-transgenic mice get aortic vascular changes, implying that Aβ causes the mis-metabolism of cholesterol and provokes peripheral vascular disease.

Anatol Kontush
HDL should be extremely altered in APP-transgenic animals both in CSF and plasma.

Ashley Bush
I agree.

Anatol Kontush
And this can lead to both atherosclerosis and AD.

Ashley Bush
Is there data on HDL in transgenic animals?

Anatol Kontush
It normally carries one Aβ per 100 HDL particles, but in APP-transgenics it could be 100-fold higher. The point is that Aβ is considered to be "free" by those who work with transgenics. And they do not bother about HDL.

Alexei Koudinov
Very possible, again, returning to HDL in the CSF of AD patients: Aβ gets bound to ApoE and ApoJ. In normal CSF, we did not see this interaction even under crosslinking. This may well be related to thermodynamic preference for Aβ to get off HDL and aggregate.

Ashley Bush
The transgenics massively overproduce Aβ, so that may overwhelm the clearance mechanism, e.g., no room left on HDLs, leading to elevated "free" Aβ.

Anatol Kontush
I completely agree; you just overload the particle with something you need, but not that much, and this something just diffuses away to make "free," i.e., aggregated (?) Aβ.

Ashley Bush
Makes sense. Then the liberated Aβ reacts with Cu and Zn.

Alexei Koudinov
Remember Aβ depositions in cardiovascular pathology, data from a decade ago.

Ashley Bush
When you drop the Zn levels (ZnT3 knockouts), it can still escape from the brain. The "free" Aβ diffuses and I do not think it can aggregate in the absence of Cu or Zn. Every plaque is full of Zn.

Anatol Kontush
Yes, Cu and/or Zn precipitate Aβ.

Alexei Koudinov
…Or the particle properties can be changed, by lipid metabolism changes (one possibility that of especial interest to us) or lipoprotein-peroxidation/oxidative stress changes (another possibility, arrayed by Anatol/Ashley), forcing Aβ to dissociate from the particle, self-aggregate, or bind to other apolipoproteins (also detected in plaques, along with cholesterol).

Anatol Kontush
Yes, peroxidation can provide metals to aggregate Aβ.

Ashley Bush
Mice and rats do not normally get amyloid deposits because their Aβ does not precipitate or react as much with Zn and Cu.

Anatol Kontush
The difference between mice and humans explains why the vaccine does not work. But it is too complex.

Ashley Bush
Looks like we are drawing to a close.

Anatol Kontush
You are right; we need to close.

Ashley Bush
Anyone with any further questions is welcome to e-mail me at bush@helix.mgh.harvard.edu.

Anatol Kontush
AD is complex, but I guess we should at the very least finish by clearly saying that Aβ possesses a physiologically important function and is not a biological waste, as still many think.

Ashley Bush
I completely agree.

Anatol Kontush
Thank you, Ashley, it was great talking to you.

Ashley Bush
Well, it's been a hoot, folks. Time to get back to my igloo in Boston.

Alexei Koudinov
Remember several APP mice that failed to show brain Aβ? The models that succeeded have mutated Aβ, that may well have physicochemistry different from non-mutated Aβ, having less affinity to HDL, and possibly creating a systematic research error in the major mouse models for AD.

Ashley Bush
Okay. Great talking to you all. Ciao. Ashley.

 

Background

Background Text
By Ashley Bush, Massachusetts General Hospital, Boston, Massachusetts

 

Introduction

Long considered a fringe approach in the field of Alzheimerology, the attempt to treat AD by targeting specific metal-Aβ complexes reached a clinical milestone last year when a phase 2 trial was completed, (see ARF related news story). The historical timeline below, by Ashley Bush, lays out how this approach developed over the past 20 years.

1984: Zn2+ is released in an exchangeable ionic form in neocortical glutamatergic. synapses. This pool of zinc represents about 15-30 percent of brain zinc.(1,2)

1988: Cu2+ released by synaptic transmission.(3)

1992: Proteolytic processing of APP by a zinc metalloenzyme.(4) Subsequently, zinc-metalloproteinases have been identified as α-secretases,(5) and of these activities, tumor necrosis factor α converting enzyme (TACE) and ADAM-10(6) appear to be responsible for the majority of α-secretase activity in cell culture. Also, occupation of Aβ by Zn(II) protects the substrate from α-secretase-like cleavage,(7) hence the effects of Zn2+ upon APP processing are pleiotropic.

1993: Zinc binding site on cysteine-rich ectodomain of APP and APLP1 and 2.(8,9) Promotes heparin binding, and inhibits oxidation induced by Cu2+ in the ectodomain binding site.(10,11)

1994: Zinc forms amyloid from human but not rat Aβ in vitro.(7,12)
1994: Cu2+ binding site in the cysteine-rich N-terminus of APP.(13)

1996: Cu2+ binding site in the cysteine-rich N-terminus of APP reduces to Cu+.(10)

1997: Zn2+-induced amyloid aggregation is dependent upon -helical structure, and is reversible with chelation.(14)

1998: Copper and iron precipitate Aβ (Aβ42>Aβ40>>ratAβ) under conditions representing mild acidosis; Co induces precipitation analogous to Zn, Ni analogous to Cu in vitro. Only Cu and Zn are released in an exchangeable form at the synapse.(15)

1998: Zinc, iron and copper are highly enriched in Aβ plaques,(16) later found to actually bind to Aβ.(17,18)

1999: Aβ is highly redox active (Aβ42>Aβ40>>ratAβ) reduces copper and iron, generates hydrogen peroxide from oxygen, which mediates toxicity in cell culture.(19,20)
1999: Amyloid can be resolubilized from human brain using zinc and copper: selective chelators. (21)
1999: APP knockouts have increased copper levels.(22)

2000: The copper binding site on Aβ42 has attomolar affinity (Aβ40 has pM affinity), making it among the strongest affinity copper binding sites in nature, akin to superoxide dismutase 1.(23)
2000: Trace metal contamination is responsible for initiating Aβ nucleation in the seeding of Aβ fibril formation. Aβ fibrils do not form in the absence of metals.(23)
2000: Cu2+ selectively oxidizes Aβ leading to oligomeric species,(23) and other modifications such as methionine sulfoxide Aβ, which becomes released from lipid membranes.(24)

2001: Aβ may act as a physiological antioxidant in CSF and plasma lipoproteins, functioning by chelating transition metal ions.(25,26)
2001: The copper/zinc binding site on Aβ resembles that of SOD, confirming the possibility that Aβ might be an antioxidant. The structure is a membrane-embedded hexamer with histidine/metal bridges.(27)
2001: 50 percent decrease in amyloid accumulation in APP transgenic mice treated orally with clioquinol, a USP antibiotic with copper/zinc binding properties. No effect of TETA, a traditional chelator that does not cross the BBB(28) (see ARF related news story).

2002: Genetic ablation of ZnT3, the ionic transporter that loads Zn2+ into synaptic vesicles, decreases brain zinc concentrations by approx. 20 percent, but markedly (>80 percent) inhibits amyloid deposition in Tg2576 mice(29) (see ARF related news story).
2002: Tg2576 and CT100 transgenic mice have decreased brain copper levels. Brain copper and iron levels rise with age in mice, and this increase is opposed by the expression of these transgenes.(30)
2002: Biological reducing agents, such as cholesterol, fuel the Cu2+-dependent generation of H2O2 and neurotoxicity of Aβ, which can be rescued with Cu2+ chelation.(17)

2003: Raising brain copper levels by genetic(31) or dietary(32) means decreases Aβ accumulation in two strains of transgenic mice, and restores diminished SOD1 activity. This possibly is compatible with Aβ being situated on the export side of copper homeostasis (see ARF related news story).
2003: Promising double-blind, placebo-controlled, phase 2 pilot clinical trial of clioquinol in patients with Alzheimer's disease (arrest in cognitive decline, lowering of plasma Aβ levels)(33) (see ARF related news story).

2004: Estrogen decreases zinc transporter 3 expression and synaptic vesicle zinc levels in mouse brain.(34)

References:
1. Assaf SY, Chung S-H. Release of endogenous Zn2+ from brain tissue during activity. Nature. 1984 Apr 19-25;308(5961):734-6. Abstract

2. Howell GA, Welch MG, Frederickson CJ. Stimulation-induced uptake and release of zinc in hippocampal slices. Nature. 1984 Apr 19-25;308(5961):736-8. Abstract

3. Hartter DE, Barnea A Evidence for release of copper in the brain: depolarization-induced release of newly taken-up 67copper. Synapse. 1988;2(4):412-5. Abstract

4. Bush AI, Whyte S, Thomas LD, Williamson TG, Van Tiggelen CJ, Currie J, Small DH, Moir RD, Li QX, Rumble B, et al. An abnormality of plasma amyloid protein precursor in Alzheimer's disease. Ann Neurol. 1992 Jul;32(1):57-65. Abstract

5. Roberts SB, Ripellino JA, Ingalls KM, Robakis NK, Felsenstein KM. Non-amyloidogenic cleavage of the beta-amyloid precursor protein by an integral membrane metalloendopeptidase. J Biol Chem. 1994 Jan 28;269(4):3111-6. Abstract

6. Buxbaum JD, Liu KN, Luo Y, Slack JL, Stocking KL, Peschon JJ, Johnson RS, Castner BJ, Cerretti DP, Black RA. Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem. 1998 Oct 23;273(43):27765-7. Abstract

7. Bush AI, Pettingell W.H, Jr., Paradis MD, Tanzi RE. Modulation of A beta adhesiveness and secretase site cleavage by zinc. J Biol Chem. 1994 Apr 22;269(16):12152-8. Abstract

8. Bush AI, Multhaup G, Moir RD, Williamson TG, Small DH, Rumble B, Pollwein P, Beyreuther K, Masters CL. A novel zinc(II) binding site modulates the function of the beta A4 amyloid protein precursor of Alzheimer's disease. J Biol Chem. 1993 Aug 5;268(22):16109-12. Abstract

9. Bush AI, Pettingell WH, de Paradis M, Tanzi RE, Wasco W. The amyloid beta-protein precursor and its mammalian homologues. Evidence for a zinc-modulated heparin-binding superfamily. J Biol Chem. 1994 Oct 28;269(43):26618-21. Abstract

10. Multhaup G, Schlicksupp A, Hesse L, Beher D, Ruppert T, Masters CL, Beyreuther K. The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I). Science. 1996 Mar 8;271(5254):1406-9. Abstract

11. Multhaup G, Ruppert T, Schlicksupp A, Hesse L, Bill E, Pipkorn R, Masters CL, Beyreuther K. Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide. Biochemistry. 1998 May 19;37(20):7224-30. Abstract

12. Bush AI, Pettingell WH, Multhaup G, Paradis Md, Vonsattel JP, Gusella JF, Beyreuther K, Masters CL, Tanzi RE. Rapid induction of Alzheimer A beta amyloid formation by zinc. Science. 1994 Sep 2;265(5177):1464-7. Abstract

13. Hesse L, Beher D, Masters CL, Multhaup G. The beta A4 amyloid precursor protein binding to copper. FEBS Lett. 1994 Jul 25;349(1):109-16. Abstract

14. Huang X, Atwood CS, Moir RD, Hartshorn MA, Vonsattel J-P, Tanzi RE, Bush AI. Zinc-induced Alzheimer's Abeta1-40 aggregation is mediated by conformational factors. J Biol Chem. 1997 Oct 17;272(42):26464-70. Abstract

15. Atwood CS, Moir RD, Huang X, Bacarra NME, Scarpa RC, Romano DM, Hartshorn MA, Tanzi RE, Bush AI. Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem. 1998 May 22;273(21):12817-26. Abstract

16. Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR. Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci. 1998 Jun 11;158(1):47-52. Abstract

17. Opazo C, Huang X, Cherny R, Moir R, Roher A, White A, Cappai R, Masters C, Tanzi R, Inestrosa N, Bush A. Metalloenzyme-like activity of Alzheimer's disease beta-amyloid. Cu-dependent catalytic conversion of dopamine, cholesterol, and biological reducing agents to neurotoxic H(2)O(2). J Biol Chem. 2002 Oct 25;277(43):40302-8. Epub 2002 Aug 20. Abstract

18. Dong J, Atwood CS, Anderson VE, Siedlak SL, Smith MA, Perry G, Carey PR. Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence. Biochemistry. 2003 Mar 18;42(10):2768-73. Abstract

19. Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, Scarpa RC, Cuajungco MP, Gray DN, Lim J, Moir RD, Tanzi RE, Bush AI. The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry. 1999 Jun 15;38(24):7609-16. Abstract

20. Huang X, Cuajungco MP, Atwood CS, Hartshorn MA, Tyndall J, Hanson GR, Stokes KC, Leopold M, Multhaup G, Goldstein LE, Scarpa RC, Saunders AJ, Lim J, Moir RD, Glabe C, Bowden EF, Masters CL, Fairlie DP, Tanzi RE, Bush AI. Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction. J Biol Chem. 1999 Dec 24;274(52):37111-6. Abstract

21. Cherny RA, Legg JT, McLean CA, Fairlie D, Huang X, Atwood CS, Beyreuther K, Tanzi RE, Masters CL, Bush AI. Aqueous dissolution of Alzheimer's disease Abeta amyloid deposits by biometal depletion. J Biol Chem. 1999 Aug 13;274(33):23223-8. Abstract

22. White AR, Reyes R, Mercer JF, Camakaris J, Zheng H, Bush AI, Multhaup G, Beyreuther K, Masters CL, Cappai R (1999) Copper levels are increased in the cerebral cortex and liver of APP and APLP2 knockout mice. Brain Res 842, 439-44. Abstract

23. Atwood CS, Scarpa RC, Huang X, Moir RD, Jones WD, Fairlie DP, Tanzi RE, Bush AI. Characterization of copper interactions with alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42. J Neurochem. 2000 Sep;75(3):1219-33. Abstract

24. Barnham KJ, Ciccotosto GD, Tickler AK, Ali FA, Smith DG, Williamson NA, Lam Y-H, Carrington D, Tew D, Kocak G, Volitakis I, Separovic F, Barrow CJ, Wade JD, Masters CL, Cherny RA, Curtain CC, Bush AI, Cappai R. Neurotoxic, redox-competent Alzheimer's beta-amyloid is released from lipid membrane by methionine oxidation. J Biol Chem. 2003 Oct 31;278(44):42959-65. Epub 2003 Aug 18. Abstract

25. Kontush A, Berndt C, Weber W, Akopyan V, Arlt S, Schippling S, Beisiegel U (2001) Amyloid-beta is an antioxidant for lipoproteins in cerebrospinal fluid and plasma. Free Radic Biol Med. 2001 Jan 1;30(1):119-28. Abstract

26. Kontush A, Donarski N, Beisiegel U. Resistance of human cerebrospinal fluid to in vitro oxidation is directly related to its amyloid-beta content. Free Radic Res. 2001 Nov;35(5):507-17. Abstract

27. Curtain C, Ali F, Volitakis I, Cherny R, Norton R, Beyreuther K, Barrow C, Masters C, Bush A, Barnham K. Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits. J Biol Chem. 2001 Jun 8;276(23):20466-73. Epub 2001 Mar 27. Abstract

28. Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I, Fraser FW, Kim Y-S, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE, Masters CL, Bush AI. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron. 2001 Jun;30(3):665-76. Abstract

29. Lee J-Y, Cole TB, Palmiter RD, Suh SW, Koh J-Y. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice. Proc Natl Acad Sci U S A. 2002 May 28;99(11):7705-10. Abstract

30. Maynard CJ, Cappai R, Volitakis I, Cherny RA, White AR, Beyreuther K, Masters CL, Bush AI, Li Q-X. Overexpression of Alzheimer's disease amyloid-beta opposes the age-dependent elevations of brain copper and iron. J Biol Chem. 2002 Nov 22;277(47):44670-6. Epub 2002 Sep 04. Abstract

31. Phinney AL, Drisaldi B, Schmidt SD, Lugowski S, Coronado V, Liang Y, Horne P, Yang J, Sekoulidis J, Coomaraswamy J, Chishti MA, Cox DW, Mathews PM, Nixon RA, Carlson GA, George-Hyslop PS, Westaway D. In vivo reduction of amyloid-beta by a mutant copper transporter. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14193-8. Epub 2003 Nov 14. Abstract

32. Bayer TA, Schafer S, Simons A, Kemmling A, Kamer T, Tepests R, Eckert A, Schussel K, Eikenberg O, Sturchler-Pierrat C, Abramowski D, Staufenbiel M, Multhaup G. Dietary Cu stabilizes brain superoxide dismutase 1 activity and reduces amyloid Abeta production in APP23 transgenic mice. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14187-92. Epub 2003 Nov 14. Abstract

33. Ritchie CW, Bush AI, Mackinnon A, Macfarlane S, Mastwyk M, MacGregor L, Kiers L, Cherny RA, Li QX, Tammer A, Carrington D, Mavros C, Volitakis I, Xilinas M, Ames D, Davis S, Beyreuther K, Tanzi RE, Masters CL. Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting Abeta amyloid deposition and toxicity in Alzheimer disease: a pilot phase 2 clinical trial. Arch Neurol. 2003 Dec;60(12):1685-91. Abstract

34. Lee J-Y, Kim J-H, Hong SH, Lee JY, Cherny RA, Bush AI, Palmiter RD, Koh J-Y. Estrogen decreases zinc transporter 3 expression and synaptic vesicle zinc levels in mouse brain. J Biol Chem. 2003 Dec 17 [Epub ahead of print] Abstract

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References

News Citations

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  6. Coping with Copper—Minute Amount of Metal Plagues Rabbit Brain
  7. CNDR 2nd Annual Retreat: Metal Complexing Agents as Therapies for AD

Webinar Citations

  1. Clioquinol Trial Postmortem: Does Blocking Metal-Aβ Interactions Work?

Paper Citations

  1. . Release of endogenous Zn2+ from brain tissue during activity. Nature. 1984 Apr 19-25;308(5961):734-6. PubMed.
  2. . Stimulation-induced uptake and release of zinc in hippocampal slices. Nature. 1984 Apr 19-25;308(5961):736-8. PubMed.
  3. . Evidence for release of copper in the brain: depolarization-induced release of newly taken-up 67copper. Synapse. 1988;2(4):412-5. PubMed.
  4. . An abnormality of plasma amyloid protein precursor in Alzheimer's disease. Ann Neurol. 1992 Jul;32(1):57-65. PubMed.
  5. . Non-amyloidogenic cleavage of the beta-amyloid precursor protein by an integral membrane metalloendopeptidase. J Biol Chem. 1994 Jan 28;269(4):3111-6. PubMed.
  6. . Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem. 1998 Oct 23;273(43):27765-7. PubMed.
  7. . Modulation of A beta adhesiveness and secretase site cleavage by zinc. J Biol Chem. 1994 Apr 22;269(16):12152-8. PubMed.
  8. . A novel zinc(II) binding site modulates the function of the beta A4 amyloid protein precursor of Alzheimer's disease. J Biol Chem. 1993 Aug 5;268(22):16109-12. PubMed.
  9. . The amyloid beta-protein precursor and its mammalian homologues. Evidence for a zinc-modulated heparin-binding superfamily. J Biol Chem. 1994 Oct 28;269(43):26618-21. PubMed.
  10. . The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I). Science. 1996 Mar 8;271(5254):1406-9. PubMed.
  11. . Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide. Biochemistry. 1998 May 19;37(20):7224-30. PubMed.
  12. . Rapid induction of Alzheimer A beta amyloid formation by zinc. Science. 1994 Sep 2;265(5177):1464-7. PubMed.
  13. . The beta A4 amyloid precursor protein binding to copper. FEBS Lett. 1994 Jul 25;349(1):109-16. PubMed.
  14. . Zinc-induced Alzheimer's Abeta1-40 aggregation is mediated by conformational factors. J Biol Chem. 1997 Oct 17;272(42):26464-70. PubMed.
  15. . Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem. 1998 May 22;273(21):12817-26. PubMed.
  16. . Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci. 1998 Jun 11;158(1):47-52. PubMed.
  17. . Metalloenzyme-like activity of Alzheimer's disease beta-amyloid. Cu-dependent catalytic conversion of dopamine, cholesterol, and biological reducing agents to neurotoxic H(2)O(2). J Biol Chem. 2002 Oct 25;277(43):40302-8. PubMed.
  18. . Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence. Biochemistry. 2003 Mar 18;42(10):2768-73. PubMed.
  19. . The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry. 1999 Jun 15;38(24):7609-16. PubMed.
  20. . Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction. J Biol Chem. 1999 Dec 24;274(52):37111-6. PubMed.
  21. . Aqueous dissolution of Alzheimer's disease Abeta amyloid deposits by biometal depletion. J Biol Chem. 1999 Aug 13;274(33):23223-8. PubMed.
  22. . Copper levels are increased in the cerebral cortex and liver of APP and APLP2 knockout mice. Brain Res. 1999 Sep 25;842(2):439-44. PubMed.
  23. . Characterization of copper interactions with alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42. J Neurochem. 2000 Sep;75(3):1219-33. PubMed.
  24. . Neurotoxic, redox-competent Alzheimer's beta-amyloid is released from lipid membrane by methionine oxidation. J Biol Chem. 2003 Oct 31;278(44):42959-65. PubMed.
  25. . Amyloid-beta is an antioxidant for lipoproteins in cerebrospinal fluid and plasma. Free Radic Biol Med. 2001 Jan 1;30(1):119-28. PubMed.
  26. . Resistance of human cerebrospinal fluid to in vitro oxidation is directly related to its amyloid-beta content. Free Radic Res. 2001 Nov;35(5):507-17. PubMed.
  27. . Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits. J Biol Chem. 2001 Jun 8;276(23):20466-73. PubMed.
  28. . Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron. 2001 Jun;30(3):665-76. PubMed.
  29. . Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice. Proc Natl Acad Sci U S A. 2002 May 28;99(11):7705-10. PubMed.
  30. . Overexpression of Alzheimer's disease amyloid-beta opposes the age-dependent elevations of brain copper and iron. J Biol Chem. 2002 Nov 22;277(47):44670-6. PubMed.
  31. . In vivo reduction of amyloid-beta by a mutant copper transporter. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14193-8. PubMed.
  32. . Dietary Cu stabilizes brain superoxide dismutase 1 activity and reduces amyloid Abeta production in APP23 transgenic mice. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14187-92. PubMed.
  33. . Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting Abeta amyloid deposition and toxicity in Alzheimer disease: a pilot phase 2 clinical trial. Arch Neurol. 2003 Dec;60(12):1685-91. PubMed.
  34. . Estrogen decreases zinc transporter 3 expression and synaptic vesicle zinc levels in mouse brain. J Biol Chem. 2004 Mar 5;279(10):8602-7. PubMed.
  35. . Idebenone treatment fails to slow cognitive decline in Alzheimer's disease. Neurology. 2003 Dec 9;61(11):1498-502. PubMed.
  36. . An iron-responsive element type II in the 5'-untranslated region of the Alzheimer's amyloid precursor protein transcript. J Biol Chem. 2002 Nov 22;277(47):45518-28. PubMed.
  37. . Association of aortic atherosclerosis with cerebral beta-amyloidosis and learning deficits in a mouse model of Alzheimer's disease. Am J Pathol. 2003 Dec;163(6):2155-64. PubMed.

Other Citations

  1. Ashley Bush

External Citations

  1. Maverick Scientist Is Winning Converts On Alzheimer's
  2. SFN 03 abstract

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