Summary

David Allsop and Ashley Bush led this live discussion on 20 December 2004. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Transcript:

Live Discussion led by David Allsop, Ashley Bush, and co-moderator Dominic Walsh on 20 December 2004.

 

Participants: David Allsop, Lancaster University, United Kingdom; Ashley Bush, Massachusetts General Hospital; Dominic Walsh, University College Dublin; Fredrik Haeffner, Brown University, Rhode Island; Tom Fagan, Alzheimer Research Forum; Alain Boom, Medical School of Brussels, Neuropathology Laboratory; Patrick Brunelle, University of Calgary, Alberta, Canada; Keith Crutcher, University of Cincinnati, Ohio; Dara Dickstein, University of British Columbia; Rachel Karlnoski, University of South Florida; Leopold Liss, Ohio State University, Department of Neurology; Andrew McCaddon, University of Wales; Kim Sciaretta, University of Chicago; Mark Smith, Case Western Reserve University.

 

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

 

Tom Fagan
Hi, all! I'm Tom Fagan, filling in for Gabrielle Strobel, and I will probably be moderating today. We have a few more folks in the "foyer" waiting to join in, so we'll give them a few minutes.

Mark Smith
Hello, all.

David Allsop
Hello, Mark. How are you?

Mark Smith
Hi, David...great, aside from 12 inches of snow outside!

Tom Fagan
Mark, where are you located? We have about five inches of snow right now, but maybe more is on the way (Boston).

Mark Smith
Cleveland, Ohio (the Heart of it ALL!).

Ashley Bush
I just battled through a blizzard to get here! Boston is being battered.

Tom Fagan
Hi, Ashley; we were just talking about the snow.

David Allsop
Hi, Ashley; glad to see you are here.

Ashley Bush
Glad to see y'all.

Fredrik Haeffner
Hi, I'm Fredrik Haeffner and I use quantum chemistry to explore Aβ-Cu mechanisms in collaboration with Ashley. I'm looking forward to an interesting discussion.

Ashley Bush
Hi, Fredrik.

David Allsop
That sounds far too complicated for me!

Tom Fagan
Okay, I think we have a quorum. David, do you want to get the ball rolling with a brief statement about what we should be discussing today? That'll give Ashley a chance to find his way around the interface.

David Allsop
Hello, Ashley and others out there, and compliments of the season to you all! To get the discussion started, perhaps we should focus initially on the ability of β amyloid to generate hydrogen peroxide, and some of the characteristics of this process. Ashley has shown this by colorimetric and fluorimetric methods. Our method converts hydrogen peroxide to hydroxyl radicals (by the Fenton reaction), which are then detected by ESR (electron spin resonance) spectroscopy and spin-trapping. I wonder how closely our data correlate with each other. Perhaps a good question to start with would be exactly what type of aggregate has the ability to generate hydrogen peroxide. Can you comment, Ashley?

Ashley Bush
Good question. The data so far suggest that something bigger than a monomer (dimer plus) is needed. The most potent oligomer is not yet known. Fredrik has been looking at the activity based on oligomers.

Fredrik Haeffner
Ashley, are we sure now that the SOD (superoxide dismutase)-like dimer exists? [Aβ has been shown to form dimers linked via disulfide bridges, see Multhaup et al., 1996]

Ashley Bush
Kevin Barnham in Melbourne has gathered data to support the SOD[-like disulfide] bridge. We can see the SOD structure in vitro (see, for example, the paper by Tickler et al., 2005, which was published after this transcript went live).

 

Fredrik Haeffner
Great, then we could have the cholesterol mechanism paper in press soon!

David Allsop
Our data suggest a short "burst" of hydrogen peroxide formation early on in the aggregation process, followed by a gradual decay to zero. The most active phase of hydrogen peroxide formation coincides with the presence of early aggregates, and not fully formed fibrils. In fact, mature fibrils are inactive.

Ashley Bush
David, I could believe that easily. Like many redox active "enzymes" Aβ/copper can be destroyed by hydrogen peroxide. I also agree with you, David, about the redox inertness of fibers.

David Allsop
To my mind, there are two possible interpretations. One is that a certain type of aggregate is the "active species" in terms of its ability to generate hydrogen peroxide. The other is that hydrogen peroxide is actually a "by-product" of the aggregation process itself, and is generated to a maximum extent during the most active phase of aggregation. Maybe the energy involved in driving the aggregation process is needed for the production of hydrogen peroxide.

Ashley Bush
Could be…the electron could come from the side chains, in which case the hydrogen peroxide generation will be stoichiometrically limited or the electron could come from reducing agents, in which case the peptide will not be consumed necessarily, but it could get oxidized by the hydroxyl radical.

Tom Fagan
David, if it was generated as a by-product, then it would probably be stoichiometric. Has the stoichiometry been done?

David Allsop
It is difficult to get a good estimate of the stoichiometry.

Ashley Bush
We have evidence of both stoichiometric generation and catalytic generation.

Fredrik Haeffner
David, yes, you could be right. H2O2 is driving dityrosine formation. Hydroxyl radical would form from formed hydrogen peroxide.

Ashley Bush
Yes, H2O2 reacts with the Cu center. I think that toxicity arises from catalytic H2O2 generation using substrates like cholesterol [as electron donors]. So here's a question: Why does it not happen in health? We believe that Aβ is a normal peptide. That it does not generate H2O2 in health. The reason: it only becomes soluble in AD.

Fredrik Haeffner
I'm putting together calculations to show that H2O2 breaks apart [the bond] between methionine 35 radical cation and Cu2+ and forms the sulfoxide and hydroxyl radical. Methionine 35 must be close to the copper center, which contradicts what Pogocki claims (for example, see Pogocki, 2004 and Shconeich et al., 2003).

Ashley Bush
Okay, so methionine can only get close to Cu if the peptide is soluble.

Fredrik Haeffner
Methionine 35 acts as a "substrate" wherein Aβ is being cross-linked via dityrosine (tyrosine 10-tyrosine 10).

Ashley Bush
Barnham and Curtain in Melbourne believe that Aβ normally forms a membrane-embedded hexamer when binding metals. This hexamer is redox-silent when the methionine is buried. When the methionine emerges (for example, as a result of oxidation) the complex becomes redox-active and capable of generating H2O2. In the normal brain, the Aβ is 100 percent in the membrane fraction.

David Allsop
Ashley, what about peptides with no methionine? In our system, we can see generation of hydrogen peroxide with peptides such as the NAC peptide, which has no methionine. (NAC, or non-amyloid-β component peptide found in amyloid plaques, is actually a segment of α-synuclein shown to be fibrillogenic; see Han et al., 1995 and Iwai et al., 1995).

Ashley Bush
David, see Ciccotosto et al., 2004.

 

Fredrik Haeffner
David, as long as there is another reducing substrate present, you will produce H2O2. So, if there's no methionine, but maybe ascorbic acid, you will see H2O2 form.

David Allsop
Fred, in our experiments, the peptide is not incubated in the presence of any substrate.

Ashley Bush
So David, do you [predict] ROS (reactive oxygen species) in the absence of metal?

David Allsop
We can always block the ESR spectrum with a good metal chelator.

Ashley Bush
Excellent. I agree completely. Many people do not realize that the chemical origin of 99 percent of ROS is from metal (Cu and Fe).

Fredrik Haeffner
David, do you see dityrosine form in α-synuclein when you see H2O2?

David Allsop
Fred, we haven't looked.

Fredrik Haeffner
2 Tyr-OH + O2 ---> dityrosine + H2O2!

Mark Smith
David/Ashley, does this affect our previous conclusions vis-a-vis Butterfield's shrapnel hypothesis?

Tom Fagan
Mark, can you elaborate on that for the uninitiated?

Mark Smith
I defer to the experts. Ashley and David!

Ashley Bush
Mark, yes. The hydrogen peroxide can generate hydroxyl radicals which would attack the peptide to give peptidyl radicals, but this is different from Butterfield's "shrapnel" hypothesis.

Mark Smith
Yes, but I'm thinking about Aβ by itself generating radicals (without metals). Is that true?

Ashley Bush
The amyloid-β peptide (Aβ) is toxic to neuronal cells, and it is probable that this toxicity is responsible for the progressive cognitive decline associated with Alzheimer disease. However, the nature of the toxic Aβ species and its precise mechanism of action remain to be determined. It has been reported that the methionine residue at position 35 has a pivotal role to play in the toxicity of Aβ. We examined the effect of mutating the methionine to valine in Aβ42 (AβM35V). The neurotoxic activity of AβM35V on primary mouse neuronal cortical cells was enhanced, and this diminished cell viability occurred at an accelerated rate compared with Aβ42. AβM35V binds Cu2+ and produces similar amounts of H2O2 as Aβ42 in vitro, and the neurotoxic activity was attenuated by the H2O2 scavenger catalase. The increased toxicity of AβM35V was associated with increased binding of this mutated peptide to cortical cells. The M35V mutation altered the interaction between Aβ and copper in a lipid environment as shown by EPR (electron paramagnetic resonance) analysis, which indicated that the valine substitution made the peptide less rigid in the bilayer region with a resulting higher affinity for the bilayer. Circular dichroism spectroscopy showed that both Aβ42 and AβM35V displayed a mixture of α-helical and β-sheet conformations. These findings provide further evidence that the toxicity of Aβ is regulated by binding to neuronal cells.

David Allsop
Ashley, what about other types of amyloid? Have you looked at anything other than Aβ?

Ashley Bush
So far only amylin (negative) and other proteins like α-synuclein, SOD, aB-crystallin, prion protein (positive).

David Allsop
Ashley, glad to see that you agree with us! We have also had positive results for British dementia peptide (ABri).

Ashley Bush
David, that is excellent. Tough peptide to work with! Have you done head to head comparison with Aβ?

David Allsop
Ashley, not sure what you mean.

Ashley Bush
David, have you compared the rate of H2O2 formation from Aβ versus that from ABri?

David Allsop
Ashley, difficult to be certain about absolute rates, but ABri aggregates and produces hydrogen peroxide much more rapidly that Aβ.

Mark Smith
Ashley, Aβ is much less toxic if you get rid of associated metals (Rottkamp et al., 2001).

Ashley Bush
Yep, without Cu or Fe it is not toxic. When you add peptide to cells in culture, it picks up Cu or Fe from the culture medium.

Dominic Walsh
Do metal-induced aggregates form true amyloid fibrils?

Ashley Bush
Dominic, we believe that metals are needed for the initial seeding for certain. We have a paper in press showing that chelators abolish fibril formation.

Dominic Walsh
Yeah, but what evidence is there that addition of metal ions leads to fibril formation? Has anyone done atomic force microscopy (AFM)?

Ashley Bush
Same (and other) papers show that the presence of trace concentrations of metals initiates fibril formation (see, for example, Huang et al., 2004). But Dominic, fibrils, I think, are passé.

Dominic Walsh
I think all Aβ assemblies are important. I want to know is there any structural handle on the metal-induced aggregates.

Ashley Bush
Dominic, as we discussed earlier, data suggests that Cu/Fe is needed for Aβ ROS generation and toxicity. No one can yet prove that Aβ oligomerizes even in the absence of metals.

Alain Boom
What do you think about the high concentration of Zinc (1mM) in amyloid-β deposits?

Ashley Bush
Alain, knockouts of the Zn transporter ZnT3 abolish amyloid in transgenic mice.

Fredrik Haeffner
Zn won't [lead to formation of] dityrosine; Cu and Fe will. Any comment on this, Ashley? What aggregates are we talking about? Covalent or non-covalent? Is this important?

Ashley Bush
Soluble oligomers/dimers are more linked to the disease problem.

Fredrik Haeffner
So Zn is not causing the trouble, then?

Ashley Bush
The observation that low Cu levels increases amyloid is linked to decreased SOD activity. This indicates oxidation as an upstream pathogenic event in the whole picture. Zinc comes much later (see ARF related news story and ARF news story on the complex relationship between copper and AD).

Fredrik Haeffner
Maybe Zn interacts with dityrosine-linked Aβ?

Alain Boom
Zinc seems to play a role in the PKB transduction pathway leading to tau phosphorylation. Do you know anything about this hypothesis?

Ashley Bush
Alain, indeed. Zinc is all over the phosphorylation pathways for tau. A large literature (see, for example, Harris et al., 2004, An et al., 2003, and Yu and Fraser, 2001).

Fredrik Haeffner
If dityrosine formation is the problem, what about trapping the tyrosyl radicals before they cross-link using spin-traps such as MNP (2-methyl-2-nitrosopropane)?

Dara Dickstein
Is there anything known about Fe transporter knockout mice and amyloid deposits?

David Allsop
Ashley, what about clioquinol? How do you think this is working when it isn't a particularly good metal chelator? Do you have any new data?

Ashley Bush
David, the data on clioquinol builds rapidly. We published how it abolishes Aβ SDS-resistant oligomers. See Barnham et al., 2004.

Dominic Walsh
I have a question on the flip side of clioquinol. If clioquinol clears amyloid in Tg2576 mice, does injection of Cu increase amyloid deposition?

Ashley Bush
Manipulating Cu has potent effects, but not the way you'd think. Decreased brain Cu increases amyloid pathology in Tg mice groups (see Phinney et al., 2003 and Bayer et al., 2003).

Dominic Walsh
Have you tried intrahippocampal injection of Cu?

Ashley Bush
Dominic, we haven't tried that. It would be too unphysiological—too easily lethal.

Tom Fagan
David, Ashley, do we have any idea what percentage of total ROS produced in the brain might come from Aβ, NAC (nascent polypeptide-associated complex), etc.?

David Allsop
No idea! Unless Ashley can comment.

Ashley Bush
We did in vitro microdialysis in Tg brain; it shows H2O2 elevation in Tg many fold greater that non-Tg.

Dominic Walsh
What about the clioquinol-treated Tg2576 mice? Have you looked at behavior or synaptic plasticity? Does clioquinol treatment improve these?

Ashley Bush
Dominic, clioquinol behavioral data indicate marked improvement. We have no data yet on synapses.

Dominic Walsh
That's encouraging—what assays and what treatment regimes were employed?

Ashley Bush
Dom, clioquinol at 30 mg/kg/day orally (gavage).

Rachel Karlnoski
Ashley, did clioquinol cause any neurotoxicity or neuronal loss in the Tg mice?

Ashley Bush
Clioquinol at the dose given is harmless on cell count and various toxicology measures. Clioquinol is in human trials now.

David Allsop
Ashley, what concentrations of clioquinol can you achieve in the brain with this type of dose?

Ashley Bush
Clioquinol concentration in the brain is equivalent to that in the plasma. I don't have numbers in front of me, but I think it is in the low micromolar range.

David Allsop
We find that relatively high concentrations of clioquinol are required to inhibit hydrogen peroxide formation from Aβ (10-20 micromolar clioquinol for 100 micromolar Aβ peptide).

Ashley Bush
David, since clioquinol binds to Aβ/metal on a stoichiometric basis, I am not surprised that you need a concentration of clioquinol that is within an order of magnitude of that of Aβ.

Dara Dickstein
Melanotransferrin (or p97) is found at high levels in Alzheimer patients (see Kim et al., 2001; Yamada et al., 1999; Feldman et al., 2001). It is a natural protein related to transferrin and has been shown to bind iron, copper, and to a lesser extent, zinc. It is also found on reactive microglia associated with plaques. Could it be a natural chelator?

Ashley Bush
Dara, p97 could be [a chelator], like ApoE and several other proteins.

Dara Dickstein
The difference is ApoE is not upregulated during AD.

Ashley Bush
Dara, I am not excluding p97, but the isoform's affinity for metals inversely corresponds to the risk for AD. There are clearly also problems with Fe metabolism in AD, but so far only Cu and Zn are known to bind Aβ in vivo. For example, APP translation is Fe-dependent (Rogers et al., 2002).

Mark Smith
Dara, in vivo, amyloid plaques are associated with redox-active iron (Smith et al., 1997).

Ashley Bush
Dara, plaques have Fe in them (mainly in neurites), but Aβ itself is not binding Fe. Here's a reference: it's Smith himself! See Dong et al., 2003. Here is a model for the Cu-Zn role in AD. Cu2+ levels rise with age in specific subcellular compartments of the brain. Aβ becomes overwhelmed in its attempt to contain or transport Cu2+ and becomes oxidized by Cu2+. This leads to protease-resistant dityrosine species, as well as oxidation of methionine 35 on Aβ, whereupon Aβ can escape its constitutive membrane compartment. This drives up the levels of soluble Aβ (monomer and oligomer) in the brain. When bound to Cu2+, these forms are toxic due to their redox activity and the catalytic generation of H2O2. These soluble Aβ species drift into the interstitial spaces of the brain where they are driven out of solution by the exceptionally high concentrations of Zn2+ in flux at the glutamatergic synapses and the perivascular spaces. While the Zn2+ partially quenches Aβ-mediated redox activity, the amyloid deposits are still sites of considerable H2O2 formation and oxidation.

Dominic Walsh
Ashley, have you tried depleting Tg2576 mice of antioxidants, for example, vitamin E or selenium?

Ashley Bush
Dominic, that's a good experiment. We haven't done it. But so far, antioxidants have failed to impress in AD clinical trials—probably because each one has only a limited scavenging ability, chemically.

Dominic Walsh
Yep, I know the studies on the use of vitamin E and selenium are weak, but perhaps it's because they are given too late. That's why manipulation of Tg mice could be useful.

Ashley Bush
Dominic, I agree.

Andrew McCaddon
Hi, Ashley and Dominic. I have seen some promising effects with NAC (N-acetylcysteine) in patients, albeit in a small number of case-control studies to date.

Dominic Walsh David, in your ESR studies, H2O2 production was blocked by a chelator or catalase—did these treatments also prevent fibril formation?

David Allsop
Dominic, we can add catalase at the end of the peptide incubation period and still block the ESR spectrum, so this can't be an effect on fibril formation.

Ashley Bush
Our view is that Aβ is the font of ROS in AD and that it is best to shut off production at its source with something like clioquinol.

David Allsop
If Mark Smith is still out there, maybe we can discuss some of his ideas. Mark, if you still think that oxidative damage comes before amyloid deposition, then what is the source of the ROS?

Alain Boom
It might be possible that after glutamatergic transmission, the accumulation of zinc in the plaque interferes with tau phosphorylation. So a relationship between plaques and tau phosphorylation could be drawn.

Mark Smith
David, our best bet is mitochondria (though not direct), certainly not amyloid, since there is an inverse relationship between amount of amyloid and oxidation! Amyloid is clearly good!!

Ashley Bush
Mark, amyloid probably has a function. We reckon it is involved in Cu export.

Mark Smith
Could be. Whatever its function is, the more of it there is, the less oxidation there seems to be—ergo, amyloid is good! See Nunomura et al., 2001.

David Allsop
Mark, what strange ideas you have!! The amyloid peptide may be an antioxidant in its soluble form, but it generates ROS as soon as it aggregates, and so becomes damaging. The phase of optimal damage may be before overt plaques appear in the brain.

Mark Smith
Strange, I agree, but not incorrect!

Dominic Walsh
David, I agree. I don't think plaques are ever good; they cause so many secondary effects, for example, microglial infiltration.

Mark Smith
Dominic, let's see what happens when pharma gets rid of them.

Ashley Bush
Mark, our data indicates that in AD, soluble Aβ correlates with disease markers.

Mark Smith
Ashley, it works if you look at insoluble or soluble amyloid—amyloid is still good!

Ashley Bush
Mark, you need Aβ to live as a neuron, but it is not soluble Aβ. Mark, we also published that amyloid plaque burden inversely correlates with oxidative damage, but even in the most burdened brains there is plenty of oxidation. See Cuajungco et al., 2000. Another Smith paper! Remember, amyloid plaques are not the cause of the disease, although I agree they are not normal.

Mark Smith
I know there is oxidation, but there is no positive relation between amyloid (in any guise) and oxidation—the reverse only.

Ashley Bush
Mark, I agree. Dominic, what's the latest with dimers? Are they a product of increased oxidation?

Dominic Walsh
We are still working very hard to achieve molecular characterization, i.e., mass spectrometry (MS).

Ashley Bush
Dominic, we found that dimers don't fly well on MS. We digested the dimer and found dityrosine.

Dominic Walsh
I was very interested in your report that Cu can cause dityrosine cross-links, and we have tried out the dityrosine-specific antibodies on our dimers and trimers, but no joy—so who knows?

Ashley Bush
Dominic, beware of dityrosine antibodies. There are very few [good ones] available. We looked, too. You should speak to Robert Cherny in Melbourne. Also look at Barnham et al., 2004.

Dominic Walsh
Ashley, thanks for the reference; I'll definitely check it out.

Fredrik Haeffner
Dominic, you missed my talk at University College Dublin chemistry department in October.

Dominic Walsh
Sorry, Fred, I was in Boston.

David Allsop
Dominic, have you ever checked whether your "soluble oligomers" or "protofibrils" generate hydrogen peroxide? How about collaborating with us on this! Also, what about so-called annular protofibrils?

Dominic Walsh
Obviously in AD there are multiple pathological mechanisms working in concert. ROS may contribute to different phases of the disease and within different brain structures at different times. The most important questions are: 1) do ROS initiate the process and 2) do they lead to an irreversible phase of the disease?

Fredrik Haeffner
What about Ca2+ ion-channels made from β amyloid (or maybe made from di-tyrosine-linked dimers)?

Ashley Bush
Fredrik, I keep an open mind about it. We published that Aβ/CuZn forms a membrane-embedded hexamer which could be the basis for a "pore." See Curtain et al., 2001.

Fredrik Haeffner
Yes, they would then be in the right position to be welded together via di-tyrosine links.

Mark Smith
I gotta go have my chelation therapy. Happy holidays to all and let's get this disease cracked in 2005. Cheers!

Ashley Bush
Cheers, Mark.

Tom Fagan
Folks, we are near the end of our hour, but this is so interesting I hope we go on a bit. But before people start heading off, I'd like to propose that our chat leaders address what work needs to be done in the near future. Obviously, Ashley is working hard with clioquinol, but what are the other key questions/experiments that need to be addressed? Anyone?

Ashley Bush
Tom, number one, the function of Aβ.

Tom Fagan
 Indeed!

David Allsop
We are looking to see which of the many amyloids can generate ROS. Is this a property of most amyloids, or just a small subset of them that can interact with metals? Bye, Dominic.

Dominic Walsh
Bye, everyone, and happy holidays and don't forget to register for our meeting in March 2005.

Ashley Bush
Bye, Dominic.

Dara Dickstein
Thank you for an interesting conversation. Happy holidays.

Ashley Bush
Thanks, Dara. Happy holidays

David Allsop
Sorry, but I have to go now. Bye to you all and Happy Xmas!! Brian Tabner also says goodbye!

Ashley Bush
Cheers, David and Brian

Fredrik Haeffner
Have a great New Year! Goodbye.

Andrew McCaddon
Bye, all.

Ashley Bush
Okay, lunch! Bye!

 

Background

Background Text

We thank Bentham Science Publishers, Ltd. for providing the full text of reviews from David Allsop's (Tabner et al., 2003) and Ashley Bush's labs (Curtain et al., 2003) that serve as background for this live discussion, which is part of an ongoing series spurred by the recent special issue of Current Medicinal Chemistry-Immunology, Endocrine & Metabolic Agents on misfolded protein in disease.

See Allsop Full Text (.pdf)
See Bush Full Text (.pdf)

Introduction

Evidence for the role of metals in the toxicity of fibrillogenic proteins has grown dramatically over the last decade or so. While dietary aluminum was once touted as a potential risk factor for Alzheimer disease (a theory that still has support, see our hypothesis pages for a new twist to the aluminum debate), there is now much better evidence linking endogenous copper, iron, and zinc with the biochemistry and toxicity of amyloid-β (Aβ) and other fibrillogenic peptides, including α-synuclein. Work from Ashley Bush's lab at Massachusetts General Hospital has shown that Aβ1-42 binds Cu(II) with extremely high affinity (attomolar dissociation constant), and that completely removing the metal from solution prevents aggregation of Aβ (see Atwood et al., 2000). Similarly, Dave Schubert at the Salk Institute, together with Mordechai Chevion at the Hebrew University, has shown that iron is required for the toxicity of Aβ (Schubert and Chevion, 1995). Zinc has also been shown to precipitate aggregation of Aβ (see Bush et al., 1994). Such observations are not limited to in vitro experiments. Larry Sparks at the Sun Health Research Institute recently reported that minute amounts of copper in the drinking water can induce aggregation of Aβ in rabbit brain (see Sparks et al., 2003 and ARF related news story), while the absence of synaptic zinc in mice expressing human AβPP carrying the Swedish mutations has been correlated with reduced plaque load (see Lee et al., 2002 and ARF related news story).

How do transition metals affect Aβ biochemistry and toxicity? Do they simply provide some additional glue to keep Aβ fibrils together, or do they have a more sinister role? What about redox chemistry? Though zinc has little or no redox activity, both ferric [Fe(II)] and cupric ions [Cu(I)] can take part in the Fenton reaction, the reduction of hydrogen peroxide to hydroxyl ion and hydroxyl radical. The latter is extremely reactive and can oxidize macromolecules at rates that are comparable to diffusion speeds. In other words, hydroxyl radicals are formed and wreak havoc in an instant. Could fibrils somehow catalyze the Fenton reaction?

At Bush's lab, Xudong Huang used a chemical assay to reveal that Aβ, in the presence of Fe(III), produces hydrogen peroxide (see Huang et al., 1999), one of the Fenton ingredients. This was confirmed in David Allsop's lab by use of electron spin resonance spectroscopy (see Turnbull et al., 2001). In these experiments, Allsop's group was able to demonstrate that toxicity and production of hydrogen peroxide go hand in hand. Aβ1-42 and Aβ25-35, for example, are neurotoxic and peroxide generators, whereas AA1-15 (a fragment of the Amyloid A protein) is neither even though it aggregates. Allsop’s group has also found that α-synuclein and certain toxic forms of the prion protein also generate peroxide (see Turnbull et al. 2003 and Turnbull et al. 2003 ), suggesting that there may be a toxic mechanism common to all these fibrillogenic proteins. Cu(II) can also contribute to production of hydrogen peroxide by Aβ, but because zinc does not, it has been suggested that formation of Aβ-Zn aggregates may offer some rudimentary protection against the Aβ toxicity (see Lovell et al., 1998). All told, the evidence points to iron- or copper-mediated production of hydrogen peroxide by Aβ, followed perhaps by Fenton chemistry to produce hydroxyl radicals.

These findings are also suggestive of a therapeutic intervention-metal chelation. Bush has pioneered the use of clioquinol, which was originally developed as an antibiotic, but which also acts as a metal chelator. It is much superior to common chelators such as EDTA (ethylenediamine tetraacetic acid) because it is small and hydrophobic, and readily crosses the blood-brain barrier. In fact, recent work from Bush's lab has shown that clioquinol can substantially reduce the plaque load in APP2576 transgenic mice (Cherny et al., 2001; see also ARF related news story). It has also been shown to protect mice in an experimental model of Parkinson disease (see Kaur et al., 2003 and ARF related news story). The drug is currently in clinical trials (see our clinical trial data page).

In this live discussion, we will debate the current theories and developments pertaining to metal-related biochemistry and neurodegenerative disease, including, but not limited to:

  • Exactly what forms of aggregates produce ROS?
  • Is aggregation-mediated ROS production widespread, or limited to just a few proteins, such as Aβ, α-synuclein, and prions?
  • How can the impact of redox and non-redox metals on sporadic neurodegenerative diseases be quantitated?
  • Why are only certain neurons affected?
  • Production of hydrogen peroxide by Aβ, and Aβ toxicity—coincidence, or cause and effect?
  • Why is clioquinol, a relatively weak chelator, so effective in animal models? Is it due to a specific interaction with Aβ, and if so, can this information be used to develop more potent compounds?

 

We thank David Allsop and Ashley Bush for jointly leading this discussion, and we encourage you to read their recent reviews (see Background text above).—Tom Fagan.

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References

News Citations

  1. Coping with Copper—Minute Amount of Metal Plagues Rabbit Brain
  2. Synaptic Zinc Fingered As Critical In Plaque Formation
  3. Two Ways to Attack Amyloid: Metal Chelator and Antibody
  4. Ironing out the Role of Metals in Neurodegenerative Diseases
  5. Copper to the Rescue?

Webinar Citations

  1. Role and Control of Metal-Mediated Fibril Toxicity

Paper Citations

  1. . Direct Production of Reactive Oxygen Species from Aggregating Proteins and Peptides Implicated in the Pathogenesis of Neurodegenerative Diseases. Curr Med Chem Immun Endoc & Metab Agents. 2003 Dec;3(4):299-308.
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  3. . 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.
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  1. . IEX-1L, an apoptosis inhibitor involved in NF-kappaB-mediated cell survival. Science. 1998 Aug 14;281(5379):998-1001. PubMed.