Posted 22 August 2006

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Live Discussion led by Bruce Kagan on 2 May 2005.

Participants: Tom Fagan, Alzforum; Bruce Kagan, UCLA Neuropsychiatric Institute; David Corbin, New York; Wonmuk Hwang, Texas A&M University; Nico Stanculescu, Alzforum; Charlie Glabe University of California, Irvine; Larry Nault, Matrix Integrated Linkage LLC; Ratnesh Lal, University of California at Santa Barbara; Dave Teplow, University of California, Los Angeles; Ben Albensi University of Manitoba; Rose Xiao, Memory Pharmaceutical; Yuri Sokolov, University of California, Irvine; Ryan Lee, W3C/MIT CSAIL.

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

Tom Fagan
Hi, Bruce! Hi, Charlie!

Bruce Kagan
Hi, Tom!

Tom Fagan
Okay, if you didn't guess by the microphone icon, I'm Tom Fagan and I'll be moderating today when necessary. Bruce is going to start us off with a brief introduction and then let the fun begin. Take it away, Bruce!

Bruce Kagan
In 1993, Arispe et al.,1993 reported that Aβ formed ion channels, and proposed that channels could cause cellular pathology and toxicity. Abundant evidence now supports this view, including the facts that virtually all amyloids form channels and that Aβ can inhibit LTP, or long-term potentiation (see ARF related news story), depolarize neurons, allow cytochrome c efflux from mitochondria (see Kim et al., 2002), and kill cells. Channel formation readily explains most amyloid pathophysiology, including memory disturbance, calcium dysfunction, membrane depolarizations, increased reactive oxygen species (ROS), sensitivity to toxins, and apoptosis.

Larry Nault
Bruce, do these channels have a hypothetical geometry. A pipe or a duct? Bounded by what?

Bruce Kagan
Larry, Arispe and Guy proposed several possible models for the pore. Recently, Arispe has published evidence that specific regions of the peptide seem to line the pore (see Arispe, 2004).

Tom Fagan
Bruce, how many channels does it take to kill a cell?

Bruce Kagan
Tom, it depends on the size of the cell. The Aβ channels are large, electrically, and would create a significant leak. A single pore could reduce the sodium (Na+) concentration by about 10 micromolar per second.

Tom Fagan
Bruce, how does that compare to normal leakage?

Bruce Kagan
Tom, it again depends on the cell. For a neuron, which must maintain a tight membrane for signaling, a single pore would pose a significant but not lethal leak.

Wonmuk Hwang
How specific are these channels? I understand that they have a distribution of sizes, which likely cause them to be nonspecific.

Bruce Kagan
Won, the channels have been described as permeable to a variety of cations including sodium and calcium. There is little selectivity amongst cations.

Tom Fagan
This fits in with what Charlie reported recently, right Charlie?

Charlie Glabe
We see fluorescent dyes that are about 600 Daltons (Da) leaking; it is not ion-specific (see ARF related news story).

Tom Fagan
Charlie, 600 Da is pretty big; do you mean such molecules are getting through these pores?

Charlie Glabe
Yes, going both ways; I'm not sure of the mechanism. Large proteins like lactate dehydrogenase (LDH) do not leak detectably.

Bruce Kagan
Charlie, we see non-electrolyte leakage of sugars with a diameter up to about 10-12 angstroms. Of course, we should temper this with the observation that we see many species of channels and they may all have different sizes and selectivities.

Tom Fagan
Bruce, so are these channels in a state of flux? Can their size change dynamically with time, and might there be large and small coexisting in the same cell?

Bruce Kagan
Tom, Charlie sees a very different result from ours, but Joseph Kourie has described at least three different Aβ channels with varying kinetics, selectivity lifetime, and so on (see Kourie et al., 2002).

Tom Fagan
Everyone, any ideas on how these channels can be stoppered? Would this be a therapeutic strategy?

Charlie Glabe
We haven't seen any known channel blockers that inhibit the permeabilization; but that doesn't mean that there aren't any.

Bruce Kagan
Tom, the only blockers we have so far are very nonspecific, such as zinc (Zn2+) or tromethamine ions (e.g., Tris buffer). The development of more specific blockers could prove useful as a therapeutic strategy. Zn2+ does block the toxicity of Aβ on fibroblasts (see Zhu et al., 2000).

Wonmuk Hwang
About Charlie's reply: How about the oligomer-specific antibodies that you published? Don't they block permeabilization?

Charlie Glabe
Won, not known, but that is a reasonable hypothesis. We find that you need to start with oligomers first. I'm not sure that they insert into the hydrocarbon region in a traditional fashion, either.

Ratnesh Lal
Calcium uptake has been blocked specifically by anti-amyloid antibody and zinc in both liposomes and cells (Rhee et al., 1998; Lin et al., 1999; Lin et al., 2001; Bhatia et al., 2000).

Tom Fagan
So how does the channel hypothesis fit with the slow progression of the disease? Given the degree of leakage, it would seem that channels would be pretty lethal and that the disease should progress more rapidly, or am I being too simplistic?

Charlie Glabe
Maybe a small leak just contributes to chronic stress as the cell has to pump more to keep up with the leak.

Bruce Kagan
Tom, Dennis Selkoe's group has shown that oligomers can impair memory in a reversible fashion. Since we know Aβ channels can inhibit long-term potentiation (LTP), this might be the first step in the pathology (see ARF related news story and ARF news story; also Wang et al., 2002).

Tom Fagan
Bruce, but in the LTP experiments, basal transmission is okay, which seems odd, given the degree of leakage you could get. I wonder if there is something modulating the channels?

Bruce Kagan
Tom, the LTP experiments require very small doses (less than toxic) of Aβ. I suspect that they are altering membrane potential in a subtle way to inhibit LTP, but not enough to derail normal transmission.

Tom Fagan
Ah! Possibly.

Wonmuk Hwang
About channel formation: Is it known whether they are formed in solution, then incorporated into the cell membrane, or is it more likely that channels are formed through interaction with the membrane or the substrate (in case of in vitro experiments)?

Ratnesh Lal
Evidence is published suggesting that both preformed oligomers can insert in the membrane or monomers can insert and oligomerize to form ion channels.

Bruce Kagan
Won, there is clear evidence that the presence of lipids or membranes affects the folding of Aβ and other amyloids, tending to promote β-sheet and oligomerization (see, e.g., ARF related news story on lipid rafts and ARF news story on the effects of lipid anchors on prion amyloid formation).

Wonmuk Hwang
Bruce, that is consistent with the finding that fibrils form in partially denaturing conditions—possibly the hydrophobic environment of the lipid tails enhances the condition for fibril formation.

Charlie Glabe
We find that monomers are pretty inert. It isn't necessarily easy to make pure monomers.

Bruce Kagan
Charlie, we find that monomers are inert also, but that with time in aqueous solution, oligomers form and insert into the membrane.

Ratnesh Lal
As mentioned above, there is published work showing that monomers can induce cell toxicity as well as calcium uptake in both cells and liposomes.

Charlie Glabe
Ratnesh, how were they sure they were monomers? They may start out that way, but things change pretty rapidly even in the absence of membranes.

Bruce Kagan
Ratnesh, how can you be certain that the peptide remains as a monomer throughout the experiment?

Ratnesh Lal
I don't think they remain monomers throughout the experiments. Instead, they do oligomerize. We have imaged those monomers in real time using atomic force microscopy (AFM). We have shown that for an extended period of time, in physiological buffer and in physiologically relevant concentration, they do not form oligomers. Nevertheless, as I mentioned above, monomers will oligomerize once in the association with lipids in the bilayer/liposomes.

Bruce Kagan
Ratnesh, so that would be consistent with all the recent work suggesting toxicity of oligomers?

Ratnesh Lal
Yes, that is all we have published in the last 7 years.

Wonmuk Hwang
Ratnesh, I think that Aβ peptides are produced throughout the lifespan.

Ratnesh Lal
Membrane-induced conversion from inert to an active form will be consistent with the expected conformational change in many amyloids associated with misfolding diseases. Won, I agree with you and have made that point in our earlier publications. Aβ peptides are produced throughout the lifespan and in many, if not all cell types.

Ben Albensi
All, so is it just the induction of LTP that is affected or later stages, as well?

Bruce Kagan
Ben, I believe it is just the induction, but I could be mistaken. Dave, do you know?

Tom Fagan
All, how many monomers does it take to form the simplest channel—in theory even, if there is no experimental data?

Ratnesh Lal
Tom, a model presented by Durrel et al.,1994 suggests that it can be anywhere from tetramer to octamer and more (perhaps up to 12).

Tom Fagan
Ratnesh, is a tetramer big enough to form a pore and span the membrane?

Bruce Kagan
Tom, Aβ42 tends to form hexamers, and those would be excellent candidates for the smallest channels.

Charlie Glabe
It isn't clear that it has to span the bilayer in the traditional sense. How do you explain the fact that polyQ permeabilizes membranes in the same fashion?

Tom Fagan
Charlie, I don't know. Any ideas?

Charlie Glabe
Maybe the same reason that highly charged, polar peptides like HIV tat and Antennapedia can cross the bilayer with large passenger molecules attached to them? (See review on cell penetrating peptides).

Tom Fagan
Charlie, are you suggesting we abandon "the channel" hypothesis and call it something else?

Charlie Glabe
I don't know; this is something we are still trying to sort out.

Ratnesh Lal
There is a model by Mobley et al. suggesting that monomers may or may not span the whole membrane. If they do not, it can still form an ion channel (like porins or others); otherwise, one would need an additional four monomers from the other membrane leaflet (and in that case it would be eight monomers total), or six or more, depending upon whether they form tetrameric, pentameric, hexameric, or higher order oligomeric channels.

Bruce Kagan
Charlie, the polyQ story is striking in that there is a minimum length for pathology, yet this minimum varies from illness to illness. For Aβ peptides, we have shown that the minimum length to obtain channels is nine residues, which would span the hydrophobic core of the bilayer in β-sheet form.

Wonmuk Hwang
How about the pure dimensional argument? The hydrophobic region of a lipid bilayer is about 3 nm, and if the channel diameter is about 2 nm, the oligomer channel must have about 36 nm^2. Given the size of Aβ, we can guess how many monomers are needed to span a bilayer like a channel. But as suggested above, even if the oligomers occupy only one leaflet, it is still possible for there to be a hole, through fluctuation of the lipids. In this case, the hole will be highly dynamic rather than statically open.

Ratnesh Lal
Won, the pore diameter of 2 nm is quite big for any channel. The outer diameter of the oligomeric channel diameter would be consistent with what we had published earlier (see Lin et al., 2001). The inner (pore) diameter will be considerably smaller (~1-1.5 nanometer).

Wonmuk Hwang
Ratnesh, I was referring to Hilal, Lansbury and colleagues' work on torroids—their electron microscopy (EM) images suggest that the inner diameter is about 2 to 3 nm (see, e.g., Rochet et al., 2004).

Ratnesh Lal
Won, Lashuel's work is done on annular pores; peptides were never associated with any membrane (either before or after). For their actual channel conformation, they refer to our work (Lin et al., 2001).

Charlie Glabe
If peptides that do span the bilayers are extended β structures, you would expect that they would have to form H bonded β barrels, which typically have ~20 strands.

Bruce Kagan
Charlie's point is well-taken. This would predict very large channels indeed.

Dave Teplow
Caution to all—there is a distinct possibility that Hilal's work, while relevant in in vitro systems, is not representative of what occurs in vivo.

Tom Fagan
Charlie, I was just going to ask about molecular models that would have a pore. If the pore idea is correct, then wouldn't it be possible to estimate how many monomers would be needed, and how would that fit with current ideas on trimers, dodecamers, etc., that seem most toxic?

Charlie Glabe
Uli Aebi tried to visualize pores or channels by AFM using preparations and conditions that caused dye leakage, and he did not observe "donuts." Instead, he saw defects in the membrane radiating from the oligomers (see Green et al., 2004).

Ratnesh Lal
Charlie, there is a serious difference between Aebi's work and other channel work. In his work, amyloid was added to membranes that were preformed and adsorbed on a substrate, a common practice for AFM work. On the contrary, when you reconstitute in liposome or bilayer and then image with AFM, you do see channels (Lin et al., 2001).

Wonmuk Hwang
In relation to Tom's question, what is the shortest length of a model peptide tried that forms channels?

Bruce Kagan
There is a report of an Aβ31-35 fragment forming channels. I don't know what to make of this unless there is one peptide in each leaflet of the bilayer (see also Le and Qiao on Aβ31-35 effects on LTP).

Tom Fagan
Charlie, Bruce, have either of you examined the effect of different lipids on leakage? Sorry, I'm not remembering the data off the top of my head.

Charlie Glabe
Yes, not much specificity. Yuri can jump in on this if I have misspoken.

Bruce Kagan
Tom, cholesterol inhibits. Negatively charged lipids are required for activity. Not much other specificity.

Ratnesh Lal
There are some publications from the Arispe group on the effect of cholesterol on amyloid insertion and channel activity (see Arispe and Doh, 2002).

Bruce Kagan
Charlie and Yuri, does membrane fluidity affect your results?

Yuri Sokolov
Bruce, we did not study the effect of membrane fluidity. But recently we found very strong lipid-dependence of amyloid-induced conductance in bilayers.

Tom Fagan
So what about other channels besides Aβ ones? What have we learned from those? Do they clarify anything or just make things more confusing?

Bruce Kagan
I am struck by how similar the channels are that we see from a variety of unrelated peptides. Their physiologic properties are nearly identical. They are all "leakage" channels.

Charlie Glabe
I second that. All amyloids are pretty similar.

Larry Nault
Must close out. Interesting hypothesis. Clearance of building channel blocks may be the key to defeat of progression. Building channels over time and diving then through the bilayer is a process that might be interrupted with agents like zinc or by clearing essential oligomer building blocks.

Ratnesh Lal
There is published work on many other amyloid proteins showing channel-like activity (Kagan's lab, Arispe's lab, Kourie's lab) and we have structural data from AFM study to confirm channel-like structures.

Tom Fagan
Ratnesh, what do you mean by "channel-like."

Ratnesh Lal
Tom, as the AFM work is purely structural and we do not do electrical recording simultaneously (I mean not on the same structures because we don't have an appropriate recording system), we take a conservative view that it is channel-like. However, in parallel studies on the same batch of specimen, electrical recording shows channel activity.

Bruce Kagan
Tom, in the case of our work and that of Arispe and Kourie, these are clearly channels by all the standard criteria. Charlie's work clearly shows permeability without "channels."

Tom Fagan
Ratnesh, I thought you were referring to a channel-like structure rather than activity.

Ratnesh Lal
Tom, we do both: image the three-dimensional structure and in parallel do electrical recording. We are hoping to do both together someday soon.

Tom Fagan
Great, Ratnesh; keep us posted!

Ratnesh Lal
Thanks, Tom. We will be in touch.

Bruce Kagan
Tom, one thing we have learned from complement and toxin studies is that "holes" that one sees in EM do not always correspond to the electrical pore pathways.

Tom Fagan
All, we are nearing the end of our hour, but we can keep chatting as long as we like. But before folks start rushing off, I just want to thank you all for coming, and Bruce for agreeing to host this chat.

Wonmuk Hwang
Is there any evidence that the channel-like structure must have torroidal morphology? How about spheroids? And thanks to all for useful conversations.

Tom Fagan
So before we go, what are the crucial experiments that need to be done to advance our understanding of what is going on? Anyone, everyone?

Bruce Kagan
I think there are two crucial experiments to be done. First, specific channel blockers need to be developed and tested in cell and animal models. Second, channels (or permeability changes) need to be found in animal models of disease.

Dave Teplow
Bruce, are there any meta studies that could be done examining patients taking channel blockers for other diseases that would answer your question?

Bruce Kagan
Dave, that's a great idea! However, for the easiest ones—the calcium channel blockers—we have already tried them without success.

Tom Fagan
Bruce, tried them in vitro?

Bruce Kagan
Tom, right, we tried them in vitro against our Aβ and other channels.

Tom Fagan
All, we talked about channel blockers, but are there other strategies to stop the leakage of these "channels"? Also, are there synthetic channels that could be made to mimic the natural ones, and how might that be useful?

Ratnesh Lal
Antibodies do block, and one can use peptides to block, as well (a practice useful in blocking gap junction ion channel activity).

Bruce Kagan
Tom, yes, one could devise treatments that made the bilayer less susceptible to channel insertion. Arispe did this recently with robust effects on toxicity. He stiffened the cell membrane with cholesterol (see Arispe and Doh, 2002).

Yuri Sokolov
Jim Hall and I found that an increase of bilayer thickness inhibits the effect of amyloid-β in bilayers.

Bruce Kagan
Yuri, what's the latest on what ions and other molecules can go through your Aβ-treated membranes?

Yuri Sokolov
Bruce, looks like everything we tested. HEPES, Tris, TEA....

Tom Fagan
Yuri, you do this by using longer chain lipids?

Yuri Sokolov
Tom, looks like with longer chains we have only preliminary data. But definitely the effect of amyloid-β is completely blocked by the saturation of bilayer with decane. We are not going to use decane as a treatment, but it is good enough to study the mechanism.

Tom Fagan
Yuri, yes, for sure, anything that sheds some light on what's going on....

Bruce Kagan
Tom, there are other ways to increase membrane thickness, such as changes in dietary lipids.

Tom Fagan
Bruce, that's where I was headed. But you said that you tried different lipids and saw no specific effects, right?

Bruce Kagan
Tom, yes we did try many lipids and didn't see major effects except of charge and stiffness, but we did not try to alter membrane thickness (one needs lipids with long tails). We should go back and do that. Good idea!

Tom Fagan
Or you could use shorter ones to see if the problem is any worse.

Bruce Kagan
Right. I know these manipulations to change lipid composition can be done in animals, but I don't know if they have ever been tried in humans. An interesting prospect! It is interesting to note that increased membrane thickness would inhibit standard channels as well as Yuri's permeability increase.

David Corbin
Bruce, have you also considered the systemic effects in Alzheimer's of impaired calcium transport by fibroblasts (see Peterson et al., 1985)?

Bruce Kagan
David, I'm not familiar with this paper. Do you think it might be evidence of Aβ channels in the fibroblasts?

Ratnesh Lal
At least in in vitro studies, we have shown Aβ-channel-mediated toxicity in fibroblasts (Zhu et al., 2000).

David Corbin
Bruce, I do not know. I was just looking at some of the early research on cell membrane abnormality in Alzheimer's to see what others have observed, such as decreased microviscosity.

Bruce Kagan
David, I'm going to look up that paper. Thanks for bringing it to my attention. Goodbye to all.

Tom Fagan
Okay, I think we should probably wrap up. Thanks, Bruce, most interesting. Thanks to everyone for joining in; I hope it was useful.

Nico Stanculescu
Thanks, Bruce, and thanks, Tom!

Bruce Kagan
Nico and Tom, thanks so much. I enjoyed this immensely!

Ratnesh Lal
Thanks all!

Nico Stanculescu
Splendid! We will be forwarding the transcript for review!

David Corbin
Thank you.