26 Jun 2003

Alzforum scientific advisor Vincent Marchesi is currently director of the Boyer Center of Molecular Medicine at Yale University School of Medicine. When his wife, a doctor herself, was diagnosed with Alzheimer's Disease eight years ago, Marchesi developed an abiding interest in AD research. Marchesi brings stellar scientific credentials to his position as independent observer, and he has no grants, manuscripts, or study section seats to protect. Read what this friend and occasional critic of the AD field has learned about the status of AD research.

Q&A

ARF: First, can you tell us about yourself, what your past and present research interests have been?

VM: I have been mainly working on membrane proteins for many years since the 1970s. Even before that, I worked on inflammation, so I have a feel for a lot of different processes. I'm also a pathologist, and was the Chief of Pathology at the Yale-New Haven Hospital for some years, so I'm familiar with human disease as it appears clinically. But my main focus has been protein analysis, protein chemistry with emphasis on the cytoskeletal proteins that support the red cell membrane. I also worked on a protein called glycophorin, which is one of the first transmembrane proteins to be sequenced back in the 1970s. What is going on in many of the areas in Alzheimer's research are basically variations of things that we worked on in simpler systems years ago.

My interest in Alzheimer's came from personal reasons, but I feel comfortable looking at a lot of the work going on in the field as an informed outsider. I'm not pretending to be an expert coming in to tell everybody what's going on, but I do think that there's a certain kind of rigor and validation which should be done by people like me, who have done comparable things in other fields and do not have an investment in this area.

ARF: Complex transmembrane proteins, changes in membrane composition and trafficking, and inflammation, are central these days in Alzheimer's research, and they are difficult to study well.

VM: You're right. Presenilin and g-secretase still puzzle me, and these are problems I used to think about years ago. We were very concerned with how lipids interacted with proteins in membranes. In fact, a long time ago we really worked out the first interactions between transmembrane components. I'm amused because many of the things we worked on are turning up again now, but I don't think there's enough of a connection between what people are doing today and what has been done in these other, simpler systems.

ARF: Because people aren't harking back to old literature?

VM: I don't want to sound like an old fogy, but one of the problems in the daily reality of doing science nowadays is keeping track of what others have done. Never mind the "ancients," I mean people who have done things last week! There is just so much being published every week.

In fact, one thing I'm most interested in doing with the Alzheimer Research Forum is to help figure out new ways we can use information, to store, recall, analyze it. This is my big obsession right now. We are doing a huge amount of experimental work in many areas, and much of it is going unnoticed in the sense that it isn't being used by people other than those who actually make the observations. This is a mistake. Also, it really amounts to squandering valuable assets. The United States public spends $30 billion a year on biomedical research, and more effort should be made to mine the information that now exists. If I make a contribution to the AD field, it could be in that area more than anything else.

ARF: Information management and finding what you need, we're working on developing new tools for that.

VM: That's what attracted me to Alzforum. I just happened on it and couldn't believe how interesting, informative, and how wide-ranging it was. I think this is the best disease site I've ever seen—and I look at many sites. I spend a lot of time looking at databases and online sites.

ARF: Wow! Well, thank you. We're trying to make it better. There's still so much more to do.

You have observed other disease research fields over the years. What strikes you as particular to AD?

VM: I think two diseases most relevant would be atherosclerosis and the muscular dystrophies. Atherosclerosis is interesting because it had a huge boost with Brown and Goldstein's discovery of the LDL receptor—and the role it plays in homozygous form of hypercholesterolemia-which won them the Nobel Prize in 1985. That really led to this notion that lipids are the prime movers and basis for the atheroma.

It's interesting that recent findings have completely changed the picture now. It's beginning to look as if the atheroma is an epiphenomenon or a secondary reaction, and it's quite likely that cholesterol is going to be a minor player as inflammatory reactions become much more important. It's possible, even likely, that the fibrils of the Alzheimer's plaque may turn out to be comparable to the cholesterol ester crystals that accumulate in atheromas, with the accumulations of fibrils being secondary to some other pathogenic mechanism.

The muscular dystrophies are also interesting, and, incidentally, they are still very much underinvestigated. I lecture on protein folding disorders in a course for Yale undergraduates, so I've spend a lot of time reading in these other fields. Dystrophin is one of the first genes associated with muscular dystrophy, by Lou Kunkel of Harvard. Dystrophin is exactly like spectrin, a protein I co-discovered. We worked out a lot of the details of spectrin/actin interactions with red cells.

If you now look at the work on dystrophin and all its interactive proteins, you'll see that the proteins that make up the muscle membrane skeleton are quite similar in character to those of the red cell membrane skeleton. The players are different and they are greater in number, but everything is the same in concept. Lou discovered the gene for Duchenne's muscular dystrophy, in which people are born without dystrophin and suffer atrophy from the beginning. But there are forms with other kinds of variations—many different variations, by the way-where you see something comparable to Alzheimer's disease in that the disease process develops more gradually. The muscle cells slowly die over the course of years, not months or weeks, and people who have these types of mutations last up to 15 years. Their problem is that the muscle proteins become unstable. The membranes are unstable and can't connect to the outside connective tissue. It's a slow process reminiscent of what people think is happening in Alzheimer's-the so-called progression, the age-dependent change. The muscular dystrophies would suggest that just AbPP is far too simple a window to look through. I know there are now four proteins that are connected together.

ARF: Yes, presenilin, nicastrin, aph-1 and pen-2 make a complex that generates the enzymatic activity.

VM: I think there's going to turn out to be many more than four, eventually, when people start looking more carefully at it; there'll be a lot more complexity. The idea of the presenilin sitting in the membranes up against the APP protein and chopping it up into pieces is inadequate when you think about the complexity of, say, the muscle membrane protein complexes or the huge number of proteins that are believed to be interacting with the T cell receptor. Do you know of the so-called immunologic synapse, where you have the T cells interacting with the helper cells interacting with the dendritic cells?

ARF: Yes.

VM: If you look there, you'll see dozens of proteins interacting with each other in the course of the lymphocyte activation, the whole NFAT (nuclear factor of activated T cells) chain of events. So the principle of multiprotein interactions being pivotal needs more attention in the Alzheimer's field.

ARF: What are the methods that have been productive in studying multiprotein complexes in these other areas that the Alzheimer's field could import more into their own work?

VM: That's a good question. One of the problems of the Alzheimer's field is that we're looking at a very complex clinical problem as if it were largely an anatomical change in a tissue that's extremely inaccessible. Focusing on anatomy is like the nineteenth century in some ways. We're using anatomical markers such as plaques, tangles, and neuronal loss, and trying to relate them to a very complex clinical course. It is really very complex—you could easily say there are multiple different diseases going on here, or that Alzheimer's is a series of different causes that could wind up having a common final pathway.

Getting back to your question: it's going to be hard because, for one thing, scientists nowadays are addicted to simplicity and are definitely inclined to study things that work. There's nothing wrong with that. No smart investigator would work on a problem that's insoluble. How would you get a research grant? Not to be facetious about it, but I think this mentality really is dictating a lot of scientific research these days. People are working on projects that are less risky than they might have in the past. The worries about getting funding are terrible nowadays. It means that the question you're asking—which is a really interesting one—is too hard to approach right now. A lot of people are actually looking more at surrogate markers, not at the real thing. I'm concerned about this amyloid oligomer story, for example. Do you recall that?

ARF: I was going to ask you about it.

VM: I can hardly believe most of the papers that are published in this field. Spectrin, a protein I worked on for years, also forms oligomers. You can measure them, you can identify them, you can analyze them, but none of those criteria are satisfied in the present AD oligomer story. Thioflavin binding—very non-specific, hardly used in other fields—is one of the principal assays. People are trying to find some inroads, so they're using these surrogate markers, and they're getting signals. The trouble is, some of these signals agree too well with some of the theories. So, if you wind up having thioflavin positivity and you say, ah, this reflects hydrophobic peptide packing, and then you look at the CD spectrum and find the pattern is consistent with b-sheet conformation, you conclude that you have specific aggregates of b peptides. None of that would have been accepted in the days when you really knew what you were looking at. So I'm skeptical of the oligomer story.

ARF: Skeptical of exactly what? That the oligomers are pathogenically relevant? That they are neurotoxic? Because if a monomeric protein fibrillizes, there must be dimers, oligomers, and protofibrils along the way—that's how they polymerize. So what part of the story do you find unconvincing?

VM: I don't understand how these intermediates have been identified, and how anyone can say that they have preparations of oligomers that don't have monomers or higher forms in them, also. Have you seen the present issue of Science?

ARF: Are you referring to Charlie Glabe's antibody? We've covered it, with comments by other scientists (see ARF related news story).

VM: I didn't speed-read it. I spent three hours studying it, including all figures and online material. I'll bet not too many people have done that. Not to sound cynical, but it's easy to read an abstract and the last few lines and say, wow, this is interesting. To go back and look at the data takes a lot more work. I am very intrigued by that idea, probably because it's so outrageous, that there is some kind of protein conformation that hasn't got amino-acid specificity. If it really is true it would be a fantastically important discovery and extremely important to track down. But really, one can't believe it yet on the basis of what they're showing in the paper. They haven't characterized that antibody well enough.

ARF: In your view the data don't support the conclusions?

VM: Sufficient data aren't shown.

ARF: What do you think should have been in the paper?

VM: For example, they didn't actually show that they could block the antibody with the original peptide. That's not in the paper or in the online material. The reviewer should have asked for that. The dot blot in my opinion is a poor man's way of doing immunochemistry, but maybe it works. Sometimes you have to do less than beautiful experiments; I understand that. In any case, you're obliged to show that the original peptide—and not just the peptide, by the way; they're claiming that it's a peptide in a particular conformation-blocks.

They state in the paper that monomers and protofibrils are not reactive, but that has to be shown. Plus, they're claiming that this is true for the insulin peptide, the amylin peptide. They should have shown that the oligomeric forms of those peptides block the dot blot. Now, I can't believe they didn't do it, the authors are just as competent as I am, so I wonder what was the result? They're also claiming that the other amyloidogenic peptides do the same thing. But they didn't describe how they prepared the other peptides; remember, the oligomer has to be prepared in a certain way. Using the gel filtration scheme that they show, they didn't say that they did that for the other peptides. It's puzzling for them not do those experiments and claim they did them, or to have done them and not put them in the online background material.

Moreover, they immunized rabbits 12 times. It is unusual to inject that often, and the synthetic peptide they immunized with was described as being 95 percent pure. That means they immunized a dozen times with something that has 5 percent contamination. It is extraordinary that they wound up with an antibody that is so specific. I believe this result still needs more substantial evidence.

Finally, there are lots of ways to look at any of these antibody complexes with synthetic peptides and really make a strong case for specific binding. This, of course, is a huge amount of data validating binding that you wouldn't expect to see in the first report. And yet, because their claim is so extraordinary—probably the most extraordinary claim in recent years—such rock-solid data is important. If such an unusual structural epitope really exists, it would be at the same level of importance as the prion concept, where one peptide is changing the conformation of a second. In fact, that is eventually what their concept is getting at, though they didn't state that explicitly. And it is a good idea, since there is literature showing that amyloidogenic peptides will actually induce others to do this. An interesting paper by Per Westermark shows this (Lundmark et al., 2002). So, this is such an extremely important claim that I would like to see substantiated.

ARF: There are also papers showing that in in-vitro experiments a-synuclein and tau synergistically enhance each other's fibrillization, and of course there's overlap in the pathology of various tauopathies and dementia with Lewy bodies that have both types of proteins aggregated and deposited. Masliah has done that, John Trojanowski and Virginia Lee, and Kurt Jellinger, and other groups are studying that at different levels (see, for example, ARF related news story).

VM: a-synuclein is a very interesting protein because nobody has ever been able to establish its conformation. It's turning out that there are a number of so-called natively unfolded proteins that seem to be always unfolded or partially folded. (See Alzforum live discussion) That itself has its implications, because it almost guarantees that there are going to be non-specific interactions at some point, unless there are no chaperones around. That's another thing: People have ignored the chaperone concept, and I don't understand why more work hasn't been done in that area, because we're talking about proteins that are unfolding and exposing hydrophobic moieties, and that's what chaperones do. They prevent hydrophobic peptides from aggregating together. So, I think chaperones are going to be one of the next big, hot areas.

ARF: I already see them coming up in the neurodegeneration literature quite frequently these days. Our last interview was with Sue Lindquist, who also thinks about this quite a bit.

VM: Yes, she's good. She's doing yeast work, where you have a chance to do good genetics and make some manipulations. Have you seen the latest thing they've done? They now make fibrils, artificial fibrils, of those proteins.

ARF: The nanotubes made out of amyloid? They used them to cast silver wires� Two papers just came out on that topic and we solicited comments on them, see at Reches and Gazit, 2003.

Where do you think that the AD field has been especially successful and made real progress in recent years? Does any area impress you particularly?

VM: Don't draw the conclusion that I think all is bad. To tell you the truth, I'm amazed. I knew little about Alzheimer's disease until my wife came down with it, even though I knew George Glenner when I was at NIH. In fact, Glenner told me about these Ab peptides in 1970. I thought he was nuts at the time.

ARF: Many people did.

VM: I didn't know anything about this field four years ago, and I'm amazed at how much has been done. I don't know everything, but I know a lot. I've spent a lot of time going through much of the recently published work. Alzheimer's research is my principal interest right now. You asked me before what I do. I'm very interested in the regulation of calcium channels. That's my other criticism, by the way, of some of the material on Alzforum. I have read some old-fashioned concepts about calcium there.

ARF: I was going to ask you about calcium...

VM: You have some contributors writing about calcium in an outdated way. There's a whole lot of excellent work going on with calcium now, on how the channels are regulated and redistributed. There are good people working in this field, Mark Mattson is one of them. But some of the comments I've seen by others are not that accurate. The idea to use non-specific calcium channel blockers, for example, is an old-fashioned notion.

ARF: Point taken. Can you give the interested scientists in the field, who may look to us for ideas from time to time, some calcium-related angles or connections that investigators could pick up on?

VM: I'd have to think about that before answering properly. One thing is that everybody's realizing now that exocytosis is a big issue. The process of exocytosis occurs in synaptic vesicles but also in T cell killing, for example. All those processes involve secretion. The crux in exocytosis is where the membrane vesicles fuse with the surface membrane. It's always been thought that the process of fusion is the only thing calcium is involved with but even then, it's always a guess as to what's going on. I think that field is changing, and there's lots of interesting things unfolding with how the exocytosing vesicles get to the surface, what are the connections, and how the cytoskeletal proteins on both sides of membranes mediate each other. I think there's going to be a real story there. I don't see people thinking along those lines in Alzheimer's, but perhaps that's just not visible in the literature quite yet.

ARF: What would be the relevance for Alzheimer's? That if something went wrong there, synaptic transmission would be affected early on?

VM: Yes, the thing that Dennis Selkoe has been reinforcing lately-let's go back and look at synaptic transmission—I think that's right (Selkoe, 2002). That may be the early step to seeing functional changes, which we can see in Alzheimer's before the cells die, and then we see the amyloid deposits.

Now, some people say-and that's the other issue linking back to the oligomer story—that the oligomer is toxic. I don't think the data supports that. Even the Glabe paper we talked about, they use LDH release and a cookbook-type of test for cell viability. One really has to look at the cells themselves carefully and ask: what is happening ? Like the cells in that paper that were rescued by the antibody; that's an amazing phenomenon, but what do those cells look like? What are the antibodies interacting with? So, I think people have got to dig in more and look more carefully at what they're talking about when they say these oligomers are toxic.

There's a whole new story in apoptosis about the apoptosome, a huge protein complex. Again, this goes back to the idea of large complexes, the proteasome, the apoptosome. All these somal bodies involve multiple protein interactions. I think that is where you've got to dig in more than just looking at the release of some non-specific intracellular protein, or just look at cytochrome C release. That is what I'm saying: If you want to claim you've identified a toxic component, first of all, you should really know what that is, if possible (though that may not be easy). Secondly, you should be looking at the dynamics of the process, not just the most convenient endpoint.

ARF: But experimentally, how do you approach that? As you say, the target tissue is inaccessible. It's not like with an erythrocyte, where you can study the complexities of a cytoskeleton and how it changes in response to whatever is happening at the other side of the membrane. You can study that quite nicely in an isolated cell, and in case of the blood cell that has real physiologic meaning, but how do you do it in the adult brain? What are the models? What are the systems to approach the question?

VM: I don't think you can do it in the adult brain, unless you're able to do sophisticated imaging techniques, but you probably can do it neuronal cultures that now exist. I think much better use can be made of neuronal cultures. There's a lot of good work now showing that neuronal cultures survive. But again, Gabrielle, it's easier to just use a neuroblastoma cell because you can buy it and grow it up, but if you really want to find out what's happening in neurons, you've got to look at neurons. You can investigate synaptic vesicles, you can investigate all the proteins that regulate synaptic reactions—I'm not saying it's easy but it's doable. The gravity of the problem justifies the extra effort. It's too important to be studied in a casual way. It's a harsh way to put it, but that's basically how I feel.

ARF: A majority of researchers consider the amyloid hypothesis the leading contender to explain the pathogenesis, and it seems that pharmaceutical companies are framing their strategies around that. How strong does the available evidence look from your vantage point?

VM: This is obviously something to be pursued, just as you shouldn't eat a dozen eggs every morning. I think amyloid generation can be a factor just as cholesterol-atheroma formation is a factor in vascular disease. And yet I also think there could be a lot more going on coincidentally with that, which is equally important. It might even be more therapeutically accessible. I'm not saying let's toss out what people have done. I'm saying look at what you've done more carefully, be more rigorous, and be more critical of what you've done, in the hopes of finding truth and also something new.

For example, going back to the scientists who wrote the Science paper: They may have stumbled onto something fascinating with the antibody they have prepared. It's quite possible there's some factor they've generated that is actually very toxic, and they have an antibody to it, but they may be missing it because they're assuming that it's some funny conformational form of a known protein. So all I'm saying is, be careful, be rigorous, and work harder at it.

ARF: On the calcium work, in Alzheimer's two areas come to mind. One, for example, is work suggesting that the intracellular fragment of AbPP regulates intracellular calcium and that presenilin mutations, in addition to generating more Aß, cause a calcium overload in the ER. Then there's work saying that increased intracellular calcium activates calpain and starts cell death pathways. Do you think there are therapeutic targets around calcium that may be more profitably exploited?

VM: I certainly do. Again, as I said before, there's a tendency to use these non-specific calcium blockers like verapamil. But there are about a dozen different calcium channels. They have many different subunits, and so forth. There are a lot of possibilities for specific intervention, but you've got to dig them out. The heart is a great example. That's where the calcium story is best studied-the sarcoplasmic reticulum and and the ryanodine receptor-I think it's a huge area of great interest, but it's complicated.
Also, the use of fluorescent calcium indicators is going to be fascinating. I read a series of amazing papers by Howard Petty at Wayne State University in Detroit. They show calcium bursts in phagocyte cells and the effects of binding to the IgG receptor, (see, for example, Worth et al., 2003). The technology is demanding, but it's possible to do these things. What he has done with phagocytes you could easily do with neuronal culture and see whether these amyloids really do change the flux patterns or modify wave configurations.

The trouble is, it's going to take people who have the amyloid oligomers to couple up with guys like Petty, and that kind of serious collaboration goes against the grain of typical academic research. We do not have a great culture of collaborating in the United States. It goes back to the system of "you've got to be independent and you're not going to get promoted unless you are." Its possible that some of the foundations that support research in AD might be able to promote specific interactions.

ARF: This merging of techniques and applications does happen quite easily at the level where an established Alzheimer's lab will attract a postdoc from a hotshot fluorescent marker or imaging lab. But a serious commitment to collaboration between independent labs is more difficult to sustain.

VM: That's helpful but not good enough. You get a postdoc who doesn't really know the technology that well, manages to get a few signals, but then leaves for a new position. You've got to try to get the original groups to work together.

ARF: You've also worked on apoptosis, have you not? From your experience, does it look to you like the neurons in Alzheimer's die by some form of apoptosis? Lysosomal proteases are also implicated in the death. There's quite a bit of debate about how neurons die. And do you think the precise mechanism of neuron death is really a research priority?

VM: It's possible that the dying is something that happens later on, that the neurons first become non-functional. The brain is a strange beast. We call it an organ, but it has so many different little parts and these defects are very strange. My wife can hardly talk but in every other way she's okay. Sometimes she has trouble walking, so there are very specific things going on in different parts of the brain. Also, sometimes what she cannot do one day, she can do the next day.

ARF: That's hard to explain.

VM: So there's a lot of stuff going on and the brain is so complicated. If a cell is dead, it's not going to come back a week later and its function may have been taken up by another cell. But we're so primitive in our pathophysiological thinking of this disease, we just focus on the anatomy, on the death of the tissue, or these plaques. That is a handicap, because if we can figure out how to study the physiology a little better, then we might be finding some really interesting things going on that are not now evident. I don't think we can do it in intact adult brains, but I think we can start on neuronal cultures. The other option is going into models like Drosophila neurodegeneration, maybe going back and looking at the C. elegans. The worm has only got about 90 neurons. We may not be able to figure out what they're thinking, but they're very interesting systems. The apoptosis story really broke in C. elegans, as you know.

ARF: Yes—by Bob Horvitz and others.

VM: Caspases started out that way.

ARF: There's a lot of work now on caspases, of course, in Alzheimer's, and models of other neurodegenerative diseases, but there's a debate about apoptosis being a rather fast process and neurodegeneration being slow.

VM: We don't know that. That's an interesting question itself: Is neurodegeneration a slow process or is it a fast process that comes on episodically as people age?

ARF: A person's cognitive decline is slow; it takes years.

VM: Right, but we don't know whether that is because the process has been taking years or whether something happens periodically over time, and the instance of whatever it is just increases incrementally.

ARF: Can I ask you how you become aware that your wife had a serious problem?

VM: For example, she began to burn coffee pots and write the same check multiple times when paying bills. It was easy to attribute these mishaps to the hectic life of a woman who was raising six children and working full time, but a painfully revealing moment came when she was lecturing to Yale medical students. She stopped talking in mid-sentence, not knowing what to say next. As she later related to me, the students shuffled around, whispered to each other, and eventually left the room, leaving her standing alone at the podium. She soon retired, at age 62, with a medical disability and stayed at home with me for a couple of years, before entering a nursing home.

ARF: When your wife was diagnosed with the disease, you were thrust into the role of caregiver, care seeker, coordinator, and patient advocate, in an academic system you'd belonged to for many years as a research scientist. You suddenly found yourself on both sides of the coin. What was that experience like, and did it change your perspective of the academic medical center at all?

VM: Yes, it really did. It changed my perspective of doctors, although I wanted to be a doctor myself ever since I was in fourth grade, and I am an MD. Effective physicians have risen in my estimation, although I've always held them in high regard. Being a doctor is a tough business. I became very conscious of what the doctors were like who were taking care of my wife. We went through many. We went through all these diagnostic procedures in order to eliminate every conceivable possibility, even including such unlikely things as AIDS, Lyme disease, everything. Going through all those tests was an awful experience. So I was watching, for the first time, the other side. You're right; it's interesting. I would say the profession came out pretty well, but with lots of variation in performance. I'm happy with how she's been treated in general, and I decided that I would just try to figure out whether I could help out in the field in some way.

ARF: Your experience seems to have been good so far, overall. Still, can you comment on what do you feel is most lacking in terms of patient care?

VM: We were lucky because I'm in a special situation here. Also, I had a job that allowed me to come home. I had a "child" at home, so I came home early often. I'm the director of this center, so my lab more or less closed down during the period when I was doing this. I could come in at ten and leave at three for a while. If you can do that, it's not a great problem to take care of someone, but otherwise sustaining a normal life becomes very challenging. Then again, my wife was young when she was diagnosed; it's not as if it's a 90-year-old person.

ARF: I'm so sad to hear it. That's terrible. Did you consider enrolling in clinical trials?

VM: Oh, yes. In fact, I tried. I called up the Elan people about the vaccine that just came out, and talked to the person who ran the program. Now I'm glad I didn't succeed. I didn't anticipate that the inflammation was going to happen; you never know. The vaccination appears to be somewhat like the statin treatment in that it's certainly reducing the amyloid burden, which is probably good. But it is going to be hard to figure out how to deal with the consequences.

ARF: Currently, the attempts are either to use passive vaccination or use an Ab epitope that doesn't activate a T cell response, and be careful in the choice of adjuvant. Do you think that will be sufficient? Of course, I can always ask your opinion on that, but until people actually try it's hard to know.

VM: I'm so keen on having anything happen, I'm reluctant to be pessimistic.

ARF: When the Elan trial failed, there was a lot of "I told you so" afterwards. Prior to that, nay-saying wasn't obvious in the literature but it was palpable. As someone who is eager for a cure, do you think we are too risk-averse when it comes to clinical trials, too concerned about side effects in Alzheimer's? In cancer people tolerate horrendous side effects.

VM: You are right. I don't know the answer to that. If I'd gotten my wife in the trial and she'd developed encephalitis, I would have been pretty unhappy. I think the difference is, Alzheimer's is such an awful disease that to make it any worse is much more painful than to just have someone get nauseous. You can somehow figure out ways to deal with side effects of other therapies, but not this.

ARF: Cancer is also an awful disease that kills some people early, and AD kills many people at only slightly older ages than does cancer.

VM: Yes, but cancer doesn't dehumanize them. They can be people until the end. This disease takes away their minds. It's a different thing. I've seen a lot of diseases, you know. This is the worst.

ARF: Shouldn't that create the greatest urgency to run clinical trials?

VM: This is absolutely right. That's one of the things I'm trying to do at Yale. I'm trying to promote more research and clinical activity. This is a very important issue. I think clinical trials have to be pushed. Yes, we should take more risk. I so agree with that.

ARF: If you had all the money you wanted, how would you go about finding a good biomarker for Alzheimer's?

VM: Another interesting question. Why do we imagine this disease is just involved with the central nervous system? There have been a lot of early claims that there are markers in the serum, and this and that. Definitive work has not been done-people who have done this work in general have been trying desperately to find something of value, and you get into this business of "anything that works, I want to try it or I need it." There is, then, a natural tendency not to be sufficiently rigorous, skeptical, demanding. As a result, many of these early observations have been ignored because they haven't been done well.

The other thing is, you can't get funding to do this kind of work now. There are a lot of judgments being made by peer-review groups, and appropriately so, I might add, but you actually asked the question what would one do if we had a significant amount of money. These questions are interesting to think about in some detail. Off the cuff I'm not sure. We don't have a handle on the pathophysiology. Remember, we're talking about anatomical changes, and this is a hell of a burden to do. A good example of what can be achieved if one uses functional markers to study a complex disease is the progress that Rick Lifton of Yale has made analyzing the genetics of high blood pressure. He's identified 30 genes that either up- or downregulate high blood pressure or confer low blood pressure. You can do that if you have real functional markers, and that is desperately needed in Alzheimer's.

ARF: Good functional markers.

VM: Yes, to get a sense about what is happening in this disease. I don't know who's studying AD biomarkers, by the way. Some people think it's a problem for the protein chemists, or the pathologists who can look into the brains and make all kinds of presumptions-Don Price is really very good-but there's no pathophysiological approach. That's what's missing now. If I were going to support some new effort, I would say, try to find people who do pathophysiology, who are trying to study decline of function, hoping to find the functions that are going wrong very early on.

ARF: How do you measure that? Are you thinking in terms of better psychometric instruments, neuropsychology tests, or are you thinking of brain imaging?

VM: I think brain imaging is the next big thing. Some groups should be encouraged to look at this more carefully. There's a lot of interesting stuff that can be done. The brains light up when people think of specific words during experiments. That used to be just simply blood flow increase, but now it's turning out to reflect specific biochemical reactions, and a whole new area of looking at metabolic activity is now possible. Creative people should figure out how you can study calcium dynamics in living brains or living neurons. I think that should start with neuronal cultures and move on with very sophisticated analyses. And by the way, these can't be simply looking at "bright spots" in cells, they have to be statistical and quantitative, so there's got to be real biophysics involved. I may sound overly critical because I so care about the result. But I actually am optimistic and positive.

ARF: You've mentioned areas that need more attention and that the general rigor of the studies needs to rise, and that you don't think it's up to par with what's happening in other areas. But we haven't found a way to do that, to improve the quality of this tremendously large output that we see every week.

VM: I acknowledge the problem is much more complicated than, say, studying a red blood cell. So I don't expect people to produce in the first instance the clarity of work that was done years ago in those areas. But I do think the subject is so important that we must support those efforts and people must push themselves harder. We have to be more rigorous, more skeptical, more demanding of what we do, and when we have leads they should really be pursued vigorously. People should be willing to help other people with their work, there must be more sharing.
I can't believe the amount of work that has already been done. Of course I only know what's in the published literature. That's only a fraction of what really is going on. Remember, people only publish what they think is positive. People don't publish negative results. (But see related news story.) There's a lot more probably known than we know in the published literature.

ARF: Is there anything you'd like to add?

VM: I've given you my positive view, and I've given you the idea that we need more rigor. I'm very keen on looking at data and output. I think we have to be a little careful of genetics in this regard. It's now become such a powerhouse technology, so we're very much obsessed by AbPP and presenilin and the ApoE apolipoprotein isoforms, but they could be reflecting very advanced, severe problems that are a subset of the larger issues. We've got to be careful about that.

ARF: So, you don't find convincing the argument that all the mutations that have been found so far for inherited forms converge on the same result, namely, increasing levels of Ab?

VM: I think they're persuasive, but they only represent a small fraction of the people. We're making extrapolations to the rest of the patients. Very similar to the LDL story, the homozygous hypercholesterolemia story, which in many ways proved misleading in retrospect. If you looked at the genetics of hypertension, another area of so-called multigenetic complex disease, every one of the genes first identified involved functions of the kidney. We used to think that adrenergics also played a role in high blood pressure. But now, all the genes that have been identified involve blood volume. Remember, high blood pressure is a combination of cardiac output and blood volume, so there's a great possibility that there's a huge area of genes involved with hypertension that are not kidney-related and we haven't seen them, yet. That could apply here, too. There could be a whole series of other genes. Actually, I'm fond of the notion that polymorphisms of inflammatory genes play a role. I saw that on Alzforum (see related ARF news and meeting report).

ARF: Also, when people look at SNPs of various pro-inflammatory genes, some find a link to longevity, or a kind of chronic low-grade inflammatory state in the elderly that may make them more vulnerable, see news story. But then, just as many other studies don't replicate those leads in their populations, and there is no general consensus yet.

VM: I think there's something to that, and I think we haven't identified all the genes involved in inflammation, these chemokines, chemotactic receptors, and others. There are about 20 different chemokines and about 50 receptors. These are all individual genes, so there's a lot of potential that may be hard to unravel.
All I'm saying is the genetics of AbPP and presenilin are so powerful for what they report that they may lead us to overlook a lot of other genetic changes.

ARF: At the moment AD genetics seems to be stuck with their four genes and the problem that they're trying to find other contributory genes that have a weak effect. They can't seem to really pick them out reproducibly. There are lots of candidates, including alpha-catenin and IDE, but then other studies don't confirm those, so the field seems to be treading water.

VM: The other thing that complicates genetics is that it is still unclear who is sick. You don't know what the disease really is. And, of course, you don't know whether you're looking at the same disease in different populations or different families. You can see why you wouldn't pick up subtleties.

ARF: And there are no large kindreds by the time people have it. The parents are gone, most of the siblings are gone.

VM: True. I enjoyed this conversation. Keep up the work at the web site.

ARF: Thank you for this interview. Keep us on our toes, and best wishes for your wife.

Gabrielle Strobel Interviews Vincent Marchesi

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