Will quantum physics help us cure Alzheimer's disease?

There's an interesting bit of writing out in the journal ChemMedChem by Jean-Louis Kraus, a medicinal chemist in France who has worked on drug discovery for Alzheimer's disease. The article is essentially a summary of Kraus's pessimistic outlook towards current therapies and approaches addressing Alzheimer's disease. Kraus has worked for a long time in medicinal chemistry and his words reflect experience and not just opinion. The article starts off with some well-founded skepticism but ends up sounding...let's say a little questionable.

For the most part I agree with Kraus that Alzheimer's research in the last decade has seen one disappointment after another and that we are still largely groping in the dark. He refers to the fact that all the concerted research into the disease and the billions of dollars in funding have resulted in only a handful of drugs and a handful of protein targets. None of the drugs even partially repair the damage and none of the proteins have been shown to be decisive as targets for treatment. Beta-secretase, gamma-secretase, the NMDA receptor, acetylcholine esterase; all of these have seen their day in the sun, followed by a disappointing set of results usurping them from front stage. That doesn't mean that none of them are important, it's just that targeting them with therapies seems to have no direct causal connection with treating the disease.

But what is even more disappointing is that the basic hypothesis driving the field - the amyloid hypothesis - has now been seriously questioned. A series of high-profile clinical trial failures have sent researchers scurrying back to their benches and while amyloid almost certainly has an important connection with the disease, it's now not clear at all whether actually targeting the infamous protein aggregates will bring any benefits. What seemed like a promising and rather direct direction of research has devolved into a scientific mess that will need at least a few years to be sorted out. As far as Alzheimer's disease goes, we are still fighting with sticks and stones.

As Kraus says, much of this failure eventually is a failure to understand the basic biology of the disease, which in turn entails a failure to understand the brain on a more general level, including mechanisms of memory generation and storage. Even now, much of drug discovery fails because of ignorance of the detailed biology of the disease and its perturbation by small molecules. What is regarded as a sound mechanistic hypothesis is often thwarted by the complex realities of signal transduction. With Alzheimer's disease we seem to have been biting off more than we could chew, and we need to keep untangling the complex interplay of amyloid, the protein tau, the secretases and a multitude of other biochemical components before we can truly start developing therapies. Thus it's inevitable and essential that in addition to chemists and biologists, we will need crucial input from neuroscientists to target the disease.

But will we also need quantum physicists? Kraus's thoughts on the relevance of subatomic science to AD left me slightly nonplussed. He says that:

The theories behind black holes generally suggest that subatomic particles (electrons, protons, neutrons) are themselves black holes, in which time expands in the opposite direction of our proper (perceived) time. Huge amounts of information could be stored by the spin number of photons present in these particle black holes. Could it be possible that the organization of brain matter, in terms of the properties of subatomic particles (quantum mechanics), confers on brain matter the capacities of memory and cognition, and that these phenomena are not encountered in other types of matter structure in the human body?

Come again? I was not familiar with electrons, protons and neutrons being black holes. But even if they are, I fail to see their direct relevance to understanding memory and cognition. Sure, it's a trivial fact that it's a very specific organization of subatomic particles that leads to a brain rather than to a liver or a chair. But the real action all takes place at the level of aggregates of these particles which we call molecules. I get the feeing that Kraus is indulging in a classic reductionist fallacy here. While subatomic particles do constitute the brain, understanding the brain can only come at a higher level, that of rather old-fashioned physics and chemistry involving ionic currents and neurotransmitters.

But Kraus finds a valuable place for quantum physicists in the war on neurodegenerative disorders:

To me it has become mandatory to create an AD scientific community that includes not only medicinal chemists, pharmacologists, biologists, and medical doctors, but also quantum physicists, in order to understand how aging alters the intimate structure of brain matter, where memory and cognition are located, with the hope of finding new AD treatment research orientations.

To me this sounds suspiciously like Roger Penrose's argument in his rather startling book "Shadows of the Mind" in which he postulated a relationship between wavefunction superposition in quantum mechanics and the growth and shrinkage of microtubules as significantly contributing to consciousness. Even a cursory look at that argument raised serious doubts about the relevance of quantum behavior in microtubules and more formal analysis seemed to confirm these doubts. I am not saying that physicists won't be a valuable asset on a drug discovery team, it's just that they are probably not going to use the tools of quantum gravity to map out cognitive pathways anytime soon.

Somewhat ironically, Kraus ends his piece by extolling the role of a systems biology approach in addressing a problem as complex as Alzheimer's disease. With this I wholeheartedly agree, but systems biology is the opposite of reductionism, where new emergent phenomena provide causal explanations that cannot be reduced to the laws underlying their substrates. We do need a suite of analytical tools operating at various hierarchical levels to address the issue, but given enough time and smart people, we should be able to do the job using standard chemistry and biology, albeit at a more sophisticated level. No fancy biophoton entanglement may be necessary.

Kraus, J. (2011). Why as a Medicinal Chemist I Am Not Optimistic about the Possibility of Finding, in a Reasonable Timeframe, Small-Molecule Drugs Capable of Curing the Evolution of Alzheimer’s Disease ChemMedChem DOI: 10.1002/cmdc.201100431

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In which creationists' understanding of amyloid appears...tangled

The biophysicist David Eisenberg of UCLA recently published a paper in which his group surveyed what they called the "amylome", the set of all possible proteins that can potentially form the deadly amyloid aggregate implicated in diseases like Alzheimer's. I haven't read the whole paper yet and will describe it in another post but it has some very intriguing conclusions (see the Nature News piece).

Eisenberg's group end up finding common segments possessing amyloid-forming propensity in pretty much every protein, given the right conditions. Not surprisingly, these segments are mostly kept tucked inside protein cores; if exposed the proteins are refolded with chaperones or eliminated as non-functional. But this ties in with Chris Dobson's work which I described in a recent post. Dobson's most recent paper seemed to conclude that amyloid is actually the most thermodynamically stable state of a protein, with "normal" protein states being metastable.

All this is fascinating stuff, but you can always trust creationists to put an anti-evolutionist spin on almost any scientific funding. Someone named Cornelius Hunter asserts on his blog that the fact that the most stable state of proteins seems to be amyloid and that most real proteins don't actually exist in this state seems to be a kind of miracle or at the very least indicates the enormous difficulties attendant in creating complex biomolecular structures.

Mr. Hunter seems to be indulging in a common fallacy, that of assuming that evolution somehow tends to an ideal. This is just not the way the process works. Considering the stringent constraints and time in which evolution has to work, it can only explore the available space of solutions and not the entire possible space. It can never achieve the best possible result in solution space, only one that is good enough under the given circumstances. I don't know if it's really that hard to understand or whether creationists like Mr. Hunter want to deliberately obfuscate the issue. Evolution can only work on what's already available; it can only mix and match existing motifs, and what exists need not be perfect at all. As an aside, this function of evolution reminds me of the concept of "satisficing" or "bounded rationality" in economics; lacking perfect knowledge of all solutions and unlimited time, economic actors like us can only pick solutions optimal within current constraints, not "global" maximums on the solution landscape.

There's therefore no reason to believe that biological evolution should create the most thermodynamically stable state of a protein. Creating a state that is functionally relevant is enough, even if it's thermodynamically metastable. In fact one can even make an argument that a thermodynamically superstable state might lead to an evolutionary dead end since it will be hard to tinker with. Given such constraints it's indeed impressive that evolution creates enzymes speeding up chemical reactions by twelve orders of magnitude, but even these enzymes are few and probably not the best possible in all of enzyme space.

So yes, the fact that it's amyloid and not the normal state of proteins that is the most thermodynamically stable one is fascinating, but in no way does this present a great challenge to evolution. In fact it reinforces evolution's essential character, to hunker down and make do as well as it can under the given circumstances. Don't we all?

The barrier to amyloid formation is kinetic, not thermodynamic

One of the questions I have pondered in the past is why the functional form of a protein should correspond to its most thermodynamically stable structure. Although this assumption is built into almost all experimental and theoretical studies of protein folding, it is not at all obvious since one may imagine other forms which could have improved stability. For instance, two protein forms may differ in the presence of a hydrogen bond or two. Based on the location and connectivity of these bonds, sometimes this slight rearrangement can cause a radical change in function, but there's no good reason why it should in the general case.

The answer however is most obvious in case of amyloid, that endlessly intriguing protein form that is implicated in so many devastating neurological disorders. Amyloid is a very stable state is often highly resistant to temperature, pH and high salt conditions. It's fair to ask how stable or unstable it is with respect to functional, soluble forms of the same protein.

To answer this question, a team led by Christopher Dobson who is a world expert on amyloid performed a series of thermodynamic measurements on a diverse group of proteins in which they measured the free energy differences between the soluble and the amyloid state. The proteins included everything from the Aß protein found in Alzheimer's disease to human lysozyme and insulin. The finding was that the free energy differences (ranging from about 3 kcal/mol to 6 kcal/mol) are not terribly dependent on the exact sequence, an observation which would be consistent with the striking recently uncovered fact that amyloid formation can be induced in almost any protein independent of its sequence. In fact the free energy difference seemed to depend more on the length and seemed to be optimal for a length of 100 residues for which the amyloid form was most stable. The difference also sharply tipped away from amyloid for increasing lengths.

This observation seems to suggest that one consequence of evolving larger proteins might be steer them away from the amyloid state and is consistent with the fact that almost all amyloid proteins have relatively short lengths (for instance, the Alzheimer's disease amyloid protein Aß has a length of roughly 40 residues). The propensity toward amyloid formation also depended on the concentration and the authors derived an limiting concentration beyond which amyloid formation would be rapid. This is again not surprising since the concentration-dependence of the process has also been demonstrated.

The real surprise came when they compared these limiting concentrations of the protein to the corresponding physiological concentrations of the same proteins in plasma. Remarkably, they found that in almost every case the physiological concentration was higher than that required to achieve amyloid formation. Thus the observations clearly indicate that for many key proteins, the amyloid state is thermodynamically more stable than the native, functional state. To put it bluntly, many nicely folded and soluble proteins are actually metastable. Now, since native proteins don't constantly form amyloid and kill us all, it's clear that the barrier to amyloid formation must be kinetic. Intriguingly, the authors speculate that these barriers can be overcome when organisms are exposed to stress, mutations or aging.

This is a pretty intriguing study and seems to underscore the belief that at least for some proteins, the folded functional state is not the most stable. However in light of what we know about evolution, this should not be too surprising. Stability is just one of many factors to be optimized during natural selection and there is no reason to assume that evolution would always act to maximize this parameter at the cost of all others. It's worth always keeping in mind that evolution cannot afford to aim for the ideal but instead has to make do with what it has.

The other question in my mind is why in spite of these barriers existing in case of so many proteins like lysozyme, insulin etc. are they regularly overcome only in the case of Aß (1-42) and a select few others. Based on the speculation in the paper, this could be because these proteins are exposed to particularly harsh conditions that force them to climb past the kinetic barrier and settle into the amyloid valley of thermodynamic comfort and physiological woe.

Among many such conditions could very well be bacterial infections. A few years back I advanced a hypothesis about amyloid formation being a defense against viral and bacterial infection mediated through the production of free radicals. A kinetic barrier-surpassing mechanism of the kind speculated here might well be what allows these proteins to achieve the transition, killing the bacteria but ironically harming their owner in the process. In the context of the present study, I think there continue to be a lot of opportunities to investigate the possible infection-induced conversion of normal proteins to their amyloid form.

Hopefully someone will do the experiment.

Baldwin, A., Knowles, T., Tartaglia, G., Fitzpatrick, A., Devlin, G., Shammas, S., Waudby, C., Mossuto, M., Meehan, S., Gras, S., Christodoulou, J., Anthony-Cahill, S., Barker, P., Vendruscolo, M., & Dobson, C. (2011). Metastability of Native Proteins and the Phenomenon of Amyloid Formation Journal of the American Chemical Society DOI: 10.1021/ja2017703

Probing amyloid, one oligomer at a time

One of the more important paradigm shifts in our understanding of the Alzheimer’s disease-causing amyloid protein in the last few years has been the recognition of differences between the well known polymer aggregates of amyloid and their smaller, soluble oligomer counterparts. For a long time it was believed that the fully formed 40-42 amino acid protein aggregate found in autopsies was the causative agent in AD, or at least the most toxic one. This understanding has radically changed in the last few years, partly through elegant work done in identifying oligomers and partly through the unfortunate results of clinical trials targeting amyloid. The new understanding is that it’s not the fully formed aggregates but the smaller oligomers that are the real toxic species.

Identifying these different monomers, dimers, trimers and tetramers is a valuable goal. But until now their recognition has mainly depended on raising specific antibodies against them, a tedious and expensive process. Small molecule probes that specifically identify each oligomer have been missing. In a recent JACS communication, a team from the University of Michigan uses a simple but clever technique to develop such probes and makes a promising step in this direction.

The probes are based on the idea that the best antidote against a poison is another poison. In this case the poison is the specific sequence of amino acids that makes up amyloid. In particular, a sequence of five amino acids- KLVFF- has been found to be sufficient for aggregation and toxicity. The aggregates form by the stacking of beta sheets principally driven by hydrophobic interaction between the FF residues; each pair thus serves as a growth site for addition of further such residues. The insight then is that if one could construct a mimic of the sequence, this mimic would basically act as a competitive inhibitor and bind to the normal sequence, inhibiting further growth. In this case the strategy was to use KLVFF segments themselves which would sort of wrap around newly formed oligomers of different constitution and sequester them from further self-assembly. So the team essentially constructed two KLVFF segments joined by a linker. The linker would also serve the purpose of providing an entropic advantage to the two segments so that they would not be at an energetic disadvantage during binding. The important question was how long the linker should be.

To decide on the length of the linker the team made some clever use of molecular dynamics simulations. Since you can estimate the approximate thickness of every oligomer, you can estimate the linker length that would be required to keep two KLVFF segments at the same distance as the thickness of the oligomer. For instance, the distances between the segments needed to wrap around the oligomers were 14-15 A for the dimer, 19-20 A for the trimer and 24-25 A for the tetramer.



But the linker should also keep the segments stable at that distance. To probe this the team used MD simulations. The MD simulations revealed the length of the linker required to keep the two segments separated at the specific distances by indicating how much time the assembly spent at those distances.

To test these results, the team then generated mixtures of different kinds of KLVFF oligomers and then added each probe to the solution. A streptavidin moiety was attached to every probe. Silver staining revealed that each probe was specifically binding to an oligomer of a certain type dictated by the compatibility of the intraprobe distance and oligomer thickness. Trimers and tetramers could be clearly identified but there was more ambiguity in case of dimers, presumably because of their less ordered structure.

Most interestingly, the team then added the probes to cerebrospinal fluid (CSF). Since amyloid is part of normal physiology, it is present in CSF. Gratifyingly they found that the probes could very clearly label trimers and tetramers against a background of several other proteins and intermediates in CSF. This experiment notably demonstrates that the method can selectively detect amyloid oligomers in complex mixtures.

I think that this work is valuable and paves the way toward the development of similar small-molecule based probes for identifying the key intermediates in amyloid formation. It could also be very useful in exploring amyloid formation in normal physiology and in exploring the stages of protein self-assembly in diverse amyloid-based diseases.

Reinke, A., Ung, P., Quintero, J., Carlson, H., & Gestwicki, J. (2010). Chemical Probes That Selectively Recognize the Earliest Aβ Oligomers in Complex Mixtures Journal of the American Chemical Society DOI: 10.1021/ja106291e

Miles to go before...

A New York Times article describes a study conducted by the NIH that basically asked scientists to simply sift through the massive evidence of the past twenty years to answer one question; what factors can cause or prevent Alzheimer's disease? The answer is unsurprisingly depressing- there is basically no proven therapy, personal habit, dietary supplement or mental task that correlates with the prevention of AD. AD is still as much a disease without a cure or preventative remedy as it ever was. We have almost as much work to do as Alois Alzheimer would have in 1908.

The bad news about AD has just kept on coming in over the last few years. Part of the reason is the very disappointing verdict on the role of beta-amyloid, reached after dozens of clinical trials which targeted the clump of protein in AD brains and failed to reverse the debilitating effects of the disease. Along with these studies, there has been a panoply of articles suggesting everything from crossword puzzle solving to Gingko biloba extracts that could possibly prevent the disease.

But as the NYT article reports, most of these recommendations are based on faulty 'studies' which are typically called "observational" studies. These studies are essentially accounts of observations made after someone has started on a measure that's assumed to be preventative. In addition, most of these observations are self-reported. Thus, evidence from such observations is spotty at best and is a far cry from the double-blind controlled clinical trials required to establish efficacy. After sifting through the evidence, the NIH study group concluded that they were sure only about one measure- Gingko biloba. And here the verdict was that Gingko biloba does not prevent AD. Apart from this, most other factors touted as preventive measures- including cognitive stimulation, vitamin E and antioxidants- could not be correlated with decreased incidence of AD with any degree of certainty. There's just no good evidence.

Part of the problem is simply the amount of time patients enrolled in trials would have to be observed in order to draw any conclusion about prevention. AD typically emerges around age 50, but its effects become apparent only in the late 60s and early 70s. A true clinical trial to study prevention would probably have to start during the young years and subjects would have to be followed for at least two to three decades, an expensive and complicated endeavor.

Yet the reports cited in the NYT should not be as depressing as they appear. For one thing, many people now think that the real reason none of the therapies for AD are working is simply because they are administered too late. Two new promising studies based on PET scans and spinal taps could make it easier to detect AD earlier and start treatment immediately. Plus, it's precisely the fragmented nature of the reported observations that provides opportunity for studying them further. Also, as depressing as the amyloid-targeting trials were, at least they provide good evidence of something that does not seem to be working. In science, the misses are almost as important as the hits. Finally, it's not like long-term studies cannot be attempted; the famous Framingham study followed the inhabitants of a small Massachusetts town not just over years but over generations to establish the connection between high cholesterol and heart disease. Perhaps a Framingham-style study for Alzheimer's is due.

Until these deep questions are resolved though, AD patients and their families will keep on living their private version of hell and will keep on trying to stave off the terrible malady by trying anything that remotely seems to work. The least we can all do is keep on searching.

The beta-amyloid hyp(e)othesis; the saga continues

As Churchill would have said, beta-amyloid is a riddle, wrapped in a mystery, inside an enigma. For years now, the "amyloid hypothesis" has been widely-accepted as somehow being importantly responsible for Alzheimer's disease. A few years back researchers were cheerfully confident that amyloid was to AD what artherosclerotic plagues were to heart disease. Rudolph Tanzi, a Harvard neurologist who is a leading authority on the disease and identified the first AD gene, had this to say in an interview in 2000:

Q. How close are we to an effective treatment for Alzheimer's disease?
A. I wouldn't be surprised if five years from now we have a pretty effective drug that can slow the disease down enough so that it will be preventable in those at risk, and significantly slow down the deterioration of people who already have it.

Q. Why do you have such optimism?

A. Because, in 15 years, we've gone from knowing little about what causes this disease to having a pretty concrete idea of which biological pathways and body proteins are involved.

If you compare Alzheimer's to heart disease where cholesterol levels must be lowered, we now have our own cholesterol equivalent, which we call the beta-amyloid. The name of the game in Alzheimer's therapy is lowering the accumulation of beta-amyloid in the brain.

It's ten years later and we are no closer to finding an AD drug. Tanzi's hope was not unwarranted given what we knew about amyloid then. But as the amyloid hypothesis matured, so did our understanding of it. First we discovered that it's not the amyloid aggregates themselves but soluble oligomers that are probably responsible for neuronal toxicity. Now it has been proposed that amyloid could have a protective antimicrobial role (I myself had an evolutionary speculation on this) in which case targeting it could even be dangerous. The fact remains that there is no proof that amyloid causes AD. It certainly seems to be related in an important way and many revealing details about it have been uncovered in the last decade, but the proof of principle has been on an increasingly slippery slope and if anything the picture gets murkier and more fascinating.

An article in the latest issue of C & EN basically says that we are targeting beta-amyloid because at least for now we cannot think of anything better to do. It's true that currently, our best bet at treating AD lies in interfering with amyloid formation. But since amyloid formation has never been shown to be causative for AD, treatments targeted at it are always going to be something of a shot in the dark. The advantage of targeting amyloid formation though, as the article says, is that there are lots of points in the mechanism where one can potentially interfere. Two key enzymes responsible for formation of amyloid are beta and gamma-secretase, which clip the amyloid precursor peptide into apparently toxic fragments. Scores of articles are published every year about new chemical agents targeting these two enzymes, and yet the jungle is thicker than we think.

Gamma-secretase is actually a multiprotein complex whose structure is not known, so finding molecules that inhibit it is like finding a black cat in a dark room. More importantly, it's also involved in a second pathway called the Notch pathway which is critical in cell-signaling. Thus blocking it may lead to one of the classic problems in drug discovery whereby eliminating a harmful function also eliminates a useful one, often fatally. Beta-secretase is much more well-studied and its crystal structure has been solved, but it poses a classic structure-based design conundrum; the enzyme's active pocket is flexible and expansive and can bind many ligands in different subpockets. Thus, developing drugs that block this moving target is admittedly challenging. Throw in the requirements for safety and an ability to cross the blood-brain barrier (BBB), and we have a pickle on our hands that's almost as dense as amyloid plaques.

But the much more serious issue is whether any of these strategies will work at all. If amyloid formation turns out to be a side-show in AD progression, then all these strategies might ultimately come to naught. Unfortunately the data so far is not promising. The last few years have seen a disappointing string of late-stage failures of amyloid blocking molecules and antibodies in clinical trials. In some cases the agents have failed to clear the plaques, but tellingly in others, clearing the plagues did not put the disease in remission. Thus the scores of pharmaceutical companies that have several pipeline dwellers focused toward amyloid may be chasing an imaginary rabbit. There are serious concerns that scientists may have to go back to the drawing board and start all over again. This would be a huge setback.

However, hope need not be completely lost. One of the most reasonable explanations for the failure of these agents is simply that they arrived too late on the scene, when the disease had progressed too far to be defeated. Perhaps these drugs would have helped had they been administered earlier. Even cancers that can be treated if detected early fail to be cured in late stages, and AD should be no different. One of the big problems in the field is that detecting AD early is still a challenge and is being addressed by many promising neural imaging initiatives. Perhaps early and focused administration of these drugs could be successful.

Yet it all hinges on putting all your eggs in the amyloid basket. Another protein implicated in AD is tau, which forms tangles in the brain. But as the article says, targeting tau may be even harder than targeting amyloid since it is ubiquitous. Other processes hypothesized to be important for AD include oxidation and other neurotoxic processes of which amyloid may simply be a side-product. And as I was thinking this morning, perhaps no drug will ever be as effective in treating AD as a balanced lifestyle that includes preventive measures; and this may especially be true if amyloid is a natural part of our body's physiology. But as of now we have to keep on trying, and the amyloid hypotheis, shaky as it is, seems to be our best bet of making a dent into this devastating disease. At the very least it will lead to novel basic insights. Perhaps it's an indication of how primitive our understanding of the disease is that we continue to cling to amyloid. Under the present circumstances it seems to be the best we can do, but as another Churchillian admonition indicated, "it is not enough that we do our best; we must do what's necessary".

A small molecule probe discriminating between Aß amyloid oligomers and fibrils

One of the conceptual shifts that the study of Alzheimer's disease has seen in the past few years is the realization that the long-studied insoluble Aß (1-42) amyloid fibrils may not be the real culprits in the disease. Instead the dubious distinction may belong to soluble Aß oligomers whose morphology differs from that of mature fibrils. Thus instead of focusing on one kind of Aß species, researchers are focusing on a broad range of oligomers and fully formed fibrils that differ in their architecture. Some differences between these species are clear; for instance the oligomers seem to permeate the plasma membrane in cells much better than the fibrils. The factors that dictate the exact morphology of these species sometimes might be subtle (such as pH shifts), and I myself have worked a little on such subtle changes leading to drastically different morphologies (see here for instance).

However, a study of different amyloid morphologies will greatly benefit from probes that allow us to selectively target and isolate certain amyloid architectures. The principal method of doing this until now has been to raise species-specific antibodies that target either oligomers or fully formed fibrils. In this regard small molecules have not been very promising as they tend to indiscriminately bind to all amyloid species; for instance Congo Red which is the archetypal amyloid labeling dye does not discriminate between different amyloid species.

Now a group at the University of Michigan has discovered some very simple probes that seem to target only spherical oligomers and not fibrils. The probes consist of tryptophan and some rather simple and well known biological molecules containing tryptophan which exhibit fluorescence when bound to proteins, and especially hydrophobic regions of proteins. The team started by screening about 70 simple organic molecules that exhibit fluorescence. Most of these molecules did fluoresce, but when bound to both forms of amyloid, thus precluding discrimination between the two species. Some did not fluoresce at all. But about 10 of them showed differential fluorescent quenching; the fluorescence was quenched much more when bound to oligomers compared to fibrils, thus allowing their selective labeling and visualization. Antibody labeling and radiography confirmed that it was indeed the oligomers that were getting labeled.

Intriguingly these probes consist of tryptophan itself as well as some very simple naturally occurring molecules containing the Trp moiety, such as melatonin, tryptamine and serotonin. In my mind this raises a very interesting possibility; could these molecules also be interacting selectively with amyloid and somehow modulating its behavior inside living systems?

In any case, what is even more valuable is that the probe seems to selectively label the oligomers in the presence of the fibrils and this can be determined from fluorescence studies. This opens up some potentially very interesting applications; for instance if there was any way at all to use such probes in vivo, we could gain extremely valuable knowledge on the ratios of oligomers to fully formed fibrils. Recalling that amyloid is astonishingly a deformed version of a naturally occurring protein, insights gained from such studies could be critical in shedding light on the natural and unnatural roles of amyloid in living systems.

Reinke, A., Seh, H., & Gestwicki, J. (2009). A chemical screening approach reveals that indole fluorescence is quenched by pre-fibrillar but not fibrillar amyloid-β Bioorganic & Medicinal Chemistry Letters, 19 (17), 4952-4957 DOI: 10.1016/j.bmcl.2009.07.082

Targeting early AD oligomers with a simple dipeptide

A couple of days ago, an American man in Switzerland became the first human being to have his assisted suicide filmed and broadcasted on television. Craig Ewert was a 59 year old university professor who had been diagnosed with motor neuron disease in 2006. He had been given 3-5 years to live, but the disease took its terrible toll in just a few months. By the end of 2006 Ewert decided he had had enough. He contacted Dignitas, a Swiss organization started by a lawyer that has helped literally hundreds of people to peacefully end their lives. On a quiet, sunny morning, Ewert sipped a lethal cocktail of barbiturates completely of his own volition, and then bit on a switch that stopped his ventilator, again of his own volition. The film crew was allowed to film the entire operation except for after he passed away, when his wife wanted a few minutes of silence to herself.

Assisted suicide is an immensely controversial topic laden with moral issues. I have thought about it and discussed it with my friends many times, and I usually find myself ending up in favour of it at least in some cases where death is inevitable and the quality of life is going to assuredly become worse. Motor neuron disease is one such ailment. Alzheimer's disease is another. I personally don't think there's any other disease as cruel as AD, with loved ones trying to grasp at a person's identity as it slips away all too slowly and painfully. To me AD seems to be one of those unambiguous cases where a person who is still coherent should be able to exercise his right to die a dignified death. As horrible as it is to watch a loved one decide to willingly end his or her life, it is usually infinitely worse to watch them fade away into a wilderness of silence.

But the insidious pain of AD also makes the search for AD therapies a particularly compelling and desperate one. 15 million people are afflicted with AD, and in 20 years that burden is supposed to double. Part of the trouble for targeting AD is a still incomplete understanding of its molecular basis. While the initial amyloid hypothesis that posits the formation of insoluble amyloid protein fibrils is still very much relevant, much less is known about its exact relation to the disease, whether as a cause or symptom. The most important recent finding with respect to this hypothesis is that it's not insoluble aggregates but soluble oligomers that are the toxic species.

A recent overview in Nature (The Plaque Plan, Nature, November 13 2008) talked about the disappointments and ambiguities facing researchers in AD. Amyloid burden does not always correlate with cognitive impairment. In addition, two big clinical trials designed to target the formation of amyloid have both ended in failure and confusion. It's back to the drawing board for many embattled scientists. A major problem inherent in testing AD therapies is the late stage at which they inevitably have to be initiated in the absence of an early test for diagnosis. Thus disease progression is already advanced and perhaps that is the reason the therapies don't work as planned. The future should be as much focused on early brain and cerebrospinal fluid imaging as on new therapies. And of course, we are still in the rudimentary stages of achieving that supreme goal of understanding how memories are formed and stored.

At any rate, the midnight oil keeps burning and the search goes one, and Ehud Gazit from Tel Aviv University has published a paper in Angewandte Chemie that features a simple dipeptide for targeting and disassembling early soluble amyloid fibrils. The dipeptide is D-Trp-Aib, consisting of D-tryptophan and the unnatural amino acid amino isobutyric acid. This amino acid is rather unique and has been extensively studied because it seems to lend pronounced alpha helicity to peptides. The running hypothesis in its inclusion in the inhibitor is that it is a ß-breaker, an amino acid that helps to break the ß sheets that are a signature of amyloid. The work also focuses on the central region of the Aß (1-42) amyloid peptide that forms its aromatic core. The peptide sequence here is KLVFFAE, and the central Phe residues are thought to strongly influence the aggregation of the peptide. Thus the use of the D-Trp; apparently it would help to interfere in aromatic assembly. In addition it would also escape degradation by ubiquitous proteases.

Being a simple uncapped dipeptide, the blood-brain barrier permeability of this molecule is admittedly lousy (It has a depressing logP value of -0.8). Strategies could probably be worked up for enhancing delivery by chemical modification. However, other tests that the authors used attest to its efficacy as a amyloid-interfering agent. The compound inhibits the oligomerization of the amyloid oligomers as shown in a SDS-PAGE gel; it inhibited oligomers that can be stabilized using SDS. Interestingly its inhibition ability decreases at intermediate concentration and then goes up again. The compound also dissociated amyloid fibrils as shown in fluorescence assays with thioflavin-T. Lastly and more importantly, it seems to demonstrate long-term potentiation (LTP) in mice that may lead to an increase in cognitive retention. The molecule also showed good bioavailability and low toxicity.

In addition the researchers also did a NMR study on the interaction of D-Trp-Aib with the core of amyloid Aß (1-42). Using TOCSY and other spectra they detected differences in the chemical shifts of alpha protons of some key amino acids. Using NOE information they have also proposed a family of solution structures representing the bound conformation of the dipeptide with the sequence. I am more skeptical about this result. Small sequences of peptides rapidly interconvert in solution and give averaged NMR signals. The binding of the peptide to the sequence is probably an on-off event in which it preferentially stabilizes a particular conformation. A more detailed structural study combined with kinetics experiments would shed light on the key binding events.

How far such therapies would take us in tackling AD in human beings remains to be seen; in any case, maybe the trials should begin soon.

Other AD-related posts: water in amyloid, amyloid dimers as possible culprits, "seminal" truths about amyloid, and amyloid as a possible window into historical pathogen wars

References:
Anat Frydman-Marom, Meirav Rechter, Irit Shefler, Yaron Bram, Deborah E. Shalev, Ehud Gazit (2008). Cognitive-Performance Recovery of Alzheimer's Disease Model Mice by Modulation of Early Soluble Amyloidal Assemblies Angewandte Chemie International Edition DOI: 10.1002/anie.200802123

Thinking about Alzheimer's Disease as Historians

Head slumped forward, eyes closed, she could be dozing — or knocked out by the pharmacological cocktails that dull her physical and psychic pains.

I approach, singing “Let Me Call You Sweetheart,” off key. Not a move or a flutter. Up close, I caress one freckled cheek, plant a kiss on the other. Still flutterless.

More kisses. I press my forehead to hers. “Pretty nice, huh?” Eyelids do not flicker, no soft smile, nothing.

She inhales. Her lips part. Then one word: “Beautiful.”

My skin prickles, my breath catches.

It is a clear, finely formed “beautiful,” the “t” a taut “tuh,” the first multisyllable word in months, a word that falls perfectly on the moment.

Then it is gone. The flash of synaptic lightning passes. That night, awake, I wonder, Did Pat choose “beautiful?” Or did “beautiful” choose Pat? Does she know?

This heartbreaking and sad account by a husband of his wife's early slide into Alzheimer's Disease (AD) reminds us of how much we need to do to fight this. I personally think that of the myriad diseases afflicting humankind, AD is probably the cruelest of all. Pancreatic cancer might kill you in three months and cause a lot of pain but at least you are in touch with your loved ones till the end. But this is human suffering on a totally different level.

The search for the causes of Alzheimer's disease goes on, and I have recently been thinking in a wild and woolly way about it from an evolutionary standpoint. While my thoughts have not been well-formed, I want to present a cursory outline here.

The thinking was inspired by two books- one book which has been discounted by many, and another which has been praised by many. The lauded book is Paul Ewald's "Plague Time" which puts forth the revolutionary hypothesis that the cause of most chronic diseases is ultimately pathogenic. The other book "Survival of the Sickest" by Sharon Moalem puts forth the potentially equally revolutionary hypothesis that most diseases arose as favourable adaptations to pathogenic onslaughts. Unfortunately the author goes off on a tangent making too many speculative and unfounded suggestions, leading some to consider his writings rather unscientific. As far as I am concerned, the one thing that the book does offer is provocative questions.

On the face of it both these hypotheses make sense. The really interesting question about any chronic disease is; why have the genes responsible for that disease endured even after so many millennia if the disease kills you? Why hasn't evolution weeded out such a harmful genotype? There are two potential answers. One is that evolution simply has not had the time to do this. The other hypothesis, more provocative, is that these diseases have actually been beneficial adaptations against something in our history. That adaptation was so beneficial that its benefits outweighed the obvious harm that it caused. While that something probably does not presently prevail, it was significant in the past. What factor could possibly have existed that needed such a radical adaptation to fight it?

Well, if we think about what it has been that we humans have been fighting the most desperately and constantly ever since we first stepped foot on the planet, it's got to be a foe that was much older than us and more exquisitely adapted than we ever were- bacteria. The history of disease is largely a history of a fierce competition that humans and bacteria have engaged in. This competition plays by the rules of natural selection, and is relentless and ruthless. For most of our history we have been fighting all kinds of astonishingly adaptable bacteria and there have been millions of martyrs in this fight, both bacterial and human. Only recently have we somewhat eroded their malign influence with antibiotics, but hardly so. They still keep evolving and developing resistance (MRSA killed 18,000 in the US in 2005), and some think that it's only a matter of time before we enter a new and terrifying age of infectious diseases.

So from an evolutionary standpoint, it's not unreasonable to assume that at least a few genetic adaptations would have developed in us to fight bacteria, since that fight more than anything else has been keeping our immune system busy and our mortality high since the very beginning. But instead of thinking about genes, why don't we think about phenotypes? Hence arose the hypotheses that many of the age-old chronic diseases that are currently the scourge of humanity may sometime have been genetic adaptations against bacterial infection. While the harm that is done by these diseases is obvious, maybe their benefits outweighed that harm sometime in the past.

When we think of chronic diseases, a few immediately come to mind, most notably heart disease, diabetes, Alzheimer's and cancer. But one of the best cases in point that illustrates this adaptive tendency is hemochromatosis which is an excess of iron absorption and storage, and it was this disease that made me think about AD. A rather fascinating evolutionary explanation has been provided for hemochromatosis. Apparently when certain types of bacteria attack our system, one of the first nutrients they need for survival is iron. By locking down stores of iron the body can protect itself from these bacteria. It turns out that one of the species of bacteria that especially needs iron is Yersinia pestis, the causative agent of the black plague. Now when Yersinia attacks the human body, macrophages rally to the body's defense to swallow it. Yersinia exploits iron resources in macrophages. If the body keeps iron stores from macrophages, it will keep iron from Yersinia, which however will lead to a buildup of iron in the body; hence hemochromatosis. The evidence for this hypothesis is supposed to come from the Black Plague which swept Europe in the Middle Ages and killed almost half the population. Support for the idea comes from the fact that the gene for hemochromatosis has a surprisingly higher frequency among Europeans compared to others. Could it have been passed on because it protected the citizens of that continent from the plague epidemic? It's a tantalizing hypothesis and there is some good correlation. Whether it's true or not in this case, I believe the general hypothesis about looking for past pathogenic causes that may have triggered chronic disease symptoms as adaptations is basically a sound one, and in theory testable. Such hypothesis have been formed for other diseases and are documented in the books.

But I want to hazard such a guess for the causes of AD. I started thinking along the same lines as for hemochromatosis. Apart from the two books, my thinking was also inspired by recent research that suggests that amyloid peptide- a ubiquitous signature in AD- binds to copper, zinc, and possibly iron to generate free radicals that cause oxidative damage to neurons. Oxidative damage they may cause, but we have to note that oxidative damage is also extremely harmful to bacteria. Could amyloid have evolved to generate free radicals that would kill pathogens? Consider that in this case it's also serving a further valuable function akin to that in hemochromatosis- keeping essential metals from the bacteria by binding to them. This would serve a double whammy; denying bacteria their essential nutrients, and bombarding them with deadly free radicals. The damage that neurons suffer would possibly be a small price to pay if the benefit was the death of lethal microorganisms.

For testing this hypothesis, I need to know a couple of things:

1. Are there in fact bacteria which are extremely sensitive to copper or iron deficiency? Well, Yersinia is certainly one and in fact most bacteria are to varying extents. But since AD affects the brain, I am thinking about bacterial infections that affect the brain. How about meningitis caused by Neisseria, one of the deadliest bacterial diseases even now which is almost certainly a death sentence if not treated? Apart from this, many other diseases affect the brain if left untreated; the horrible dementia seen in the last stages of syphilis comes to mind. Potentially the brain would benefit against any of these deadly species by locking its stores of metal nutrients and generating free radicals to kill them, a dual function that amyloid could serve. I have not been able to say which one of these bacteria amyloid and AD might have evolved against. Maybe it could have been against a single species, maybe it could have been a general response to many. I am still exploring this aspect of the idea.

2. More importantly, I need epidemiology information about various epidemics that swept the world in the last thousand years or so. In the case of hemochromatosis, the causative genetic stimulus was pinned down to Yersinia because both the disease etiology and the pandemic are documented in detail. I cannot easily find such detailed information about meningitis or syphilis or other outbreaks.

3. In addition, while risk factors have been suggested for AD (for instance the ApoE epsilon4 gene allele), no specific genes have been suggested as causal factors for the disease. There is a clear problem with correlation and causation in this case. Also, the important role played by environmental factors such as stress and diet is becoming clear now; it's certainly not an exclusively genetic disease, and probably not even predominantly so.

4. Most importantly, I think it is impossible to find instances of AD clusters in history for a simple reason-the disease was simply unknown before 1906 when Alöis Alzheimer first described it. Even today it is not easy to make an assessment of it. All cases of Alzheimer's before a hundred years back would have been dismissed as cases of dementia causes by old age and senility. Thus, while the causative hypothesis is testable, the effects are hard to historically investigate.

The fact that AD is a disease of age might provide some credence to this hypothesis. Two things happen in old age. Firstly, the body's immune defenses start faltering, and this might need the body to marshall extra help to fight pathogens. Amyloid might do this. Secondly, as age progresses evolution is less worried about the tradeoff between beneficial and harmful effects because the reproductive age has already passed. So the devastating effect of AD would be less worrisome for evolution. Thus, the same AD that today is thought to reduce longevity would have ironically increased it in an age where infection would have reduced it even further.

However, if AD is an adaptation especially for old age, then it begs a crucial question; why would it exist in the first place? Evolution is geared toward increasing reproductive success, not toward increasing longevity. There is no use as such for a rather meticulously developed evolutionary adaptation that kicks in after reproductive age has passed. I think the answer may lie in the fact that while AD and amyloid do affect old people, they don't suddenly materialize in old age. What we do know now is that amyloid Aß is a natural component of our body's biochemistry and is regularly synthesized and cleared. Apparently in AD something goes wrong and it starts to suddenly agglomerate and cause harm. But if AD was truly an adaptation in the past, then it should have possibly manifested itself in younger age, perhaps not a much younger age but an age where reproduction was still possible. Consider that some dementia is much preferred to not being able to bear offspring, and so AD at a younger reproductive age would make evolutionary sense even with its vile symptoms. If this were true, then it means that the average age at which AD manifests itself has simply been increasing for the past thousand years. It would mean that AD is not per se a disease of the old; it's just become a disease of the old in recent times.

So after all the convoluted rambling and long-winded thought, here's the hypothesis:
Alzheimer's disease and especially Aß amyloid is an evolutionary adaptation that has evolved to kill pathogens by binding to key metals and generating free radicals

There are several details to unravel here. The precise relationship between metals, amyloid and oxidative damage is yet to be established although support is emerging. Which of the metals really matter? What do they exactly do? The exact role that amyloid plays in AD is of course under much scrutiny these days. And what, if anything, is the relationship between bacterial infection and amyloid Aß load and function in the body?

In the end, I suggest a simple test that could validate at least part of the hypothesis; take a test-tube filled with fresh amyloid Aß, throw in metal ions, and then throw in bacteria that were thought to be responsible for major epidemics throughout history. What do you see? It may not even work in vitro- I wonder if it could be tried in vivo- but it would be worth a shot.

Now I will wait for people to shoot this idea down because we all know that science progresses through mistakes. At least I do.

Water and Amyloid Self-Assembly

One of the most significant, interesting and yet poorly understood effect in chemistry and biology is the hydrophobic effect. An important manifestation of its role in biology is invoked by the picture of two parallel hydrophobic plates with water between them that are slowly brought close to each other. At a certain distance called the 'dewetting distance', dewetting is observed and the water suddenly gets expelled out from them. Since nature abhors the resulting vacuum, the two hydrophobic surfaces collapse and 'stick' to each other.

Bruce Berne and his colleagues at Columbia had hypothesized that such dewetting could be observed in biological systems. A remarkable dewetting transition in the protein mellitin and in other proteins in the PDB provided evidence. Now they and others investigate such possible roles of water in the formation of amyloid by molecular dynamics simulations. They focus on the central core peptide of the Alzheimer's Aß (1-42) peptide consisting of the 16-22 region. Since one of my projects involves investigation of the self-assembly of this segment, it is of particular interest to me. This oligopeptide is interesting because it seemingly is the smallest stretch of the bigger parent that also forms the characteristic cross-beta sheet structure of amyloid. Importantly, it is also soluble which means it is more amenable to structural characterization compared to the bigger peptide.

Berne's group investigates the role that water plays between two sheets of 16-22 consisting of nine peptides each. The MD simulations were run for 1 ns, a typical time for such systems. The dewetting critical distance was found to be 12.8 A. Compare this with the classic repeat distance between amyloid beta-sheets which is 9.9 A. Basically two types of phenomena were observed depending on different trajectories and conditions; dewetting, which means that water expulsion was followed by hydrophobic 'collapse', and the simultaneous occurence of expulsion and collapse. The latter phenomena can be interpreted as a kind of 'lubrication' effect that water has been hypothesized to play in the folding of proteins. They haven't been able to put a finger on which is the phenomenon in the 'real' system, but it at least seems plausible that dewetting could be observed in such systems. In the cases where dewetting is not the driving force for assembly, they dissect the interaction energies of different amino acid residues that contribute to the total energy, and find that the Phe-Phe energy contributes to the most.

From a methodological standpoint, the investigation is complicated by the fact that, as in other such studies, the results seem to depend on the methods used. This is a common characteristic of modeling and theoretical investigations. In this case, turning off the protein-water Van der Waals forces causes dewetting in every instant, perhaps not a surprising conclusion since there is now no attraction at all between the water and protein. Also, turning off the electrostatic forces caused no changes, which means that electrostatic attraction plays a small role in the system. Again probably not too surprising. A third observation is that the choice between the two phenomena depends on rather small changes in temperature.

The interpretations are also rendered ambiguous by the fact that amyloid surfaces and indeed most 'hydrophobic' protein surfaces are a poor approximation to two parallel hydrophobic plates. It should be noted that the attractive energy of interaction between two flat plates separated by a distance R is inversely proportional to the square of R. Compare this with the case of two spheres of radius R whose classical Van der Waals attractive energy is proportional to the sixth power of R. That means that in the case of the plates the attraction falls off much more slowly which can lead to significant dispersion forces even at relatively long separation.

This paper, while not leading to very novel or practical results, sheds light through detailed investigation of the possible role water can play between amyloid sheets. It again demonstrates the important effect that choice of methods and parameters can have on observations.

Krone, M.G., Hua, L., Soto, P., Zhou, R., Berne, B.J., Shea, J. (2008). Role of Water in Mediating the Assembly of Alzheimer Amyloid Aß (16-22) Protofilaments. Journal of the American Chemical Society, 130(33), 11066-11072. DOI: 10.1021/ja8017303

Aß Dimers- The Long-Sought Minimal Culprit in Alzheimer's Disease?

Following on the heels of the headline-making Nature publication that demonstrated that NSAIDs (Non-steroidal AntiInflammatory Drugs) uniquely targeted a substrate (APP) rather than an active site of the gamma-secretase complex involved in plague formation in Alzheimer's (see Discount Thoughts for a great summary) comes a paper that may turn out to be one of the important papers in the history of Alzheimer's disease (AD) research.

Since 1905 when Alois Alzheimer first detected the symptoms of what we today call AD and identified the characteristic plaques that form in the brains of AD patients, the "amyloid hypothesis" has become almost synonymous with AD. For decades now, insoluble amyloid plaques, later found to consist of 40 (Aß 1-40) and 42 (Aß 1-42) residue oligopeptides, have been thought to be the hallmark of AD. Indeed, amyloid has become the poster boy for diseases caused by protein misfolding. Say "protein misfolding", and college students will pipe up and say "Alzheimer's"

However, the truth as usual has been complicated. In the last few years, attention has been shifting from the insoluble Aß to soluble forms of the peptide that are apparently in equilibrium with the aggegated beasts. Many oligomers have been isolated through antibody labeling and their toxicity has been demonstrated to various extents under various conditions. The "amyloid hypothesis" has become much more complex than before, and one of the original questions- whether these insoluble plaques are really the cause or just a manifestation of AD- has raised its head even more.

Recently exciting progress has been made in the field, with everything from metals to free radicals being implicated in the dementia and neuronal death that AD causes. On a wall in my room I have a Sigma Aldrich poster displaying a huge schematic of the principal species and pathways involved in AD, and one look at the poster clearly indicates how convoluted the whole scenario is. One of the continuing main reasons for slow progress has been the lack of structural information, with amyloid itself not being crystallizable and soluble species by definition being hard to structurally nail down.

But in light of the connection to soluble oligomers unearthed for AD, one lingering question has been foremost on everyone's minds- What is the minimal soluble species responsible for the symptoms of AD? Now it seems that a paper might go a long way in answering this question.

The short answer is "dimers dimers dimers". For the long answer, read the Nature Medicine paper. Charles Selkoe, Ganesh Shankar and others at Harvard separated different Aß species, insoluble and soluble, from the brains of AD patients. They then performed detailed characterization through immunoprecipitation, Western blots and other techniques, and then injected these fractions into rats, documenting which species can be identified as being the minimal as well as dominant contributors to the pathophysiology of AD.

I am no neurologist (paging Retread) but the researchers seem to have focused on three indicators of "brain damage"- an adverse effect on LTP (long-term potentiation); Wikipedia defines this as "the persistent increase in synaptic strength following high-frequency stimulation of a chemical synapse" which seems to indicate the fidelity of synaptic communication and a contributor to memory, LTD (long-term depression) which is the weakening of a synapse, and a decrease in dendritic spine density.

The researchers clearly find that dimers displaying a mass band of 8kD (confirmed by mass spectrometry) provide the greatest effect on these three parameters. Monomers and other soluble oligomers were not just less toxic but inactive. They also performed the interesting experiment of treating insoluble Aß cores with formic acid, this causing some of it to dissociate into dimers. This concoction proved deadly for rat brains, while the original untreated assembly did not prove as toxic. To make sure that the dimers were pure, they also used synthetic Aß dimers and obtained the same results. These set of results are pretty conclusive in demonstrating the toxicity of dimers.

As an interesting sidepoint, the authors also demonstrate the role of the metabotropic glutamate and NMDA receptors in facilitating the symptoms.

The significance of these results are clear. The authors themselves say "Our findings fulfill an essential requirement for establishing disease causation in Alzheimer’s disease". Many questions still remain though. We still don't know the molecular mechanism through which these dimers finally lead to neuronal death. Do they exert their effects by binding to metals like copper or iron? Do they slide into neuronal membranes and cause them to disintegrate? What other species do they actually go through before they cause harm? All these effects have been suggested as part of the list of effects responsible for neuronal damage. Which effects do Aß dimers fit into?

But all this is later. For now it's a significant achievement that we seem to have a handle on the minimal species responsible for AD. It's a staggeringly simple (or not...) structure involved in the progression of a set of maddeningly complex events. This finding seems to open a whole new window of experiments, conjectures and principles related to Aß dimers and AD in general.

My compliments to the team.

1. Shankar, G.M., Li, S., Mehta, T.H., Garcia-Munoz, A., Shepardson, N.E., Smith, I., Brett, F.M., Farrell, M.A., Rowan, M.J., Lemere, C.A., Regan, C.M., Walsh, D.M., Sabatini, B.L., Selkoe, D.J. (2008). Amyloid-ß protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nature Medicine DOI: 10.1038/nm1782

2. Halliwell, B. (2006). Oxidative stress and neurodegeneration: where are we now?. Journal of Neurochemistry, 97(6), 1634-1658. DOI: 10.1111/j.1471-4159.2006.03907.x

3. Bush, A.I. (2003). Copper, ß -amyloid, and Alzheimer's disease: Tapping a sensitive connection. Proceedings of the National Academy of Sciences, 100(20), 11193-11194. DOI: 10.1073/pnas.2135061100

4. Kukar, T.L., Ladd, T.B., Bann, M.A., Fraering, P.C., Narlawar, R., Maharvi, G.M., Healy, B., Chapman, R., Welzel, A.T., Price, R.W., Moore, B., Rangachari, V., Cusack, B., Eriksen, J., Jansen-West, K., Verbeeck, C., Yager, D., Eckman, C., Ye, W., Sagi, S., Cottrell, B.A., Torpey, J., Rosenberry, T.L., Fauq, A., Wolfe, M.S., Schmidt, B., Walsh, D.M., Koo, E.H., Golde, T.E. (2008). Substrate-targeting gamma-secretase modulators. Nature, 453(7197), 925-929. DOI: 10.1038/nature07055

AD, metals, and evolution

There always seems to be something new emerging in Alzheimer's disease research. In the last few years, at least one widely accepted hypothesis, the amyloid hypothesis, has come under scrutiny. In AD, it was thought that peptides aggregate into beta-amyloid fibrils (Aß) which causes toxicity to neurons.

However, many reports in the last two years are suggesting that it's not the aggregated beta-amyloid, but its soluble precursors that seem to be more toxic. There were some indications that this was true before too. For example, neuronal damage and memory impairment did not always correlate with Aß levels.

Another big hypothesis that has been widely prevelant in the AD community is that metals such as copper, aluminium, iron, and zinc, might correlate with Aß aggregation. New reports also suggest that these metals may in fact bind to the soluble and toxic forms of Aß, and help them to aggregate. One very interesting aspect of metabolism emerging from this research seems to be that it's not necessarily a larger amount of these metals from our environment or diet that's causing the problems, but an age-related breakdown in metal metabolism in the brain, which is an especially capacious reservoir of metals. Thus, perhaps we need not be especially worried about getting more copper or zinc from our diets.

One more thing that the metals might be doing is to help in free-radical generation, that might damage neurons. Again, there is still a correlation vs causation problem with this hypothesis, but free radicals are being increasingly found in vitro, when metals are added to Aß fibrils. While aluminium seems to have been largely a scare idea, zinc and copper are emerging as more plausible agents for free radical production and neuronal damage.

Based on evolutionary concepts, I cannot help but think that there may actually have been some protective function of amyloid in ancient human civilizations. Maybe it sequestered copper and provided a ready source when there was a lack of copper (or iron for that matter) in the diet. Or maybe it recruited copper to fight microorganisms and especially bacteria, at a time when there were no defenses against them except for natural ones. In this way, it could have dealt a double blow; denying essentials metals to the bacteria, and recruiting them to produce free radicals which would kill the little beasties.

In any case, AD research at the molecular level seems to be advancing rapidly, and we can expect some interesting discoveries in the years to follow.

References:
1. A nice review on AD and amyloid, free radicals, and oxidative damage
2. A recent Angewandte Chemie paper on the influence of copper concentrations on Aß geometry, morphology, and aggregation.