Tuesday, October 31, 2006

FROM THE LAB.4 - Nov 2006

by Dr. Malcolm Leissring, Scripps Florida

Q: What’s so special about insulin-degrading enzyme?
A: Let me count the ways….

For those new to this blog, this is the latest entry in a series describing what scientists know about Alzheimer’s disease and how that knowledge is being applied to the development of new treatments. We’ve come a long way, describing how the main culprit is the excessive accumulation of a small protein fragment called beta-amyloid, how this accumulation is based on an imbalance between its production and its destruction, and how the key players involved in the destruction of beta-amyloid are enzymes known as proteases, which chop it into pieces.

Last time I gave a laundry list of proteases that can degrade beta-amyloid, and this month I am going to explain why my lab is especially interested in one protease called insulin-degrading enzyme (IDE). Last month, grant deadlines precluded me from adding a blog entry, but—to make up for it—I have two VERY exciting recent developments to tell you about.

Insulin-degrading enzyme (IDE) was actually the first protease to be reported to be able to destroy beta-amyloid, way back in 1994. The original paper received scant attention however, probably because the study relied on brain tissue that was basically put in a blender—hardly a refined experiment!

It turned out however, that the original investigators were on to something. A few years later, Dennis Selkoe, my post-doctoral advisor at Harvard Medical School, discovered that IDE was the main protease responsible for degrading beta-amyloid even in intact cells grown in a dish.

This did not halt the skepticism however, for a very important reason. Why? Because IDE is mostly present on the inside of the cell (the “cytosol”), whereas beta-amyloid is secreted outside the cell. Many, many papers had shown that IDE was either present on the cell surface or secreted like beta-amyloid, but this did not convince the skeptics—they basically thought that what was really happening was a few cells were breaking open and spilling their guts, and IDE along with them, meaning that people were basically studying an artefact. Scientists thought this, because we know the mechanism by which most secreted proteins get out of the cell, and IDE does not have what it takes to be secreted by this route.

This leads me to the first exciting and surprising discovery, which was published in the October 20th issue of Cell, one of the most prestigious journals in all of science. This finding was from outer space: IDE, it turns out, is the “cellular receptor” for varicella zoster, the virus that causes chicken pox and shingles! A “cellular receptor” is basically a handle on the cell surface that the virus grabs onto, which allows it to enter into the cell.

Why does this disprove the skeptics? Because (a) the virus is far too large to enter the cell on its own, and (b) cells that may have accidentally released IDE by breaking open are dead and gone, and so cannot be infected by the virus. The only remaining possibility is that IDE really is on the cell surface of living cells. Never would I have guessed that the final definitive proof would come from virology--Wow!

Let’s go back to why IDE is particularly important beta-amyloid protease. The work of Dr. Selkoe and others on IDE got the wheels turning inside the minds of another kind of scientists—the geneticists. Genetics is the study of how various traits—such as eye color or susceptibility to diseases—are transmitted through our DNA. Geneticists are heroes in Alzheimer’s research, because they discovered specific mutations in specific genes that cause Alzheimer’s with 100% certainty in a few families that are scattered around the globe. Their discoveries showed beyond any shadow of a doubt (at least in my opinon and that of most Alzheimer’s researchers) that too much beta-amyloid (specifically a long form composed of 42 amino acids) is the cause of Alzheimer’s disease. When the geneticists turned their attention to IDE—lo and behold—they found a significant link! The first papers describing this genetic connection appeared in the prestigious journal Science in the year 2000, led by the laboratories of Rudy Tanzi and Steve Younkin.

The next piece of evidence implicating IDE in Alzheimer’s disease came from a study led by Wes Farris, my close colleague while I was in Dr. Selkoe’s lab. Wes, together with a number of colleagues including Rudy Tanzi and Suzanne Guenette, examined mice that had the IDE gene turned completely off. These mice had significantly higher levels of beta-amyloid in their brains than mice with an intact IDE gene. Quite significantly, Wes looked directly at beta-amyloid degradation in neurons taken from the mice lacking IDE, and found that the amount of degradation of extracellular beta-amyloid was reduced by nearly 95%. That means that IDE is far and away the most important protease involved in the degradation of extracellular beta-amyloid in neurons, though we should not discount the importance of proteases that can degrade beta-amyloid before it is secreted outside the cell.

Hot on the heals of this result, I led a study wherein we generated mice that have twice as much IDE in their brains as normal mice. When these mice were crossed to a mouse model of Alzheimer’s disease, this resulted in a ~50% reduction in the amount of beta-amyloid, even a greater reduction in amyloid plaques—and even prevented the premature lethality that these mice are vulnerable to. This work has been described in previous posts, so I won’t elaborate further.

So is that enough reason to favor IDE?

Let me give you yet another reason, which is the second major (and incredibly exciting) advance that I eluded to. But I need to give you some background first.

How do we know what we know about all these different proteins that we study? Well, its mostly through indirect means. We can purify proteins, and if they are enzymes like IDE, we can measure their ability to do what they do, which in the case of IDE is to cut small peptides like beta-amyloid into pieces. We can measure their mass and shine different kinds of light on them, which can give us clues as to how they might work. But what we really want—the ultimate goal—is to know exactly what the enzyme looks like in 3-dimensional space, down to the individual atoms.

Now, how could we possibly do this? I mean, enzymes are incredibly complex creatures—IDE, for example is composed of far more than 2,000,000 atoms! Peering into microscopes would never ever give us that level of detail. It just ain’t gonna happen.

It turns out there is a technique that can accomplish this amazing feat. It’s called X-ray crystallography. Let’s break it down. Crystals. We all know about crystals. Take salt, for example, or diamonds. The characteristic feature of crystals is that they form a highly regular pattern in 3-dimensional space. Salt crystals are square. Different gem stones, for example, can take on a variety of shapes, like hexagons, tetrahedrons, dodecahedrons, etc.

The regular spacing of atoms within a crystal allows us to determine the relative positions of each atom in space. Exactly how they do this is pretty complex (and beyond me, frankly), but basically they shine light at the crystal of a particular wavelength that is approximately the same size as the atoms in the crystal. The light that you and I see all around us is just a tiny slice of all the wavelengths within the so-called “electromagnetic spectrum.” Microwaves, for example, are a form of electromagnetic radiation that just happen to be perfectly sized to heat water molecules. It turns out that X-rays are the perfect size to look at atoms. What happens is they shine X-rays on the crystal, and some of the light will bounce off the atoms and be deflected. Then they spin the molecule around on one axis and measure all the light that is reflected. Somehow (don’t ask me how), they can use this information to reconstruct the exact 3-dimensional configuration of the molecules in the crystal.

But, relative to common table salt (comprised of only 2 atoms), proteins are HUGE, made of hundreds of thousands to millinos of atoms, and also quite flexible, so it is not a simple matter to get them to form crystals. Basically, scientists just try out hundreds and thousands of different conditions (different salts, pHs, temperatures, etc.), more or less randomly at first, to see if any of them yield crystals. My lab was working vigorously to try to get crystals of IDE. We actually got some crystals, and even took them to an X-ray source, which is a humungous, miles-wide physics facility of which there are only a few in the world. Unfortunately, the data we got were far to “fuzzy” and so could not be interpreted. So it was back to the drawing board.

Well, it turns out that several groups have been trying the same thing with IDE for many years (not too surprisingly). One group got lucky, led by Wei-Jen tang at the University of Chicago. It was more than luck, I can assure you, but luck plays a very big role in science.

Dr. Tang’s group succeeded in getting not just one, but FIVE crystal structures of IDE. What is so cool is that they got structures of IDE together with 4 different peptides that IDE can degrade: insulin B chain, amylin, glucagon and … beta-amyloid! What a feat!

Dr. Tang submitted their paper to Nature (arguably the most prestigious scientific journal), and I was lucky enough to review the paper together with Dr. Selkoe. This process can be tedious, and we went back and forth for 3 different revisions, spanning several months. But I think we, as reviewers, made a significant contribution, as I will try to explain. It also gave us the opportunity to write the “News & Views” commentary accompanying the article in Nature, which—although small peanuts compared to the article itself—certainly was a terrific opportunity.

The structures show that IDE looks like a clam shell, with two bowl-shaped halves connected by a hinge. In all of their structures, IDE was “closed”, completely encapsulating the substrate. As it happened, another crystal structure of a bacterial form of IDE had been solved (but, amazingly, never published on). For this structure, the protease was cracked open. This suggested the protease could open and close, in a manner not unlike the main character in the familiar video game, “Pac Man.”

In the original version of the paper that we reviewed, Dr. Tang made the claim that IDE is normally closed. His rationale was quite reasonable: he found that the two halves of IDE formed very close contacts, which would be predicted to be held together by a physical force known as “hydrogen bonding,” which is something like a weak glue or magnet. While this was reasonable in principle, we felt that other factors might explain the fact that IDE was in a closed state in the crystal structures. One possibility is that the binding of the substrate (i.e., beta-amyloid, glucagon, etc.) held IDE in the closed state [Note, Tang’s team used an “inactive mutant” that was incapable of cutting the substrate into pieces]. Another possibility is that the crystal could only form when IDE was in the closed state—otherwise, you might not get any crystals at all.

Dr. Tang responded with an amazing set of experiments. He introduced mutations into IDE that had two effects. One, the intended goal, was to make it possible to seal IDE shut into the closed state by introducing one amino acid on either side that together could form an irreversible bond under certain experimental conditions. The bond could also be broken under another set of conditions. Thus, he could manipulate the protease at will into a permanently closed state or not. The result, which was not too surprising, was that IDE was inactive when it was clamped shut, since nothing could get in or out.

The other effect of the mutations he introduced was unintended, but—as is the pattern if not the rule in science—led to a most amazing discovery (which was in fact a validation of his original hypothesis). These mutations also disrupted the “hydrogen bonding” that he claimed held the two halves of IDE closed most of the time. The hydrogen bonding can be thought of as a “latch” that holds the two halves of the clam shell shut, much like a latch on a purse. The mutations disrupted this latch.

Here’s the punchline: the mutations accelerated IDE’s ability to degrade substrates by 4000%!!

What does this mean for Alzheimer’s disease? Recall that the transgenic mice that I made had a mere 100% increase in IDE activity. This was enough to reduce the severity of the disease by half. If the IDE inside people’s brains contained the mutations that Dr. Tang introduced, NO ONE would get Alzheimer’s disease, EVER!

But we don’t have those mutations, do we? And we can forget about mutating our IDE genes, at least in the foreseeable future. But what we can do is find DRUGS that accomplish the same feat. In fact, my lab has already discovered molecules that do much the same thing (with some important caveats), although they “only” activate IDE by ~ 350%. Not too shabby for a start, though.

There is a long way to go before this discovery could be translated to the clinic. But what it does accomplish is just as important. It silences the skeptics. It proves them wrong. Most importantly, it gives us hope.

The more donations, the more potential drugs we can test. Please pass it on.


  1. Dr. Leissring,

    Thanks for this meaty post - very exciting!

    After reading through this information, I have some questions that I hope you can address. I realize some of my questions may stem from my lack of understanding of this complex topic! Thanks for your patience.

    I thought it was interesting that IDE is a receptor for the virus that causes chicken pox and shingles. I understand that the significance of this discover was that IDE had to be present on the outside of cells. But is there a connection between the receptor aspect of IDE and the idea that Alzheimer's might be associated with a virus?

    Is there any evidence that our brain cells might produce or excrete more IDE in response to increased beta amyloid?

    Your explanation of x-ray crystallography is interesting. But do we know that the configuration of IDE in a crystal is the same of that in the body?

    And one last question for a while...if you manipulate IDE to have a higher level of activity, thus degrading more beta amyloid, will it degrade other essential substances (insulin is the obvious one)?

    Thanks for all your work...

  2. Dr. Leissring;

    Ditto on the previous comment about Alzheimer's being associated with a virus. That hit me like a ton of bricks. I'm having a hard time tracing my family history in relation to when the first symptoms of Alzheimer's appeared in one of my relatives other than my Grandmother. Depression seems to be the only consistency, is beta amyloid linked by any chance?

    I appreciate your writing on this complex issue - it is very easy for me (non medical background) to read and digest. Thank you and keep the information coming!

  3. In response to Mona's and Holly's astute questions:

    Mona wrote: "is there a connection between the receptor aspect of IDE and the idea that Alzheimer's might be associated with a virus?"

    This is a great question. The very first paper I ever published was a review on the idea that herpes simplex virus 1 (HSV1)--the virus that causes cold sores--was associated with Alzheimer's disease. Now this idea was pretty "out there" with respect to well-established theories. But I did my duty and examined the evidence. It was pretty weak, in my considered opinion. For example, if just a single case was incorrectly diganosed (not at al unprobable), then the evidence would completely fall apart. I don't mean to disparage the researchers pursuing this, but there numbers were just not good enought to make a rock-solid case.

    Believe it or not, the virus that causes chicken pox and shingles (varicella zoster) is related to HSV1. Both are so-called herpesviruses. Both infect neurons. And practically everybody is infected with both viruses-and I mean everyone! But different people show different degrees of symptoms, and in general, these symptoms return in the later years of life, when the immune system begins to fail.

    So, thatnks to this exciting new paper, we have a new hypothesis to explore. Since the shingles virus binds to IDE, it probably turns it off, preventing it from degrading beta-amyloid. What is exciting is that inhibitors of IDE prevent infection. And our lab has recently developed inhibitors that are ~1,000,000 times more potent than those used in the Cell paper. describing this result. Also, a vaccine for varicella zoster has been developed, and medical doctors are encouraging ALL elderly folks to get this vaccine because it is quite safe. Please ignore all the scary talk about vaccines floating about nowadays--most is unscientific hype. Talk to your doctor.

    Mona wrote: "Is there any evidence that our brain cells might produce or excrete more IDE in response to increased beta amyloid?"

    Some proteins go up or down dramatically--sometimes several-hundreds or thousand-fold--if you simply sneeze. Others are really stable. IDE appears to be in the latter category. The largest observed change in IDE expression was ~40%, as we reported (Leissring et al, Neuron 2003). This reduction was seen in mice over-expressing a different beta-amyloid-destroying protease called neprilysin. We interpreted this as cross-talk" between the two proteases.

    But you know what, Mona, you got me thinking. The mice with increased neprilysin expression also had dramatically decreased beta-amyloid levels. So, as you suggest, less beta-amyloid, less IDE. You've inspired me to do the experiment!! I'll report back soon!!

    Mona wrote "Your explanation of x-ray crystallography is interesting. But do we know that the configuration of IDE in a crystal is the same of that in the body?"

    Another great question. The answer is that the *relative* positions of the atoms are certialny the same, but the *exact* positions are less certain, and surely in the body the enzyme is moving around, whereas in the crystal, it is necessarily standing still. There is a lot to be gained from knowing the relative positions of the atoms, but it is an incomplete picture, perhpas on "frame" in a movie, and perhaps an unnatural one.

    Mona asked: "if you manipulate IDE to have a higher level of activity, thus degrading more beta amyloid, will it degrade other essential substances (insulin is the obvious one)? "

    This is the usual question/criticism about manipulating any enzyme's activity. There are two hopeful answers. First, if the basic problem underlying certain cases of Alzheimer's is too little IDE, then raising those levels is only going to resotre normalcy. The trick is to figure out for which cases this treatment the appropriate one. I think we'll get ther eventually, but it's a way off.

    The second hopeful answer is that IDE is really very different from a lot of proteases. The substrates (e.g., beta-amyloid, insulin, glucagon, etc.) get basically compltely entrapped within a very large chamber within IDE, and there are many points of contact that are unique for each substrate. You could in principle find a drug that affects one substrate's contacts, but not another. Believe it or not, we've already identified molecules that show that this is true. Stay tuned....

    Holly asked "is beta amyloid linked [to depression] by any chance?"

    Hard to say. Alzheimer's is relatively common. Depression even more so. Depression can lead to unnhealthy behaviors, which in general promote disease. Not uninteresting, but difficult to prove any link, because both are so common (like Alzheimer's and herpes or chicken pox).