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Research Newsletter-Spring/Summer 2006

Neurodegeneration



An interview with Joseph Beckman, Ph.D.
Professor of Biochemistry and Biophysics
Ava Helen Pauling Chair, LPI

Q. How did you get interested in ALS?

A. It began when I watched the national news one night in 1993, when it was reported that mutations to an antioxidant gene cause ALS. The mutations affected a protein that I had been using to protect the brain against stroke and head trauma. My original research was in treating stroke, which started when I was in the army. We modified an antioxidant enzyme called superoxide dismutase, the same enzyme linked with ALS, by chemically coating it with polyethylene glycol to keep the kidneys from filtering the enzyme from blood too quickly. We found that the modified protein protected animals from stroke. This was surprising because we injected superoxide dismutase into the bloodstream and it shouldn't have crossed the blood brain barrier. These results led to clinical trials on head trauma. In the early stage of phase 2 trials, the treatment looked very promising. I was told that some people admitted to the hospital with fixed and dilated pupils recovered from severe head trauma and their intracranial pressure appeared to be decreased. However, with slightly larger doses, the effect went away. In animals, slightly higher dosages of superoxide dismutase were also not protective—the brain actually swelled more. The paradox was that the protein was not toxic even at much higher dosages in animals. I was puzzled about how a good protein could go bad.

Q. How did you make a connection between superoxide dismutase and ALS?

A. In 1989, I had discovered a damaging reaction that was catalyzed by superoxide dismutase. I thought that there must be a disease in which this adverse reaction would occur. Listening to the news that evening in 1993 about a gene implicated in ALS, my interest peaked when Tom Brokaw said, "and this protein is an antioxidant enzyme, raising hopes that the disease may be treated with antioxidants." I then thought that I knew exactly what the enzyme was and what bad thing it was doing to cause ALS. Sure enough, the mutations were affecting superoxide dismutase. So I wrote up my short hypothesis paper on ALS, superoxide dismutase, peroxynitrite, and protein nitration and published it in Nature in August 1993. I have been testing that hypothesis in ALS for the past 13 years. My initial ideas were too simple, but overall the concept is holding up.

Q. How does the body get rid of superoxide?

A. Superoxide is removed by superoxide dismutase. There are three different forms of the enzyme—one inside the cytosol of the cell, one in the mitochondria, and one outside of cells. Detoxifying superoxide is clearly important—one-half percent of the soluble protein in your body is made up of this enzyme. My own research focused on understanding how this enzyme works to protect brain tissues. It scavenges superoxide, but it wasnít clear that superoxide was particularly toxic. The existence of superoxide was predicted and named by Linus Pauling in the early 1930s. He was thinking about reactions between potassium and oxygen and realized that a very unusual compound would be formed in that the oxygen would have a three-electron bond. He knew that potassium superoxide was unstable and even dangerous in the laboratory and called this new form of oxygen superoxide. But when you dissolve superoxide in water under conditions that occur inside the body, superoxide actually is a weak reducing agent. In the body, it is not the "super" oxidizing agent promised by its name.

Q. What does superoxide do in the body?

A. It is produced in large amounts by inflammatory cells, where it helps kill pathogens. But it has a more subtle role that affects most disease processes because of its rapid reaction with nitric oxide. In 1988, I read that the endothelium-derived relaxing factor (EDRF) had been identified as nitric oxide. I had been following this story because EDRF was an unknown and mysterious factor produced by the cells that line all blood vessels. It turned out to be an inorganic molecule that's a common air pollutant. In an old chemistry textbook I had bought from a used book store while in high school, I found a reaction showing that nitric oxide reacts with superoxide to form peroxynitrite and that the peroxynitrite molecule breaks down to form two damaging free radicals—hydroxyl radical and nitrogen dioxide. In other words, superoxide—not a particularly dangerous chemical in vivo—could make very damaging oxidants by reacting with nitric oxide. We characterized these reactions and showed that this actually does happen in many different human diseases. By accident in the lab one night, I added peroxynitrite to bovine superoxide dismutase, which is isolated from cows. The bovine enzyme turned bright yellow and stayed yellow for years. Eventually we crystallized it and determined its molecular structure. We found that a nitro group was added to the sole tyrosine, an amino acid, on superoxide dismutase. We were fortunate to have used bovine superoxide dismutase instead of the human form because the human superoxide dismutase has no tyrosines. We discovered the process of tyrosine nitration, which has been implicated in cardiovascular disease, arthritis, lung injury, cancer, organ rejection, Parkinson's disease, Alzheimer's disease, and ALS.

Q. What's special about tyrosine?

A. Tyrosine is an aromatic amino acid that's important in allowing proteins to make contact with other proteins. It is also important for regulating many processes that control the major functions in cells, such as whether to divide, to differentiate, or to die. It's one of the more uncommon amino acids, occurring in only 3-5% of proteins. There are many proteins that have no tyrosines at all, so human superoxide dismutase is not an exception.

Q. Are other antioxidants like uric acid or coenzyme Q10 useful in ALS?

A. Uric acid may work because the pathologic process weíve implicated in ALS can be inhibited by uric acid. Uric acid is being used as a promising treatment for multiple sclerosis in an animal model. The problem is that it is very difficult to get uric acid into the brain. In our cell culture models, uric acid is protective, and Kristine Robinson in my lab is testing it in vivo right now in animal models of ALS. In some transgenic animal models of ALS, coenzyme Q10 is protective, but only weakly. Most patients are taking large amounts of it. There are studies showing that massive amounts of coenzyme Q10 are therapeutic in Parkinsonís disease and possibly in Huntington's disease.

Q. How is ALS related to other neurodegenerative diseases like Alzheimer's or Parkinson's? Is there a common etiology?

A. I think so. Much can be learned from what is common among the three diseases, and all three can affect some people. On the island of Guam, Parkinson's, Alzheimer's, and ALS can all develop in the same individual with high frequency. All of these neurodegenerative diseases affect motor systems. I believe they are manifestations of a similar disease process but affect different regions of the brain. The underlying mechanisms that we've been studying apply to all three. A particularly promising approach to this has come from my close colleague, Luis Barbeito, in Montevideo, Uruguay, who is interested in pathological changes initiated by astrocytes.

Q. What are astrocytes?

A. Astrocytes are support cells for neurons. In the brain, there are about ten times more astrocytes than neurons. They're important for providing the proper nutrition to neurons and maintaining the proper ion balances of neurons. Astrocytes are also important for defending against infections, when they can become reactive. We're finding that reactive astrocytes may have a major role in ALS, Parkinson's, and Alzheimer's. When astrocytes become reactive, they develop a star-shaped structure, and that's why they're called astrocytes ó star cells. We've worked out much of the pathway that makes astrocytes potentially damaging to neurons. This pathway involves the generation of the oxidant peroxynitrite that may be initiated by the mutant form of superoxide dismutase. Astrocytes respond differently to peroxynitrite than do neurons, which are far more likely to die. Depending upon the brain region, neurons respond differently in ALS, multiple sclerosis, or Alzheimer's disease.



Q. What part of the nervous system is affected by ALS?

A. The brain contains many billions of neurons responsible for thoughts, memories, emotions, and all of our actions, directly controlling who we are, what we perceive, and how we think. These neural networks are organized to pick up sensory information, such as information from the eye, and then process the information in the outer layers of the brain. Deeper in the brain are structures that are involved in more elaborate activity, such as controlling emotional responses. But the ability to move is determined by a remarkably small number of highly specialized neurons in the spinal cord. There, only 500,000 motor neurons control the movement of every muscle in your body. These cannot be replaced. If those motor neurons die, as happens in ALS, you will be unable to move a muscle or breathe. In the final stages of the disease, patients are even unable to blink. Yet the rest of the brain is fully functional. That's what makes this disease so horrible.

Q. How do you study these effects?

A. We study the chemistry of superoxide dismutase, culture astrocytes and motor neurons, and study animal models of the disease. We also work on autopsy samples from human patients.

Q. Do animal models of ALS or other neurodegenerative diseases accurately represent human symptomology and brain pathology?

A. Not exactly. A post-doc in my lab once told a famous neuroscientist, "Well, maybe humans are a poor model of the mouse disease!" But they're the most accepted model available and can teach us many things.

Q. Are there other substances that may be important in preventing the development of these diseases?

A. We know from epidemiological studies that nonsteroidal anti-inflammatory drugs may be protective in some forms of neurodegeneration. For example, people who have arthritis and take an NSAID or other antiinflammatory drug have a much lower risk of Alzheimer's disease. Paired-twin studies yielded similar results. People with elevated uric acid, such as those with gout, have a much lower incidence of multiple sclerosis, and scientists have found similar correlations for Parkinson's disease.

Q. If some of these substances reduce the risk for these diseases, does that suggest that they also may be useful in slowing the progression or even reversing the disease?

A. That's been studied intensely. ALS patients typically take massive amounts of vitamin E, coenzyme Q10, and vitamin C, but the effects are small once the disease has started. There are conflicting studies on vitamin E and Alzheimer's disease. The overall effect seems to be a small improvement in quality of life, but no effect in lengthening survival. But there are some important factors that have not been considered in the human trials. Maret Traber of LPI has shown that vitamin E is only absorbed if it's taken with food. In many studies no one tells the patients that, so they take vitamin E in the morning with coffee and absorb very little. That's one of the frustrating things that leads to so many conflicting and confusing results in the news.

Q. Has exposure to any environmental toxin been associated with the development of ALS or other neurodegenerative diseases?

A. The general consensus is that there's not a strong association between any particular environmental chemical and ALS. There have been lots of studies that find suggestive clues. But ALS is rare, and it is extremely hard to do good epidemiology. The exception is the Island of Guam, where, after WWII, the incidence of ALS was enormous. It's been studied extensively, but no one has figured out the cause. We're collaborating with an investigator, Chris Shay of the University of British Columbia, who has isolated some toxic chemicals from cycad nuts that may play a role. ALS patients have very slightly elevated levels of lead. We tested this with transgenic animals and got the paradoxical result that lead was slightly protective, so we've been investigating that further. Of course, this does not imply that ALS patients should take lead, but it shows us some of the difficulties in studying this disease.

Q. What's wrong with the abnormal superoxide dismutase implicated in some cases of ALS?

A. The superoxide dismutase loses its affinity for zinc, becoming toxic to motor neurons and contributing to development of peroxynitrite.

Q. Have you tried to restore normal superoxide dismutase in animals by supplementing them with zinc?

A. We've approached it from two different angles. If we deplete the animals of zinc and they carry a mutant superoxide dismutase, the animals develop the disease faster. If we supplement with zinc, the animals still develop the disease, but are slightly protected. However, large amounts of zinc caused the animals to die when they were very young. We have shown the deaths resulted from the large doses of zinc that impaired copper absorption. If copper isnít absorbed, iron cannot be inserted into hemoglobin. We have to be careful about taking zinc supplements because of this anemia. Another problem is getting zinc into the motor neuron, which is inside of the spinal cord, protected by the blood brain barrier. Zinc itself is rather toxic to neurons, so a sudden influx of zinc kills neurons in a culture dish.

Q. Can motor neurons be regenerated?

A. Unfortunately, the answer is still no. Adjacent motor neurons will sprout and take over some of the functions of dead motor neurons. We hope to stop the progressive nature of the death of one set of motor neurons after another, which may provide an effective treatment for the disease.

Q. Do you think that stem cell therapy holds promise for ALS patients?

A. Yes, but it's a long way off. I am concerned because some implanted stem cells can develop into tumors. It's still a process that needs to be studied carefully for a more complete understanding. We need to have better access to more stem cell lines to really unravel this mystery. I think our work with micronutrients will be of greater benefit much sooner.

Q. Is there something people can do to reduce the risk of developing ALS, aside from taking vitamin E?

A. Vitamin E is not proven to help, but it is the only compound that has ever been identified that might reduce the risk of developing ALS. I think maintaining adequate zinc levels might help, but there is no evidence for this. It is based only on our animal experiments. Only 2% of patients who develop ALS have mutations to superoxide dismutase, but members of their families who have not developed ALS may carry superoxide dismutase mutations. Low-dose zinc supplementation in those family members may be helpful. Zinc deficiency is also a chronic problem in the U.S. population—after magnesium, it's the second greatest mineral deficiency.

Q. Are there geographic concentrations of ALS?

A. Other than Guam and a couple of places in southern Japan, the incidence is remarkably constant around the world.

Q. Are there specific gene mutations that have been identified in Alzheimer's or Parkinson's?

A. A number of genes have been identified in Alzheimer's and Parkinson's. Some of them cause what is called earlyonset familial autosomal dominance Alzheimer's disease. Mutations to genes called presenilin 1 and presenilin 2 account for 60% of patients with a familial origin of the disease. A lipid-carrying protein called ApoE4 predisposes people to develop Alzheimer's disease. But all these mutations account for only 5-10% of the total number of Alzheimer's patients.

Q. Are motor neurons attacked in Parkinson's disease?

A. No. In Parkinson's, the striatal cells in the substantia nigra are affected. Some of the neurons turn black from oxidation of the neurotransmitter, dopa, which is indicative of oxidative stress.

Q. Is oxidative stress a common denominator in many of these pathologies?

A. Oxidative stress plays a role. So does brain inflammation, as illustrated by the activation of astrocytes. But are these events early causal agents or tombstones—something that's happened after the die has been cast and the cells are already dead?

Q. Have you had to develop special analytical tools to study peroxynitrite, motor neuron death, and astrocyte activation?

A. Absolutely. Probably 95% of our effort goes into developing analytical methods. But once working, the experiment generally moves along quickly. You have to be suspicious when you get the answer that you want and perform control after control to make sure you havenít deceived yourself. I am particularly excited by the use of mass spectrometry to isolate superoxide dismutase directly from tissues. This method allows us to determine precisely whether it has copper, zinc, both, or neither bound to it. No oneís been able to do that until now. We now have a method to measure directly superoxide produced in tissues, which is exciting because it gives us insights into what's happening inside motor neurons and astrocytes. We can also use those analytical tools to understand why vitamin E works. That's not been proven yet, but the epidemiological evidence indicates that it may have important effects in the brain. There is strong evidence that the brain is susceptible to vitamin E deficiency. For instance, horses that are deficient in vitamin E develop an ALS-like syndrome. Humans, on the other hand, develop a sensory neuropathy not usually associated with a primary motor neuropathy defect. The tools for genomics, the advances in mass spectrometry, and the technology to study how every gene in a cell is turned on or off were unimaginable 15 years ago. Now you can really investigate these processes at a level that is just truly astonishing. And yet it still comes back to a lot of the science that Linus Pauling invented when he applied quantum mechanics to chemistry. When we study the structure of superoxide dismutase and visualize it in color—all 1,200 atoms—we use the coloring scheme called CPK that Corey, Pauling, and Karplus devised many decades ago. Much of what we study was anticipated or predicted by Pauling in the 1930s and 1950s. I heard a story about a young student in a lab in Albany, New York, that was famous for developing methods using gas chromatography. The student was working at night, and there was an old man working in the corner of the lab, injecting things into the gas chromatograph. The student was really impressed with how friendly and knowledgeable the guy was. The next day he came into the lab and asked, "Who's that old guy down at the end of the bench?" "Oh, that's Linus Pauling." He had come to the lab in his 70s to learn the latest gas chromatography technology so that he could analyze metabolites in urine and plasma, a method he called orthomolecular diagnosis.

Q. What other micronutrients may be helpful in ALS?

A. We have become very interested in Tory Hagen's research on lipoic acid and acetyl-L-carnitine, two "age-essential" substances that induce genes that help you resist stress. We have seen benefits with acetyl-L-carnitine and lipoic acid in our transgenic animals. Nobody's tested these particular substances in ALS patients. Based on the evidence that Tory's gathered, we have a plausible mechanism for how they might work. Tory has observed mitochondrial dysfunction in old rats, and it's also a problem in ALS. Energy is produced in the cell's mitochondria, and Tory refers to them as the "Achilles' heel" of aging. They are fascinating organelles that also play an important role in nerve degeneration.

Q. Does peroxynitrite attack mitochondria?

A. Absolutely. Mitochondria are a key site for the generation of superoxide. Nitric oxide will diffuse into the mitochondria and combine with superoxide to form peroxynitrite, which then attacks the superoxide dismutase in the mitochondria. We're collaborating with Tory's group on this new probe that can measure superoxide. In fact, we've used it to measure superoxide in heart mitochondria from old rats. Kristine Robinson in my lab is doing most of this.

Q. Does peroxynitrite have some beneficial effects?

A. It kills pathogens and is critical for defense in the immune system. If you knock out the ability to make both superoxide and nitric oxide in a mouse, it's nearly impossible for it to survive to adulthood without dying from massive infections. However, immune cells that produce peroxynitrite almost always die as a consequence.

Q. Does peroxynitrite figure in autoimmune diseases or immunological problems?

A. It has a huge role in autoimmune diseases. In fact, immunologists have recently begun to pay attention to tyrosine nitration because if you put a nitro group on a protein, it makes it antigenic. That explains how antibodies can be developed against endogenous proteins that are then recognized by the immune system. I read a paper today showing that a huge number of antibodies to nitrotyrosine are found in the synovium of arthritis patients.

Q. Does uric acid scavenge peroxynitrite?

A. Technically, uric acid is not a scavenger of peroxynitrite. It competes for tyrosine nitration, which sounds like a subtle difference, but is biologically very important. Uric acid as an experimental tool is useful to study tyrosine nitration and to distinguish that from other actions of peroxynitrite. Uric acid seems to afford some protection in a wide range of different diseases, but the levels that you need for protection are about three- to four-fold higher than what are normally present in your plasma. In multiple sclerosis, immune cells cross the blood brain barrier, and uric acid is very effective in preventing that. But ALS involves the motor neurons and probably wouldn't be affected.

Q. What about clinical trials with micronutrients?

A. We're quite excited that these can be tested in patients. When we've tried to develop drugs, there's an eight- to ten-year process trying to get a drug company interested. Because of potential side effects and toxicity with drugs, the protocol development is slow and painstaking. With micronutrients, you can proceed quite rapidly into clinical trials because of their excellent safety profiles.

Q. Do you have collaborators with whom you work closely?

A. Yes, I work with a couple of excellent scientists in South America, particularly from Uruguay, who've been very instrumental in all of our work on ALS. They have provided most of the ideas that have driven this project forward. And they are the ones who are developing the connections between ALS, Alzheimer's, and Parkinson's disease. We also work with scientists in Zurich and all over the world. With the Internet, it's easy to share data and even look through a microscope 12,000 miles away. The world has become much smaller.

Last updated June, 2006