2009 Advances in Brain Research

Transcription

1 2009 Advances in Brain Research Conversations with five leading neuroscientists on timely topics in brain research JESSELL FELDMAN CATTERALL LLINAS BARNES News Office The Dana Foundation

2 contents 1 Harnessing Stem Cells to Solve ALS THOMAS M. JESSELL, Ph.D. Professor of Biochemistry & Molecular Biophysics Columbia University Investigator, Howard Hughes Medical Institute 5 Pitfalls and Promise on the Road to ALS Therapeutics EVA L. FELDMAN, M.D., Ph.D. Professor of Neurology University of Michigan 9 Ion Channels and Epilepsy: From Molecule to Mouse to Medication WILLIAM A. CATTERALL, Ph.D. Professor and Chair of Pharmacology University of Washington 13 Research on Brain Rhythms Stimulates New Treatment Approaches RODOLFO A. LLINAS, M.D., Ph.D. Professor & Chair of Physiology and Neuroscience New York University School of Medicine 16 Applying Insights from the Study of Normal Aging to Solve Dementia CAROL A. BARNES, Ph.D. Professor of Psychology and Neurology Research Scientist University of Arizona 2009 Advances in Brain Research Brenda Patoine, Interviewer/Writer Barbara Rich, Ed.D., Editor

3 Harnessing Stem Cells to Solve ALS THOMAS M. JESSELL, Ph.D. Professor of Biochemistry & Molecular Biophysics Columbia University Investigator, Howard Hughes Medical Institute Q: Why has Amyotrophic Lateral Sclerosis (ALS) been such a tough disease to crack? A: I think all neurodegenerative diseases are tough nuts in their own way; if they weren t we might have better therapies or even cures for some of them. I think it s a reflection of how difficult these problems are in general, whether it s Parkinson s or Huntington s or spinal muscular atrophy (SMA). There is progress, but it is slow and tough. Therapeutically there is nothing in my view that works very effectively for ALS. To some extent that doesn t distinguish it from dozens of other neurodegenerative and neurological disorders. This is a problem facing the field of neurodegenerative research in general. Despite many advances in understanding neuronal ALS is probably many different diseases with a common cellular and behavioral phenotype. development and function, we are still not yet at the point where we can intervene in an effective way. ALS is particularly tough for several reasons. For one, compared to another motor neuron degenerative disorder like spinal muscular atrophy, for example, which affects children, ALS is a very diverse disease. It isn t genetically uniform. In contrast, almost all kids with SMA have a mutation in the same gene, so there is at least a rational thought process about how to approach the disease. In ALS, probably 85 percent of individuals have a sporadic nonfamilial form, so there isn t the uniformity you see in something like SMA or Rett syndrome. This makes attempts to find a common pathway or common hypothesis all the more difficult. Even with familial cases of ALS, there are many different genes. Superoxide dismutase (SOD1), the first gene identified as a cause of ALS, has been known for 15 years, but no one yet understands why mutations in SOD1 lead to ALS. So the heterogeneity of the disease complicates matters. In reality ALS is probably many different diseases with a common cellular and behavioral phenotype. It has also been difficult to think of rational hypotheses or even to test hypotheses effectively because the neuron that is affected in ALS, the motor neuron, is so inaccessible. It s buried in the spinal cord. To some extent the basic research that is focusing on ALS at the moment is trying to overcome that problem through the use of stem cell biology. The situation is particularly stark in ALS because of the rapid progression of the disease, which typically claims lives in three to five years. There is still very little that one can do. It is a challenge to the field. The encouraging thing is because of certain basic advances, people now feel guardedly optimistic that a focus on this disease will have an impact over the next five to ten years. Q: What are the key scientific advances that are fueling this optimism? A: It comes back to the point that 85 percent of people with ALS have no obvious genetic component. So the question is, why do motor neurons die? Is it an environmental toxin? Is it a disorder in glutamate 1

4 Harnessing Stem Cells to Solve ALS continued clearance? Is it a protein defect? What is cause and what is consequence? None of these questions have really been answered. The problem is, how do you test hypotheses in an adult-onset disease that affects motor neurons? One way forward is to find a way to study in vitro large numbers of human motor neurons that carry hallmarks of the disease. If you can do that, it means you can test some things rationally and apply the advances in genomics and biochemistry to the problem. Remarkably, that is beginning to become possible now. Our interest in ALS has been primarily at the basic scientific level of trying to understand more about the normal program of motor neuron differentiation and connectivity. Our view is that, if we understood how motor neurons are generated normally, how they form connections with muscle, how central connections in the spinal cord form, and so on, then we would have a better chance of understanding what has gone wrong in a disease like ALS or SMA. So we ve spent years trying to understand the normal program of motor neuron development. A few years ago a fellow in my lab, Hynek Wichterle, decided that if one really knew enough about the normal program of motor neuron differentiation, then one should be able to take the same developmental signals and turn another cell type, such as an embryonic stem cell, into motor neurons. This would allow one essentially to generate unlimited numbers of motor neurons. Then one could think about applying ALS genetic insights onto that. That scenario has now become possible, not only in the mouse where we did it, but also in humans. There was a very important paper published in Science 1 last August by my Columbia colleagues Wichterle 1 Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science Aug 29;321(5893): Epub 2008 Jul 31. and Chris Henderson, together with Kevin Eggan at Harvard. They took advantage of the ability to generate motor neurons from stem cells, using this remarkable method for reprogramming skin fibroblasts to become stem cells that was first demonstrated by Satoshi Yamanaka. Wichterle and colleagues took fibroblasts from a patient with ALS and used the transcription factors Yamanaka has defined to reprogram those [t]here are now at least two dozen patient-specific ALS embryonic stem cell lines that can be effectively converted into motor neurons. fibroblasts into stem cells. Then they used the differentiation protocol we had developed to turn those stem cells into human motor neurons. So it was the combination of the basic research that we ve done on the motor neuron, the Yamanaka reprogramming, and Eggan s expertise in human embryonic stem (ES) cells that all had to come together to produce this. Three different backgrounds had to converge to get this result, which was the first real example of putting all those individual pieces together. Now one can generate patient-specific human motor neurons in the billions. You can do that in familial cases where you know the genetic lesion as well as in sporadic cases where you don t know the nature of the insult. Eggan has done this: there are now at least two dozen patient-specific ALS embryonic stem cell lines that can be effectively converted into motor neurons. This gives one the ability to test any hypothesis in human motor neurons that are bearing some reflection of the disease. This is very much the state of the art at the moment. 2

5 Harnessing Stem Cells to Solve ALS continued Q: You and your colleagues have also found that astrocytes play a key role in the demise of motor neurons in ALS. What is the significance of this finding? A: This work was published in 2007 in Nature Neuroscience 2 with my Columbia colleague Serge Przedborski, who was really the leader on this; my group provided the basic scientific perspective to ask the right questions of embryonic stem cells. Serge made the remarkable discovery, which was also made by Tom Maniatis s group at Harvard, that part of the reason Suddenly you can bring the world of contemporary biology to bear on this problem. motor neurons die in ALS is because when the mutant SOD1 gene is expressed in astrocytes, the astrocytes release a toxic factor or factors that act selectively on motor neurons to prompt their demise. I think these two reports together have really motivated the field to ask questions about whether this is true in humans. There were two papers published in Stem Cell in December 2008, one from Eggan s group and one from Rusty [Fred] Gage s group at Salk Institute, that have shown that the same phenomenon is true in humans, perhaps even in a more robust way. Now we can start asking questions about what actually is being released by astrocytes expressing the mutant gene that damages motor neurons. At the moment, there are various candidates that might explain this. To me, what s most important is that, with the availability of induced pluripotent stem (ips) cellderived motor neurons from individual patients, you can now test hypotheses in a rational and a rigorous 2 Nagai M, Re DB, Nagat T, Chalazonitis A, Jessell TM, Wichterle H, Przedborski S. Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nature Neuroscience May;10(5): way, including hypotheses aimed at understanding what is going on with the astrocytes. That was just not possible before. You couldn t think of doing highthroughput drug screens to find compounds that prevent the death of motor neurons, regardless of what the astrocyte toxic factor is. That is going on now. Suddenly, with this ips programming, that limitation or bottleneck in actually getting cells to do research has disappeared. This all happened in the last three to six months of Now you can try to identify what the astrocyte factor is, and you can try to identify what the mutant gene does in motor neurons or in astrocytes. Suddenly you can bring the world of contemporary biology to bear on this problem. One has to be optimistic at least I m optimistic that this rational and rigorous approach is going to change the way we think about the disease in the next five years, to the point that there will be some consensus as to common cellular mechanisms that link the familial and the sporadic cases. This will in turn provide more objective, rational ways of finding therapies. Q: How do you foresee the approach to therapeutic development changing as a result of the ability to generate motor neurons from ALS patients cells? A: Most of the drugs that have been screened to date in clinical trials have been disappointing failures. I think that s because, even though you can find drugs that have some effectiveness in the mouse model, if you don t have a better cellular assay you have only a limited shot at success in humans. So at the very least, these ES cell-derived assays are going to allow you to come up with a better set of leads for drug development. Eventually that has got to result in better therapies. You also want to get the pharmaceutical companies interested, and they don t know how to deal with mouse model assays; such assays are just incompatible with the way that pharmaceutical or biotech companies generally think. But if you can create assays based on ES cell-derived motor neurons, it will allow you to screen 3

6 Harnessing Stem Cells to Solve ALS continued chemical libraries of a million or more compounds to find lead drug targets. Then you can optimize those targets and test the most promising of them in the mouse models. You then have a sort of pipeline, or a way forward, to move from basic drug discovery to something that will work in animal models that should then allow you to identify more effective compounds for human clinical trials. The great thing about these ips cell lines is that you can derive these lines from patients with sporadic forms of the disease, where you don t know the nature of the biochemical lesion. As a result, you now have a chance to ask if the changes in motor neurons expressing the mutant SOD1 gene are the same as the changes in motor neurons from a patient with sporadic disease. By doing that with two dozen lines, you should be able to get better characterization of disease phenotypes. It may turn out that ALS is really seven different diseases, so therapies that will affect ALS disease type one may be quite different from those that affect ALS disease type five or seven. Q: The induced stem cell work as applied to ALS has been hailed as the discovery of the year by the science and lay media alike. Why so much attention on such a relatively rare condition as ALS? A: ALS is the first disease to be studied using skin cells from patients to create disease-specific stem cells for research. But I think part of the reason this has received so much attention is that you could in principle do this for any neurodegenerative disease. It s just that the methods for turning stem cells into motor neurons are much better worked out than for almost any other cell type, so ALS was a natural first target. It s a fortunate coincidence of scientific developments over the last five years that has propelled ALS to the forefront of this kind of research. There was also a paper by George Daley published in Cell shortly after the ALS work was published that did the same thing with a number of other diseases, not only neurological diseases. He showed that you could take these fibroblasts from different patients and make embryonic stem cell lines, although he didn t actually differentiate them into various cell types. I think it s still true that ALS is the only one so far where researchers have gone all the way through the program and generated well-characterized human motor neurons from an individual patient. Now we can create assays to ask what biochemical or genetic changes there are in motor neurons from ALS patients that you don t find in normal unaffected individuals. That work is going on in half a dozen places as we speak. Because this is all so recent, many researchers are just now taking the initial observations and running with them. It s happening in biotech labs, in stem cell labs, in motor neuron labs, even in biochemistry labs, which have gotten interested in the disease because suddenly you can do science in a way that you couldn t before. This is what you want. These are such tough problems that it s unclear where the next breakthrough is going to come from. If there are only two labs pursuing it, it ALS is the first disease to be studied using skin cells from patients to create disease-specific stem cells for research. is going to take longer to reach that breakthrough than if there are three dozen labs doing it, each imposing its own biases and prejudices and testing different theories. In principle, all of these theories can now be tested on the same cell line or the same set of motor neurons, so more cross-validation will emerge. This is what is needed to drive the field forward. If I were a patient or a relative of a patient, that s the way I would want the field to move, as rapidly as possible. n 4

7 Pitfalls and Promise on the Road to ALS Therapeutics EVA L. FELDMAN, M.D., Ph.D. Professor of Neurology University of Michigan Q: In the last few years, we ve witnessed significant advances in unraveling ALS and even testing potential therapies in clinical trials, but effective treatments still evade us. What s been the problem? A: The problem is two-fold. While we have made progress in understanding disease pathogenesis we re still clearly not fully there, and that has made therapeutic development difficult but not impossible. I think until we have a complete grasp on what is causing this devastating illness, we will not be able to We are left with no clear-cut therapy that is able to halt the progression of this disease to any significant degree. completely cure it. However, I don t at all think that this lack of knowledge needs to stop us from trying to develop new therapies. The second part of the problem is that the therapeutics that have been tested have all failed, probably for multiple reasons. The obvious one is simply that the therapy was ineffective. Another reason has to do with the choice of endpoint measures the clinical yardsticks by which we judge a drug s effectiveness. This is still somewhat of a controversy in the field. Should we be looking at respiratory parameters such as how long the need for a ventilator is delayed? Should we look at arm strength, or the ability to walk? How do we define efficacy? These questions continue to be debated. Clearly no drug has been a home run, because if it were, you would see a positive effect regardless of the clinical parameter used. A third reason for drug failure involves how it is delivered to the nervous system. Being able to get the therapy to the cells of interest is extremely important. It could be that a therapy might truly work if it could reach the motor neurons in the spinal cord and/or the brain area that s affected. I think this has been a significant stumbling block for therapeutics. We are very interested in this path, as are many other laboratories, and we plan to begin a small pilot trial soon to determine if stem cells may be an effective way of delivering Insulin-like Growth Factor (IGF-1) directly to motor neurons (see below). These are three key points that I think have hindered our ability to develop a therapy for ALS and bring it to fruition. We are left with no clear-cut therapy that is able to halt the progression of this disease to any significant degree. In terms of therapeutic treatment of this disorder, we re not that much further along than we were in 1939 when Lou Gehrig was diagnosed. Currently my hope for an ALS therapy is not necessarily a complete cure, though that of course remains the goal. But if we could discover a drug or intervention that would halt the progression of the disease stabilize and control it I would be very satisfied, realizing that a complete cure would still be pursued. You can liken it to diabetes: we may not be able to cure diabetes but we can use insulin to control it. Q: Have the current mouse models of ALS failed us in terms of therapeutic development? A: The ALS mouse model is very interesting. The most commonly used model is the G-93A mouse model, in which a mutated copy of the human superoxide dismutase (SOD1) gene, which is known to cause 5

8 Pitfalls and Promise on the Road to ALS Therapeutics continued familial ALS in humans, has been placed in the mouse. These mice develop a disorder over time that clearly mimics ALS both clinically and pathologically. One of the problems with the mouse model is that many therapies that have been tested in it and shown to be very effective have failed in human trials. That This picture shows embryoid bodies or an aggregation of stem cells. These cells have been differentiated as a group to become neural tissue (green). From this ball of neural cells, individual cells start to send long nerve processes out, sensing their environment and contacting other embryoid bodies. Image courtesy of Simon Lunn, University of Michigan has certainly caused a high degree of frustration among not only clinical scientists but also patients. One explanation for this apparent disconnection between mouse and human results is that the SOD1 mutation that was used to develop the mouse model represents only a small portion of human ALS cases. Only 10 percent of ALS cases are inherited or genetic forms of the disease and of those only 20 percent are due to a mutation in SOD1. The mouse model, therefore, clearly represents a genetic form of ALS and likely typifies some aspects of the 90 percent of ALS cases that are idiopathic, but it may not be a true, clear representative model of these idiopathic cases. The mouse is a good tool, but it is not a perfect tool. Q: What needs to be done to overcome these obstacles and advance progress in therapeutic development for ALS? A: Many things need to be done. The obvious need is to develop better animal models. We are clearly going to continue to need more and improved models as we try to understand what is causing the disease and test therapeutic hypotheses. Beyond that, there is this idea out there of conducting what s become known as a futility trial, an idea I m very interested in but am just starting to learn about. Essentially, you set up a trial design where you need many fewer patients for a much shorter time to test whether or not a drug may have a clinical benefit. These kinds of novel trial designs may make it feasible as long as the drug is shown to be safe to replace animal-model testing of promising drugs with a human model. I m completely serious about this. [Johns Hopkins neurologist] Jeffrey Rothstein just conducted a futility trial using the antibiotic ceftriaxone, and with only 60 patients they were able to tell that this drug may potentially offer a new therapy for ALS. Now they re going to do a larger trial with 600 patients. The idea is not only to be thinking about different animal models, but also to look at our human trial designs and how we can investigate the potential therapeutic of a drug using smaller numbers of The obvious need is to develop better animal models. patients. Let s say I find a new drug in my lab tomorrow and I show that it works in the ALS mouse. Should the next step be a big, double-blind, placebo-controlled trial in some 300 patients? If I have a promising drug, and I know it s safe, wouldn t it be better if I could say whether or not it is effective by using a small number of patients? 6

9 Pitfalls and Promise on the Road to ALS Therapeutics continued Q: You have advocated for further study of insulin growth factor (IGF-1) as an ALS therapy, yet a recent IGF-1 clinical trial failed to show a benefit. What s the best way forward in your view? A: I think that targeted drug delivery is the way to move forward with IGF-1. I am almost certain that IGF-1 could provide a high degree of neuroprotection to motor neurons under attack in ALS if IGF-1 could be delivered to the site of injury. From my point of A more practical approach to gene therapy may lie with the rabies virus. view, one of the reasons that the recent 330-patient, double-blind, placebo-controlled IGF-1 trial directed by the Mayo Clinic failed is that IGF-1 was given twice a day by subcutaneous injection. We had no clear evidence that IGF was going to reach the nervous system using this delivery route. In a mouse model of ALS, if you give IGF-1 via subcutaneous injection, it is not effective. However, if you deliver IGF-1 via genetherapy, with the idea that it can actually reach the spinal cord and the motor neurons that are diseased, it is effective in blocking the progression of ALS. The human trial was not a gene therapy trial but rather a trial in which IGF-1 was delivered subcutaneously by injection, so it is not surprising that it failed. I think gene therapy is the easiest way to do targeted drug delivery with IGF-1. There have been several approaches to doing this so far. One has used an adeno-associated viral vector, which is one step up from the common cold virus. The infectious part of the virus is removed and the gene for IGF-1 is inserted, producing a vector that is genetically engineered to produce IGF-1. You can then inject these vectors into muscle where they will be taken up by nerve terminals and transported back to nerve cells. It s a very good approach; it clearly works in animals and one can envision it working in humans. The problem is that it takes a lot of injections, and the injections have to be near motor nerve terminals. You might need to make several hundred injections to get enough IGF-1 transported back to the spinal cord for it to have an effect. While the concept is good, I think it may not be completely practical. A more practical approach to gene therapy may lie with the rabies virus. The rabies virus expresses a coat protein that has a high affinity for motor neurons. When someone is infected with rabies from an animal bite, the virus is selectively picked up by motor neurons via the coat protein and carried back to the spinal cord. The idea is to isolate the rabies coat protein and incorporate it into a viral vector producing IGF-1. In this scenario, you create a viral vector that is both making IGF-1 and that motor neurons selectively latch onto. Some people are even asking if you could deliver the IGF-1-producing This picture shows a field of human neural progenitor stem cells in close proximity to each other. These cells have started to undergo differentiation and become nerve cells. In the green area, cells that have become neural send out long nerves to sense their environment and make contact with other cells. In red, cells that have not yet differentiated maintain their flat stem cell morphology. Image courtesy of Simon Lunn, University of Michigan vector by some sort of local or topical application that is absorbed into the skin rather than injecting it, to eliminate the need for multiple injections. The approach may or may not work, but at least it s a way to be thinking toward new therapies for the future. 7

10 Pitfalls and Promise on the Road to ALS Therapeutics continued Q: You are now collaborating with a biotech company to use stem cells to deliver IGF-1. Where are you in that program? A: We are pursuing an approach to induce neural stem cells (stem cells that are committed to becoming nerve or glial cells) to make more IGF-1, then to transplant these cells into the spinal cord or brain near the diseased area. The idea is to have these stem cells produce and deliver IGF-1. It would be analogous to delivering chicken soup to a sick neighbor. This is something that I definitely think holds therapeutic promise. Toward that end, we are engaged in two research tracks. We have a rat model of ALS that carries the same genetic defect that the mouse model does, but in a larger animal. This allows us to do intraspinal transplantation of stem cells that produce IGF-1 to see if we can slow progression of the disease and prolong meaningful life in the rat. We have also applied to the FDA for approval to transplant neural progenitor cells that endogenously make IGF-1 into the spinal cords of our patients with ALS to see if there is therapeutic benefit. It s a fairly aggressive approach requiring a one-time surgery that will entail multiple injections of the stem cells. We have designed the study as a ramped Phase 1 safety trial, which means we ll do it in steps: enroll three patients, watch these patients carefully and if they do well, enroll three more and so on. If we can show safety then we of course want to conduct a larger trial. We have injected these cells safely into the spinal cords of pigs, which are very similar to humans, and the pigs did extraordinarily well. Based on that work, we believe we can safely transplant stem cells into human spinal cords. I am the principal investigator on the trial, and we are doing it in collaboration with Jonathon Glass and neurosurgeon Nicholas Boulis at Emory University, where the trial will be physically conducted. The trial is funded by a company called Neural Stem. Dr. Clyde Svensen s group at the University of Wisconsin is also looking at stem cell therapy in ALS, and has worked with Dr. Boulis to produce very promising results. Q: If approved, this will be one of the first trials in the country to investigate the use of stem cells for a neurological disease, a big step forward that some argue we re not ready for. What has been the reaction from the scientific and patient communities? A: The ALS community is amazingly collegial and supportive but there are some physicians who feel that this is too aggressive or too bold of a move right We are pursuing an approach to induce neural stem cells to make more IGF-1. now, based on the scientific data that is available on stem cells. I wouldn t say that is the prevailing wisdom among the ALS scientific community, but rather that the jury is still out. I have presented the human trial only a few times publicly and it has been embraced, but there have been investigators who are concerned about doing surgery and transplanting these stem cells into a patient with ALS, who is already debilitated. We understand that transplanting stem cells directly into the spinal cord is a very bold step, but in this aggressive disease we think that bold steps need to be taken. Of course I can t be sure that it is going to work, but I think that it holds sufficient therapeutic promise to go forward. The patients are very enthusiastic, and as long as they fully understand the potential risks, I think it is something that we should pursue. You know, right now, when I diagnose a patient with ALS, I have nothing I can offer them. I have patients who are going to China or Mexico to receive stem cell transplants. I would rather have them stay here and know that we are giving them the best possible care, which someday, we hope, will include stem cell therapy. n 8

11 Ion Channels and Epilepsy: From Molecule to Mouse to Medication WILLIAM A. CATTERALL, Ph.D. Professor and Chair of Pharmacology University of Washington Q: Your research has focused on a form of epilepsy that is caused by a defect in ion channels, but epilepsy is but one of many so-called ion channelopathies. What are the common features of these conditions? A: Ion channelopathies are diseases that are caused by mutations in the genes for certain ion channels. These mutations, while not severe enough to cause death before birth, change the function of the ion channel just enough to cause a disease state. Ion channels, of which there are four main types (potassium, sodium, calcium and chloride), are the key signaling molecules that generate and regulate electrical signals in tissue. Defects in ion channels therefore interfere with their ability to properly regulate tissue function, and disease results. Since these channels are widely distributed in every tissue of the body, diseases In a sense, the inhibitory neurons are the traffic cops of electrical signaling in the brain. of ion channels are also widespread; they may affect the skeletal muscles, kidneys, heart, and many other organs in addition to the nervous system. The first ion channelopathies to be discovered were mutations of sodium channels that cause periodic paralysis of skeletal muscles; these were first reported in In the case of epilepsy, a series of papers from 1998 to 2001 implicated mutations in different subunits of sodium channels as the root cause of two types of childhood epilepsy: a relatively mild form called Generalized Epilepsy with Febrile Seizures Plus and a devastating form of childhood epilepsy called Severe Myoclonic Epilepsy of Infancy (SMEI). We now know that mutations in one type of sodium channel cause epilepsy, while mutations in another type cause periodic paralysis and yet others cause cardiac arrhythmias. That s because different genes encode sodium channels in different tissues. There is a gene that encodes the skeletal muscle sodium channel; there is a different gene that encodes the primary cardiac sodium channel, and there are four different genes that encode sodium channels in the brain. So the same sorts of genetic defects can cause quite different diseases depending on which sodium channels and which tissues are affected. Q: What spurred your interest in studying channelopathies? A: I started working on ion channels at the beginning of my research career, when I was a post-doctoral fellow in the early 1970s. At that time, people were interested in studying the electrical signals in the brain and other cells, which are very important for regulating cell function. In the brain, electrical signals are how nerve cells talk to each other and how they send commands out to the periphery, to tell muscles to contract, for example. Sensory receptors in the peripheral parts of the body, including the eyes, ears, skin, etc., also send information back to the brain in the form of electrical signals. It s been known for more than 100 years that ion channels make those electrical signals. 9

12 Ion Channels and Epilepsy: From Molecule to Mouse to Medication continued What wasn t known, when I began working in the field, was the identity of the molecules that generate electrical signals. In the first part of my career, I worked on discovering the ion channel molecules, and we reported on those studies in a series of papers published in the 1980s. That was an important step toward understanding one aspect of how the brain works at the molecular level, because we were able to identify the molecules that generate electrical signals and study them in great detail by cloning their genes and experimenting with them. That was also the precursor to understanding ion channelopathies, because you can t discover that an ion channelopathy is due to a sodium channel mutation until you know the structure of the sodium channel gene. My work on ion channelopathies is really much more recent, having started in the last five years. We were first attracted to this question of inherited epilepsy because we were interested in making a genetic model of epilepsy in the mouse. We were intrigued by studies from human geneticists that suggested SMEI is caused by loss-of-function mutations in sodium channels that is, by mutations that prevent the sodium channels from working. This finding was a paradox, because sodium channels are responsible for driving the electrical signals in the brain and we know that epilepsy results from too much electrical signaling in the brain. Yet here was a form of epilepsy caused by a mutation that prevents sodium channels from working. We wondered why these mutations cause epilepsy, and we decided that inserting them into the mouse genome was a good way to figure it out. If the mouse developed epilepsy that was similar to human SMEI, it would be possible to study this disease in ways that are impossible in humans. We now have this mouse model of epilepsy and we have built an active research program around it. Q: What have you learned by studying the SMEI mouse model? A: The first interesting result from our work with the SMEI mouse model was the discovery that the particular gene that is mutated (called SCN1A) encodes sodium channels. These channels that are critical for the electrical excitability of inhibitory neurons in the hippocampus, an important region controlling excitability of the brain. Inhibitory neurons are one of two broad classes of neurons in the brain the other is excitatory neurons, which excite or activate other cells. Inhibitory neurons release the neurotransmitter GABA onto nearby We were first attracted to this question of inherited epilepsy because we were interested in making a genetic model of epilepsy in the mouse. excitatory neurons, which quiets them down. There is always a dynamic balance between excitation and inhibition in the brains normal functioning. One can think of this as the yin and yang of electrical signaling in the brain, where you have excitatory neurons activating circuits and signaling muscles to contract, for example, along with inhibitory neurons telling the excitatory neurons to slow down. You need to have a balance. In a sense, the inhibitory neurons are the traffic cops of electrical signaling in the brain. If they cannot make electrical signals and cannot tell the excitatory neurons to slow down, the excitatory neurons just have a party. The result is an 10

13 Ion Channels and Epilepsy: From Molecule to Mouse to Medication continued epileptic seizure, which is essentially uncontrolled, synchronized excitatory transmission in the brain. The mutations we discovered in the SCN1A gene disrupt the delicate balance of electrical signaling in the brain by impairing the inhibitory side of the yin and yang. Q: If a failure of inhibitory signaling causes seizures in SMEI, what might account for the other symptoms of this disease? A: Other aspects of this disease may be explained in the same way. Like other epilepsies, SMEI has a number of comorbidities; that is, other disease symptoms that are separate from the seizures. In SMEI for example, children are ataxic: they have walking difficulties and abnormal gait, even when they re not having a seizure. By far the most troubling comorbidity in these children is psychomotor delay: between ages one through four, their brain development and function either plateaus or regresses. They never really recover from that, proceeding through their teenage years with low IQ and poor motor skills and almost always requiring a caregiver for the rest of their lives. They have a number of other minor comorbidities as well, including sleep disorders and sensitivity to light. Our thinking is that all of the comorbidities seen in SMEI are really due to the same root cause: the failure of inhibitory neurons to fire electrical signals in a normal manner. We ve been studying this hypothesis in ataxia, which we thought might be explained by a malfunction in inhibitory neurons in the cerebellum, which controls movement. This turned out to be the case. In our mouse model, Purkinje neurons, an important set of inhibitory neurons in the cerebellum that coordinate movement, fail to fire normally. We think this is why we see ataxia in our mouse model and in children with SMEI. Knowing that, we can imagine how other comorbidities might result from this same mechanism. For example, the cognitive impairment that we observe in our mice may be due to the failure of inhibitory firing in the part of the brain used for thinking about complex things, the cerebral cortex. We have not shown that specific result yet; it is still a hypothesis based on the results that we have shown in the cerebellum. We are also looking at other comorbidities to determine if they too are a result of a failure of inhibitory firing. It s an active area of research for us, and I would guess that we will have a clearer view in a year or two. Q: SMEI is a fairly rare form of epilepsy. How might better understanding of its pathogenesis lead to progress in more common epileptic syndromes? A: SMEI is very rare, fortunately, because it is so devastating. The estimates of frequency of this kind of epilepsy have been increasing as diagnosis of it has become more widespread, and the current estimate worldwide is one in 20,000 births. I think it is significant because of its severity rather than how many people it affects. On the other hand, it is fairly common for a child to have a seizure in the context of a fever or a disease, but it seldom develops fully into epilepsy. It is becoming increasingly understood that SMEI is responsible for many cases in which a child has a febrile seizure and then goes on to develop a complicated epilepsy syndrome. Even though SMEI is rare and unique in many respects, there are two areas where we think this work may have an interesting and important impact on epilepsy research more generally. First, this sets a precedent for making mouse models of epilepsy by showing that you really can gain new insight into the basis of the disease using a mouse genetic model. We ve made a genetic change in the mouse that is exactly what happens in humans and we think our mouse epilepsy looks just like the corresponding epilepsy syndrome in humans. We think this is a step forward into how to fashion experimental studies on the fundamental basis of epilepsy. I hope it will stimulate other scientists to make genetic mouse models to better explain the pathophysiology of other types of epilepsy. 11

14 Ion Channels and Epilepsy: From Molecule to Mouse to Medication continued I am also hopeful that the basic mechanisms that we will discover of how the brain becomes epileptic in SMEI may have commonalities with epilepsy in general. I suspect that there are a limited number of ways that the brain can lose control of its excitability; evolution has presumably made it very difficult for that to occur. If we can find the exact mechanisms that cause the brain to lose control of its electrical signaling in this model of epilepsy, it is possible that these mechanisms may have some generality for other, more common forms of epilepsy that are difficult to study. Q: How is this understanding now being applied to therapeutic development? A: Our hope certainly is to provide some therapeutic benefit for children with SMEI. One reason this particular childhood epilepsy is so devastating is because it is not well controlled by the existing panel of anti-epileptic drugs. In fact, some drugs that are prescribed frequently for other forms of epilepsy can actually make SMEI worse. There are sad stories of children being incorrectly diagnosed and treated with the wrong drugs, resulting in a worsening of their syndromes. We think another opportunity for our mouse model is to explore some novel therapeutic approaches that are difficult to try in children, due to the very appropriate safeguards concerning testing new therapies in children and the small number of affected children who are available for clinical studies. In mice, we have much more latitude in the kinds of treatments we can try. We have a program that is just ramping up to do what might be called clinical trials on mice. First, we want to see if we can find combinations of known drugs that will be efficacious in the mouse model and determine if it s reasonable to try them in humans. We will investigate drugs that are already in the clinic or are in early development by drug companies but haven t yet been fully tested clinically. For example, there are drugs that can be used to enhance the actions of GABA, the inhibitory neurotransmitter. Some of these are used to treat epilepsy, and some are known in the research laboratory but are not yet used in treatment. We are trying to use novel combinations of drugs that act in different ways synergistically we hope to enhance neurotransmission by GABA neurons and therefore tune up the inhibitory electrical signals and restore the yin-yang balance of electrical signaling in the brain. We have to find a treatment that will strengthen the inhibition without strengthening the excitation. We don t know of a way to do that with drugs that act on sodium channels directly, but we think that we can do it with drugs that enhance the actions of the GABA. There are many so-called GABA co-agonists, drugs that enhance the strength of GABA signaling. The most famous is probably diazepam (Valium), and the one that is used most often in epilepsy is a relative of diazepam called clonazepam (Klonipin; Rivotril). There are other families of drugs that also enhance GABA neurotransmission in different ways but are not so widely used. We are currently treating mice with combinations of these drugs, and have found that the mice are more resistant to seizures and live longer. Based on those findings, we are optimistic. Once we find the optimal drug combinations, we plan to treat the mice with them for a long time, as a child would be treated, to see if they continue to be effective. I think within the next year we will know whether the first set of ideas that we re testing are going to bear fruit. It will be exciting for us if we can find novel treatments that are effective in our mouse model of SMEI and may hold promise for improving therapy of children with this devastating disease. n 12

15 Research on Brain Rhythms Stimulates New Treatment Approaches RODOLFO A. LLINAS, M.D., Ph.D. Professor & Chair of Physiology and Neuroscience New York University School of Medicine Q: You have spent much of your career studying brain rhythms the patterns of nerve-cell activity that underlie various behaviors or mental states and how disrupted rhythms contribute to neurological and psychiatric problems. How is it that such a seemingly simple problem, an abnormal rhythm, can produce such diverse symptoms? A: The idea has always been that psychiatric and neurological conditions are very different. But if one looks carefully at these disorders, one finds that a very similar cellular mechanism an abnormality in the rhythms of neuronal firing can generate many different types of symptoms depending on where in the brain the abnormal activity is located. One example is the aura that sometimes occurs before an epileptic seizure. The aura can be of any An abnormality in the rhythms of neuronal firing can generate many different types of symptoms. type: auditory, where you hear sounds or words; visual, where you see things; motor, where you move; psychomotor, in which you begin to think desperately about certain things, or any number of other kinds of auras. This would suggest that the mechanism underlying these auras must be very different, yet there is but one mechanism: an abnormal rhythm that precedes the seizure. The same cellular mechanism can produce a variety of functional conditions depending on the type of the abnormal rhythm and its location in the brain. Rhythm abnormalities can happen anytime a group of nerve cells in the thalamus or cortex begin to generate oscillatory [firing] activity at a lower frequency than is normal in the alert, awake brain. Specifically, the cells fire coherently and at slower wavelengths in discrete areas of the brain. A similar pattern occurs throughout the brain when you fall asleep. When that type of pattern occurs outside of sleep, the part of the brain affected functions abnormally: it becomes fixed at a low frequency and does not respond correctly to external inputs. It becomes disconnected and non-responsive, similar to what happens under general anesthesia, where low-frequency brain activity correlates with loss of responsiveness to external pain and other sensory inputs. There is also a phenomenon we call the edge effect associated with the area of low-frequency activity. Cellular activity at the edge of low-frequency activity can be altered in an opposite pattern, producing ongoing high-frequency activity. These alterations produce what are known as positive symptoms, which are continuously on. For example, deafness due to low-frequency activity in the auditory system would be considered a negative symptom, whereas a continuous sound associated with such deafness, a tinnutis, is a positive symptom; it is accompanied by high-frequency gamma bands in the cortex due to a failure of inhibitory mechanisms [that would normally tamp down the firing]. In Parkinson s disease, difficulty moving or partial paralysis are negative symptoms, whereas ongoing uncontrollable tremor is a positive symptom. Both types of symptoms are produced by the same mechanism: abnormal rhythms. This is beginning to be called thalamocortical dysrhythmia, defined as a set of neurological and psychiatric conditions produced by abnormal 13

16 Research on Brain Rhythms Stimulates New Treatment Approaches continued oscillatory activity in the major neural circuit that links the brain s thalamus and cortex. Different symptoms are produced depending on where in the brain the rhythm disruption is occurring, but the neuronal mechanisms are the same. Q: How do these understandings help explain the success of deep brain stimulation? A: This the most plausible explanation for why we are seeing such success with deep brain stimulation (DBS.) There is no question in my mind that DBS is one effective way to make progress in treating brain rhythm disorders. It s interesting that electrical brain stimulation has been developed through years of trial and error. The concepts behind it are not new; the effects of stimulating the brain have been known for a long time. The first serious DBS was done in Switzerland in the 1930s and 40s, and actually was the subject of a Nobel Prize given to Rudolph Walter Hess in 1949 for work in animals. The technology so far is not that innovative. We have been very interested in using electrical brain stimulation as a therapy for thalamocortical dysrhythmia, and are pursuing various strategies aimed at improving the technologies and the techniques that are used. We have developed patents for the use of nanowires as both stimulating and recording probes, for example. Q: To date, DBS has largely been used in Parkinson s disease and other movement disorders. Do you see this changing? A: DBS is now also being considered for depression, obsessive-compulsive disorder, and other neuropsychiatric conditions. There is the fascinating case reported by Columbia and Cleveland Clinic neuroscientists Nicholas Schiff and Ali Rezai, who treated a fire fighter who had been in a coma for nine years. They targeted the midline thalamus (intralaminar nucleus), and the man recovered consciousness as well as memories from decades before. We have to pay attention to these cases because they are giving us important clues about brain function. There is also the intriguing possibility that brain stimulation over the long-term may result in secondary changes related to plasticity. We are just beginning to understand these changes and what their implications may be. Q: Do you encounter resistance to the concept of electrically stimulating the brain? A: Sometimes, mostly because people don t understand what brain rhythms are all about. They think you just stick electrodes into the brain and look for a sweet spot and see if the patient gets better. There is a perception out there that we really don t know anything about how electrical brain stimulation works. But we know that such stimulation works by changing brain rhythms, at least for the acute results. The only thing that this electrical stimulation can do to neurons, especially in cases when patients respond immediately to stimulation, is to modify their firing patterns. I believe people s views are beginning to change, in part due to the simple fact that brain stimulation has been so totally successful. Indeed, people who are completely paralyzed, who couldn t do anything, suddenly are able to be constructive members of society and to take care of themselves and so on, all as a result of targeted stimulation of the brain. It is amazing. At the National Institutes of Health, such results have been considered among the most important breakthroughs in 20 th century neuroscience. But it really goes beyond electrical brain stimulation. There is growing evidence that localized microlesions in the brain can help modify dysrhythmias in certain cases, and there are drugs that can help as well. The fact is that now that we have a target abnormal brain rhythms we can address that target through whatever means we have. We have something that can be localized, measured and understood at the singlecell level and at the level of channels and of circuits and so on. It is very exciting. It is interesting to me that it has taken the success of electrical brain stimulation to validate this long history of basic research on abnormal 14

17 Research on Brain Rhythms Stimulates New Treatment Approaches continued rhythms, which people had really not paid much attention to previously. Q: Basic scientists and clinical scientists have historically occupied different worlds, with crossfertilization the exception rather than the rule. Do you see this changing at all? A: Yes, I think the relations between basic science and clinical science are changing very rapidly. This is a touchy issue, because clinical scientists are mostly interested in basic science only when the implications of the basic science research are clear and the applications are mature. On the other hand, it may have taken basic scientists decades of hard work to understand the principles, so they are cautious to take the same care to show how something is clinically relevant. You have this situation where the clinical scientists look at the basic scientists and say, Yeah, but is it relevant? and, the basic; scientists look at the clinical scientists It has taken the success of electrical brain stimulation to validate this long history of basic research on abnormal rhythms. and wonder if they understand the implications of the basic finding, or if they even care. It has been a difficult situation; people know what they know and want to stay in the areas that are familiar to them. Venturing into areas that may be outside the norm must be done with extreme care, and one has to be ready to be criticized by both sides. That said, it is indeed time for change. More and more these days, basic and clinical scientists are taking the extra step and trying to communicate. There is this new willingness to educate one another. Q: You have told the story of using your technique for detecting abnormal brain rhythms on a skeptical neurosurgeon from New York University, who was stunned that you were able to diagnose his tinnutis and describe it just by recording brain rhythms with MEG. Are you finding more acceptance these days for your ideas about the clinical implications of your basic research on abnormal brain rhythms? A: The case of Dr. [Patrick] Kelly was a very good case in point. He was basically saying, Look, I ve been hearing about this research for so many years; can you guys really offer me something that will help my patients? In some ways, the question he asked typified the attitude of many clinicians: Are you coming here to tell us something that we can use or what? I had been discussing this research with Dr. Kelly for many years, and the feeling he had was that there was not very much that this kind of interaction could generate. Since the tinnutis incident with Dr. Kelly, which happened just last summer, I ve been talking to my colleagues in the department of neurosurgery all the time, and many people are coming to find out what is going on. So things like that do help change attitudes, and slowly it is beginning to percolate. Q: What has driven your interest in bridging the divide between clinical and basic science? Why care? A: Well, it is true one is not required to do any of this. We basic scientists have our labs, our grants and areas of studies and so forth. But on the other hand, one may also wonder if understanding the clinical implications of what one does should not also be part of the process. Personally, the issue centers on whether there is more to basic scientific work than the immediate knowledge that it generates. It is clear that we are at a time in history when the nervous system is seriously beginning to be understood. For those of us in basic research, it is a given that the basic knowledge we are generating is crucial and essential. The question then becomes: Is it sufficient? n 15

18 Applying Insights from the Study of Normal Aging to Solve Dementia CAROL A. BARNES, Ph.D. Professor of Psychology and Neurology Research Scientist University of Arizona Q: You are examining how memory processes change with normal aging. Why focus on normal memory changes as opposed to Alzheimer s disease or other pathologies? A: One reason we are interested in studying normal age-related memory changes is that only about five percent of people over 65 have Alzheimer s disease (AD). The proportion increases in older age ranges, but the fact remains that most of us will age relatively normally and will not have a dementing illness. The question then becomes: what is normal versus what is pathological in terms of memory changes? To answer The hippocampus, the structure that s so important to memory, actually maintains a steady supply of its principal cells across the lifespan. that, you have to first know what is normal. Only then can you begin to assess what goes wrong in Alzheimer s or other dementias. That s the approach we have taken. My interest in separating pathology from normal also stems partly from my fascination with the fact that only humans get AD. No other animal gets it. What we re studying in animal models of memory loss is normal age-related memory loss. We re trying to use the knowledge we can gain from animals to then define in humans what might be expected at what age range. If we could understand that, it would have a tremendous impact. The population is growing fastest in the higher age ranges, so it would be very good for us to have a clear, long-term view of how the brain processes that underlie memory change with age. That s the basic rationale for this kind of work. The ultimate goal is to develop therapeutic interventions that could be used to optimize cognitive performance, in normal aging or in pathological states. Q: Has the view of what happens to the brain during normal aging changed in recent years? A: One critical issue revolves around the question of what is happening in the brain networks underlying memory. Do we lose neurons as we age from brain structures that support memory? The dogma until very recently has been, yes, of course, we lose many many neurons as we age. In fact, it was presumed that we lose neurons every day. That has turned out to be false. It is really remarkable that the hippocampus, the structure that s so important to memory, actually maintains a steady supply of its principal cells across the lifespan. This has now been shown across species, from mice and rats to dogs and primates. If cell loss is not the issue, then what is it that goes wrong in the aging brain? It turns out that what goes wrong involves changes in the connections between the cells. Even where there is no reduction in the number of cells, there is a loss of synaptic contacts. As a result, the ability of one cell to communicate with another is diminished. This disrupts the networks that serve memory functions. So we have two sets of broad changes occurring in the aging brain. We know that the synaptic contacts decline; that s an anatomical change. We also know that the ability of the synapses to be modified the plasticity of the synapse is altered with aging. That represents a functional change. 16