Wisconsin Public Television University Place  Myelin Wrapping of Nerve Cells  John Svaren 1/9/13 UP821   >> Welcome to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the UW Madison. I also work for UW Extension, Cooperative Extension, and on behalf of those folk and our other sponsors, Wisconsin Public Television, the Wisconsin Alumni Association and the Science Alliance here at UW Madison, thanks for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight it's my pleasure to be able to introduce to you John Svaren. He's a native of South Dakota. He was born in Sioux Falls and grew up in Arlington. He received his PhD in molecular physiology from Vanderbilt University in Nashville, Tennessee. He did his postdoc in Munich and at Washington University in St Louis. 

He came to UW Madison in 2000, where he is now an associate professor in the department of comparative biosciences in the school of veterinary medicine. He's also the faculty director of the Cellular and Molecular Neurosciences Core in the Waisman Center. Tonight he's going to talk to us about spiraling under control, gene regulation and myelin wrapping of nerve cells. I have to say it, the great twitter that I was able to put out has, Got myelin? Get's on your nerves? [laughter] It's not every day that I get to rip off the Got Milk? people. This is going to be a pretty interesting story. John gave this talk to the Plato Frontiers and Life Sciences group five or six weeks ago. It's a good thing to have nerves that work well. Please join me in welcoming John to Wednesday Nite at the Lab. [applause]  >> I really wanted to thank Tom for the invitation. It's really a pleasure to be here. 

I'm really glad he invited me back again after hearing my talk a few weeks ago. I really appreciate that. Today, or tonight, I wanted to tell you a little bit about myelin and what it does in the nervous system. The second half of the talk I wanted to tell you a little more about some translational projects that we have which are applied to sort of under-appreciated but relatively common disease which I'll tell you a little more about later. Usually I warn people when I talk that I'm going to tell you about neuroscience, even though I'm not really trained as a neuroscientist. I'm actually more of a biochemist. I've learned neuroscience on the side of things. So if you are neuroscientist, or know a lot about neuroscience, I beg your forgiveness in advance for that. Sometimes when I go to parties people ask me what I study and I say myelin, and after the blank face appears I say, well myelin is the insulation around nerves. I talk about how the fact that you're very familiar electrical system at home, of having wires that have insulation around them. 

Well, myelin is sort of the insulation around nerves that transmit impulses to your muscles within the nervous system as well. But it's a little bit more than just insulation, it's very dynamic. I want to tell you more about that, and what it's function is. This is a picture of an axon. We talk about a nerve cell sending projections to either the next nerve of to a muscle. Those projects are known as axons. Oftentimes, in both the brain and as well in peripheral nerves, those axons are associated with myelin. Myelin is something that is very critical to the function of nerve cells. There are certainly nerves impulses that travel and myelin facilitates those transmission of nerve impulses. Importantly, myelin is one of the things that really speeds that transmission. 

Of course, our brain and nervous system depends on rapid transmission of nerve signals. That really depends on myelin that's formed by different cells in the nervous system. The other thing that's very important is that myelin plays sort of a protective function. It also supports the axon itself. This axon looks kind of fragile, and it is fragile. Being associated with different cells that make myelin helps to stabilize it and helps support the function of those axons. So, one of the things I want to define ahead of time are some terms. I also want to mention, too, if you have any questions at all during the talk, I'm really happy to try to repeat something or answer any question you might have. So when we talk about the nervous system, there's really kind of two basic groups of cells. We think of the neurons, which certainly are the cells that communicate nerve impulses. 

The rest of the cells in the brain or the nervous system are referred to collectively as glia. Glia include cell types such as oligodendrocytes that make myelin in the nervous system. There are also other types of glia that play very important roles in the brain, that help respond to infections, or any kind of a nerve injury. There's been sort of an explosion of research in the last decade or so into, really, understanding the function of glia and how they really fundamentally support the nervous system. I'm going to give you a book recommendation. If you want to learn more about this topic, I would recommend a book called "The Other Brain." This is a book written by Doug Fields. He was a researcher at National Institutes of Health. He's been a myelin researcher for a long time, very well known. He's also a very good writer. He's written a number of different articles for Scientific American, and other lay scientific publications. 

So I really recommend this book. This was written for the lay press. You don't have to be a scientist to understand this book. I really recommend it to you. It really tries to go through all the profound functions of glia that have been uncovered in the last decade. There are more to be uncovered, as well. So I mentioned that myelin is very important for the speed of nerve transduction. It really makes a tremendous difference here. So if you take an axon, that is transmitting this nerve impulse, and I've tried to symbolize this here by this little blue dot that's going across the screen. Unmyelinated axons typically transmit that nerve impulse at about .5 meters per second. 

But a myelinated axon can transmit that at speeds of about 100-fold greater, roughly 50 meters per second. The reason that it can do that, is that these segments of myelin are separated by little spaces. These spaces are called nodes. The transmission of action potentials can essentially skip from node to node. They transmit at a much higher rate, compared to an axon that doesn't have myelin. So the function of the nervous system is dependant on the speed of conduction. I don't know if you've ever, of if you can remember. Oh, I'm sorry, I didn't see your hand. [inaudible] There certainly are. So, the peripheral nervous system, many of the axons that detect pain, for example, are unmyelinated. 

To me, that makes sort of sense. I don't know if you've ever put your hand accidentally on a hot stove. It's there for like half a second before you say, man, that's hot, and you have to move it away. So pain fibers tend to be unmyelinated, with small diameter axons. But larger diameter axons, that actually transmit nerve impulses to your muscles are larger diameter and they tend to be myelinated. So there are unmyelinated axons both in the peripheral nervous system and in the brain, as well. Sorry I didn't see your hand. They have lights. It reflected off my glasses. So, if you maybe recall, like I can, the days that you had a dial-up modem at home to get something over the Internet, and the patience you had to have to receive some image or something you were looking at, and the day that you got the cable modem and appreciated the speed. 

The DSL really rapidly speeded up the appearance of these images on your computer. You can appreciate the nervous system is like that. You know, that if you don't have rapid transmission of certain nerve impulses, They might as well not be there at all. They're not arriving in time. The function of the nervous system, both in the brain and in the peripheral nervous system really depends on very fast transmission of nerve impulses. So, I'm going to tell you a little bit more about myelin in the central nervous system, partly because at the Waisman Center, I'm fortunate to have colleagues. The Waisman Center has a large number of people that are very adept at imaging of myelin. You probably know of Richie Davidson, and also many of his colleagues that have done brain imaging of different types. Part of that imaging is focused at looking at what's referred to as white matter. White matter refers to regions of the brain that are very highly myelinated. 

Most of the axons are myelinated. Other parts of the brain, which are gray matter, have less myelin. One of the most critical areas of myelination is actually a structure known as the corpus callosum. The corpus callosum is really what connects the two halves of the brain to each other. I'd like to think of this as the information super highway that communicates between these two hemispheres. Certainly, that's a little bit longer distance in the brain. It's very important for those two halves of the brain to be able to communicate and coordinate their activities very rapidly. So the high speed transmission is very important. You can see this bridge shows up as a very white piece of material on this imaging that was done by MRI. This was a slide provided to me by Andy Alexander, of the Waisman Center. 

One of the reasons that myelin is white is if you section a brain, it also looks white, not only imaging, but also in the section here. The reason that myelin is white is that all this wrapping involves an incredible amount of lipid. Lipid is a very important part of myelin. It helps insulate that. One of my favorite quotes about this, is you don't want to remove all the fat from your body, because the brain is almost entirely fat. Without a brain, you might look good, but all you could do is run for public office. [laughter] So, when you're talking about cholesterol, it's good to have high cholesterol in your brain. We don't want to eliminate that. So myelin itself really depends on a tremendous amount of lipid synthesis as it's formed, and also to maintain that structure. So, in the brain, white matter plays a number of very important roles. 

I really won't have time to go through everything that happens in the brain that's facilitated by myelin, but as I said, it's very important for high speed transmission between different brain regions. There are many diseases that affect white matter in the brain. Probably the best known is Multiple Sclerosis. Multiple Sclerosis results from the destruction of myelin in the central nervous system. For people that have this, they are always in this process of breaking down the myelin and hopefully rebuilding the myelin to restore the function, but over a lifetime, this certainly affects their quality of life, as well. There are certainly brain injuries, other kinds of disorders that affect white matter. In fact, there are also white matter disturbances that have been shown by imaging studies in diseases like schizophrenia, for example. There are also genetic disorders of myelin that profoundly affect the function of the central nervous system. One of the really cool things about myelin is that it's dynamic. It's not something that's just laid down during development, during adolescence, and then never changed. 

It actually responds to neural activity. So there's been some very fascinating studies where people learn a new task, such as juggling for the very first time. People can actually detect changes in myelin, increases in myelination in certain areas of the brain. We sort of increase that ability to coordinate that sort of motor activity. Also, musicians, over time if they are very skilled, they will accumulate changes in myelin structure. So this responds to learning and the environment as well. Myelin is something that's dynamic. There was just a paper that came out just in the last month or so in Nature Neuroscience, showing that if you take mice and put them in a socially isolated environment so that they don't have social interactions, they'll have less myelin in parts of the prefrontal cortex as well. Myelin is something that responds to normal activity. If as area of the brain is not being used the myelin may deteriorate to a certain extent. 

If it gets to be re-used again, the myelin will sort of come back and help grease the skids for nerve impulses to travel. This is pretty much all I know about the neuroscience on this slide here, but one of the things that's unusual about myelin is that our bodies have actually two ways to make it. In the brain, or the central nervous system, there's one cell type which is known as oligodendrocytes. These are the cells that are affected in Multiple Sclerosis. Oligodendrocytes make myelin in the central nervous system. This includes not only the brain, but also the spinal cord. All the neurons in the spinal cord are associated with oligodendrocytes, and many of those are myelinated. In contrast, when nerves exit the spinal cord, and this is sort of a picture of nerves that radiate out through the limbs and to different parts of the body, this is referred to as the peripheral nervous system. There's a completely different cell type in the peripheral nervous system that does exactly the same thing. It makes myelin. 

These cells are referred to as Schwann cells. Actually, you can have differential effects. Multiple Sclerosis doesn't necessarily effect myelin in the peripheral system very much, and there are also specific disorders affecting PNS myelin that don't actually effect the brain. I'll tell you a little bit more about our research on this as well. Most of our work today is actually focused on the peripheral nervous system, so I'm going to spend more time talking about that. Many of the lessons that we learned, I think, are applicable not only to the cellular peripheral nervous system, but also the brain as well, because very common mechanisms are used to make myelin in these two types of the nervous system. One of the things that I think is really fascinating about the peripheral nervous system is the size of the cells. When you think about, you have nerves that are going down the back of your leg for example, they're exiting the spinal cord, so they're entering the peripheral nervous system. They can innervate muscles all the way through your leg, down even to your big toe. Those motor axons that control that are essentially one cell. 

The cell body is back in the spinal cord, and they've sent a projection all the way down through all that tissue, maybe more than a meter depending on how tall you are, to innervate those muscles all the way down through your leg here. This is just one example I borrowed from a friend of mine. If this little square up here, which you can barely see, is the cell body in the spinal cord, and if you drew then the axon to scale, based on that scale-- See all those lines going back and forth here? I don't even know how many laps there are, but that would be the length of the axon. Keep in mind that the cell body, which has a nucleus and all the other parts of the cell, is way down here. It has to transmit nerve impulses to muscles way down to the bottom of this as well. I tried to come up with my own analogy. This is my favorite one. If you're a single peripheral nerve that went from the spinal cord down to your toe with the same diameter as the Alaska pipeline, which is about one meter wide or so, it would actually stretch the length of the Alaska pipeline. It's about 800 miles. One single cell that's transmitting information from the top of this pipeline down to muscles for the very longest peripheral nerves that exist within your body. 

The integrity of that transmission is very important, and the maintenance through your lifetime of those peripheral nerves is also very important, in order to maintain our ability to keep balance and walk through the lifetime that we live. I wanted to tell you a little bit more about peripheral nerves. But I just wanted to take a minute to acknowledge some giants in the myelin field. Actually, Richard and Mary Bunge who actually used to be at Washington University in St Louis where I did post-Doc. They eventually moved down to direct the Miami Project which was involved in trying to develop cures for spinal cord injury and that sort of thing. They really pioneered many of the basic aspects of myelin structure. Richard Bunge was actually a medical student here at the University of Wisconsin. Mary Bunge was a graduate student here at the University of Wisconsin. I had the privilege of marrying-- Not marrying, but meeting Mary Bunge at a meeting just a couple of years ago. She raved about her experience at the University of Wisconsin. 

She said that she and her husband, who had unfortunately died about a decade earlier, really treasured the time that they spent at the University of Wisconsin. We have kind of a long history with myelin based on the work of these pioneers as well as other people here that are at UW. As I said, our lab has been focusing more on Schwann cells, which are actually the cells that make myelin in the peripheral nervous system. Now this is a picture here, an electron micrograph picture of axon here, which is associated with a Schwann cell here. You can see the Schwann cell sort of encompassed the diameter of this axon here, in the perimeter of that axon. Then during development what happens is this cell begins to send out this little lip of membrane. It begins to migrate along the perimeter of this axon here. This continues many, many times. This lip of membrane sort of makes, you know, many, many laps, maybe 10 or 20 laps around the cell. Then you end up with something that looks like this. 

You have compact myelin. This sort of black structure around the edge of the cell here is formed by about 10 or 20 laps of membrane that is wrapping the axon. Now Schwann cells are fascinating in that they do this sort of gymnastics, at the same time, they're not even relying on outside sources of lipid. They are actually making their own cholesterol from scratch. They're not relying on cholesterol you get from the diet or that's produced by the liver. They're actually making cholesterol at the time when they're doing this as well. So you have a tremendous number of events happening as the cells wrap around here. There's a lot of metabolic changes, a lot of lipid synthesis that's happening at this time, in order to make this compact myelin. Keep in mind this then has to be maintained over the lifetime of the individual. The Schwann cells have to keep making lipid at a certain level to sort of maintain the structure of that. 

Schwann cells are interesting for a number of different reasons. There are a number of diseases or pathologies that effect myelin in the peripheral nervous system. I'm going to tell you more in a minute about a set of disorders known as inherited myelinopathies. This is referred to as Charcot-Marie-Tooth disease. This was named after two French physicians and a British physician who co-contributed the first characterizations of this disorder. It's usually abbreviated as CMT. I'll tell you a little bit more about that in a minute. There are also other things that effect peripheral myelin. Your probably aware of diabetic neuropathies. Diabetic neuropathies effect peripheral nerves. 

They eventually effect the structure of myelin as well, so that's a very common thing that you'd expect exhibited by especially people with type 2 diabetes. Another very interesting thing to me is that actually leprosy is actually a disease of peripheral myelin as well. Leprosy, which is also known as Hansen's disease, is caused by an infection of Schwann cells by a bacteria known as Mydrobacterium leproe. That leads to demyelination and damage to the myelin in the peripheral nervous system. As a result there's a loss of feeling and other problems that people who have Hansen's disease or leprosy experience over their lifetime. There are a number of diverse sort of things that effect peripheral myelin, so I think it's a very interesting cell type. First I want to tell you a little bit about my interest in Schwann cells, then I want to go into some of the translational projects that we have going on in our laboratory. What I've been really interested in is trying to understand all the genetic events that coordinate all these important events by myelination. Oops. Almost lost that. 

We're particularly interested in how genes that coordinate myelination are turned on during the myelination process, how these are related to diseases that effect myelin. We're also very much interested in epigenetic mechanisms, or chromatin modification mechanisms that coordinate this process of myelination. Like many developmental processes there's lots of important genes that are involved. We're interested in how these gene control events are coordinated. This is a picture I like very much here. This shows and axon that's surrounded by myelin here. This is the large nucleus of the Schwann cell right here. We're very much interested in what's happening in that nucleus. What's happening on the DNA that changes all these metabolic processes and everything that lead to the formation of myelin? But I also want to then introduce some of the reasons we're interested in those sorts of processes as well. I mentioned before that there's this class of diseases known as inherited peripheral myelinopathies. 

This was abbreviated as CMT, as I mentioned before. This is actually one of the most common inherited disorders of the nervous system. It effects one in 2,500 people, which is a very high prevalence. Surprisingly, not many people know about this compared to other kinds of disorders which are even rarer. For example, ALS, or Lou Gehrig's disease, is not as common, or other disorders. This is relatively common. These disorders, and there are more severe forms here, as well known as Dejerine Settas Syndrome or congenital hypomyelinating neuropathy. These share a number of different kind of hallmarks. These result from failure to either to create or to maintain peripheral myelin. As a result, you end up having decreased nerve conduction velocities. 

These are often diagnosed clinically by a neurologist who can apply two electrodes to an arm for example, and they can measure the amount of time it takes for an electrical impulse to go between the two electrodes. They can measure the time, and those nerve conduction velocities decrease. I'm sorry. [inaudible]  Yep. [inaudible] Yeah. Thanks for bring that up. The person mentioned that there's actually two forms of CMT. One is referred to as demyelinating forms of CMT. That is mainly what I'm talking about here. This results in destruction of the myelin and slows down nerve conduction velocities. 

There's also axomal forms of CMT, and in that case nerve conduction velocities are not really effected as much as the amplitude of the signal. These axomal forms of CMT are actually problems mainly with the axon itself and not the myelin structure here. Thanks for bringing that up. [inaudible] In general, there's been tremendous progress that's been made in understanding the genetic causes of these two different forms of CMT. In general, there's very distinct genetic causes of the demyelinating form effecting Schwann cells, and the axomal form that effects the axons themselves. Occasionally there's some forms of CMT that sort of bear both hallmarks. They have slower nerve conduction velocities and also reduced axomal amplitude as well. There are some cases that kind of have hallmarks of both. But in general they are fairly well distinguishable. A neurologist can usually diagnose axomal and demyelinating based on a series of neurological tests. 

Yep. That's a great question. Flag me down. I'm having trouble seeing through the lights here. As a result of having sort of reduced function of the peripheral nervous system this results in sort of an progressive muscle weakness and atrophy. A person with CMT sometimes has particularly atrophy at the lower levels, the distal parts of the leg. There's reduced muscle mass at that level. There's been, as I mentioned before, tremendous progress made in understanding the genetic cause of this as well. We became interested in this set of disordered because we realized some of the research that we were doing was applicable to the study of CMT. What I want to tell you about in sort of the last twenty minutes or so of my talk is really some studies that have been supported by work from the Charcot-Marie-Tooth Association. 

The CMT Association is actually a disease foundation in the US that supports research into this spectrum of disorders. I've been fortunate to be associated with them for the last three or four years, and became a board member about a year ago. It's been really a pleasure and an honor for me to be involved in this endeavor, and I've really learned a lot through this. I want to tell you a little bit about what we're interested in doing in trying to take some of our research and apply that to try to identify new kinds of therapy for this type of disorder. 2012 was sort of a landmark year for CMT because one of the very first genetic causes of CMT were identified in 1992. The very first issue of this journal, which is very well known, known as Nature Genetics-- Not the first issue, but the first year of Nature Genetics, they had a series of papers by four research groups identifying the genetic cause of the very most common form of Charcot-Marie-Tooth disease. This effected a gene known as Pmp22. Pmp22 stands for peripheral myelin protein 22. What was found was not a mutation in that gene, but actually a chromosomal duplication. There's a stretch on chromosome 17 that has a Pmp22 gene. 

What happens is a certain stretch of DNA around that gene gets duplicated in tandem. So you have, on the same chromosome, two copies of the Pmp22 gene. On the other chromosome there's still a normal copy. As a result of this duplication you end up with three copies instead of two of the Pmp22 gene. That turns out to be the very most common form of CMT. I'd mentioned that one in 2,500 individuals have CMT. This actually duplication accounts for more than 35% of all CMT cases. For those where the genetic diagnosis is known, it accounts for well over half of those. Over 55% with a known form of CMT have this duplication of the Pmp22 gene. As I mentioned before, there are a series of other genes that are involved, where mutations have been identified. 

These include not only the demyelinating forms which I've been focusing more on in this talk. This is referred to as CMT1. When Pmp22 is duplicated that's CMT1A. There are other demyelinating forms referred to as CMT1B or CMT1D. The axomal forms of CMT are referred to as CMT2. There are mutations in different types of genes that are associated with the axomal forms of CMT. But our research, really we've personally been focusing on CMT1A. In part because this is really the most common cause of CMT. You may be asking, Pmp22, what is Pmp22? Pmp22 is actually a component of myelin. This myelin sheath that surrounds the axon has a lot of lipid. 

I told you about that. It also has specific proteins that are important for maintaining the structure of myelin. You can imagine that you have all these wraps around an axon, you know, something has to hold those wraps of membrane together. There are protein components like Pmp22 that are involved in maintaining the structure of myelin. These are imbedded within the cell membrane. In research since 1992 when this was identified, there's been a lot of progress that's been made. For example, how do we really know that Pmp22 is the cause of CMT1A? Having one extra copy of a gene doesn't seem to be that big a deal, but it's been shown since then that rats or mice that have been engineered to have an extra copy of Pmp22 show CMT1A-like symptoms. It's really a problem of having just a little bit too much. One extra copy of this gene is enough to cause this most common form of CMT. When we got involved with these sorts of studies, we were first of all, just trying to understand how this regulation happens, then we realized, well, maybe we can not only find out more about the problem, but try to also be part of the solution as well. 

Let me just put it to you. This is not rocket science. I wish it was more complicated. It's make me look better. But this is really quite simple. In a normal case we have two copies of the Pmp22 gene. We'll call the level of Pmp22 protein that is produced 100%. When we have three copies of the gene, we essentially have one half too much, 150% of the level here. Keep in mind that the amount of the protein that's produced from each of these genes depends on the number of the genes. Just having one extra gene changes the level of Pmp22 protein and elevates that. 

This is all we want to do, we want to dial down the output of the Pmp22 gene so that we go from 150% to 100%. So it's a relatively subtle change. Just a 50% reduction in the amount of Pmp22 protein would be enough to cure this disorder. This is essentially the job that we're working on is really trying to find a way of reducing the amount of Pmp22 protein. That would address, not the symptoms, but actually the root cause of CMT1A. When we were trying to go about that, one of the things that we're very much interested in is saying, well, the Pmp22 gene is made of the DNA codes that make up the Pmp22 protein. But there are other parts of the gene as well. These other parts of the gene establish the level at which that gene is expressed. What I mean by that is, how much Pmp22 is being made? It determines the activity of the gene. Ultimately the DNA sequences not only code for the protein, but also establish the level of that protein as well. 

So we need to really find those DNA sequences which were setting the amplitude, or the level at which Pmp22 protein is being produced. We set about to try to understand how the Pmp22 gene is regulated, how it's turned on in Schwann cells, and what sets its level, you know, at a certain level. Because we want to turn down that level in order to cure this disorder. What I'm showing here is just sort of a schematic of chromosome 17 were the Pmp22 gene is. You can zero down and see different genes that are surrounding the Pmp22 gene. Then if you look even further, you end up essentially looking at DNA. This is the DNA sequence that makes up part of the Pmp22 gene. Our job was really to try and find which parts of this DNA sequence were important for establishing those levels. When we started setting out about this we decided, well, the most important thing we could do is try to understand how the Pmp22 gene is regulated. All gene regulation depends on specific proteins, that interact with parts of a gene, with different parts of the DNA. 

And essentially turn on that gene and all the processes that result in the formation of RNA, then eventually the formation of protein that's being produced in myelin as well. Through the work of a lot of other groups we know the names of those proteins. These are affectionately referred to as Egr2 and Sox10. I won't tell you what the acronyms mean, because it doesn't really reveal anything about their function. Keep in mind that these are proteins that bind to the Pmp22 gene and turn it on as Schwann cells are making all this myelin. We're very much interested in trying to figure out, okay, what parts of the Pmp22 gene are important. Which parts do these two transcription factors bind to? This was our conundrum. How do you find which parts of the gene are important for it's activation? The solution to that is to be able to say, you take the proteins out of a Schwann cell, you fish them out, and you simply see which parts of the Pmp22 gene are associated with that. This was something that was unthinkable five years ago, or ten years ago, but there have been tremendous changes in technology that have really enabled us to do that. One of those is actually just down the hallway this way, in the Biotech Center. 

You go down this hallway and there's a facility which does what we call next generation sequencing. Next generation sequencing is something that's really revolutionized many areas of biology. They've revolutionized genome sequencing. Thanks to this, you can have your personal genome sequenced very easily. I think the cost is less than $10,000 now. Before this it was very laborious. You probably remember the Human Genome Project that wrapped up in the 90s. It took billions of dollars to be able to complete the human genome. Nowadays people can do that with $5,000 in a garage if they have the right technology that's there. I was thinking about this earlier today. 

Essentially what this involves is, we used to sequence DNA one molecule at a time. We'd send one molecule over here to the Biotech Center and they'd give us the sequence of that molecule. Nowadays, they can actually put hundreds of millions of molecules on these slides here. These are referred to as lanes here. Each of these lanes have little dots of DNA that are being sequences at the same time. What's happening then in this instrument, which is down the hall here, is that all these sequencing reactions are being imaged. These are little microbeads that are present on this slide that are being imaged as the sequencing reactions are happening. You see the sort of constellation of stars essentially, they're all being imaged simultaneously. It's not just a hundred, it's not just a thousand, but 200 million of these things are being monitored at the same time. You get massive, what's referred to as, massive parallel sequencing, to generate all these sequences of DNA. 

In our case, what we're using it for is to identify pieces of DNA that are associated with proteins of interest that we have. This is also used in epigenetics research to identify different proteins and how they package DNA, and which pieces of DNA are associated with parts of the epigenetic code as well. As a result of this kind of technology we can really identify which parts of the Pmp22 gene are very important for it's regulation here. Essentially, we took all those pieces of DNA, we identified where they are in the gene. This is just a sort of a map showing that Pmp22 gene. These peaks here correspond to sequences of DNA that are bound to these proteins that regulate the Pmp22 gene. When we had those sequences we thought, great, we have Pmp22 by its tail, so to speak. We know exactly how Pmp22 is regulated, and we can begin to use that information. We know that these sequences of DNA that are associated with these peaks are really key to the regulation of Pmp22. The next up for us was how can we actually translate this into something that's useful. 

We took advantage of a very common assay in microbiology, which is actually using assays involving light. We employ firefly genes. If you'd been looking at your backyard in July and admiring the fireflies. Some people call them lightning bugs and get them mixed up. You see the glow of the luminescence that occurs from those fireflies. That gene has been identified. Promaga in town here developed this technology in many different forms. This gene is referred to as luciferase. The way that we can us that is we can take the parts of the Pmp22 gene to determine its level and we can fuse it to the luciferase gene. The way we do this is we use genetic techniques to fuse that DNA for the luciferase gene with these regulatory DNA sequences, and that makes something that we refer to as a reporter. 

Now when I say a 'reporter' this  is a common term that we use in molecular biology, it's sort of like a meter on the side of your house. The person comes around and checks how much natural gas you've been using and how much electricity you've been using in your house. It's sort of a meter in the level of usage in your house. Now when we have a gene like this that produces light, the more that that gene is elevated, the more light that's produced, or if it's reduced, than there's less light. We can actually assess the status of the Pmp22 gene, how active it is, depending on how much light is produced from the luciferase gene. When we make this sort of reporter-- We refer to this as a reporter assay. We assay the activity of the Pmp22 depending on the amount of light that's being produced. This is how this works. We take Schwann cells that we can culture in the laboratory and these have an engineered luciferase gene that's measuring, that's reflecting, the activities of the Pmp22 gene. We put all those Schwann cells in a plate here. 

We refer to these plate, and usually these plates have many different wells of those. You many have seen actually plates that have about 96 wells, that's commonly used in laboratories. Actually the screening laboratory that we collaborate with uses plates that have 1,536 wells in them. So there are even higher numbers of wells here. What happens is the yellow in each of these wells is meant to refer to the amount of light that's being produced from the cells in each of those wells. And the amount of light, as I said, reflects the activity of the Pmp22 gene. We can then take whole libraries of compounds. These are different compounds that have been collected. Some of them are FDA approved compounds that are already used in medications or other kinds of compound libraries. We can take a little bit of each of those compounds and put them in each of these wells here, then measure the amount of light that's produced from each of those wells. 

What we're hoping to find is compounds that reduce the level of light. That's what I'm trying to symbolize here. You have a couple wells here that are not yellow. They're actually white. So there's a reduced amount of light that's being produced from them. These are compounds that we're going to flag to investigate, because they're reducing the level of light that's being produced, and probably reducing the activity of the Pmp22 gene. We've set up a number of different assays like this to be able to screen for compounds that reduce the activity of Pmp22. As I said already, these reductions are measured by reduction in the reported light from each of these wells. We're in the process of not only creating these assays and testing them, but then collaborating with people that can actually do the drug screening processes in a very high throughput fashion. Those drugs then are flagged for further testing. 

Other traditional assays that we can use to determine if they really are able to reduce the amount of Pmp22. We're very fortunate to be able to collaborate on this endeavor with a really state-of-the-art facility that is present at the National Institute of Health. There's a new institute that has formed at NIH which is referred to as NCATS. That stands for National Center for Advancing Translational Sciences. They have a state-of-the-art drug screening facility there that's dedicated to finding new kinds of therapies for various kinds of disorders. We've been collaborating with an individual there by the name of James Inglese. He actually has a lot of drug company experience, and he's brought to NIH to really help projects like ours to identify new compounds for different kinds of disorders. It's fascinating to visit this facility. They don't have people screening all these compounds. They actually employ these very high precision robots. 

You can see these robotic arms here. These are the same kinds of in car manufacturing plants to do highly precise assembly of different auto parts for example. They can also program them to, you know, move plates around. They can add small amounts of compounds to each of these wells. They can automate this process so that you can screen not just a thousand or ten thousand, but actually compound libraries number in 400 thousand. In contrast to many screening facilities, they actually are able to screen not just in one concentration, but they are able to do seven doses of the compound. You can actually see a dose curve on each of those compounds. We're very fortunate to be able to collaborate with them. They've been helping us in the screening process. What's the strategy then to identify new compounds? Really to the very thing that you can hope for is to find the compound that's already FDA approved. 

Then you can save about a billion dollars and about 10 years or so of time because it's gone through all the toxicology tests and been used in that approval process. If you've found a compound that's been designated in some other treatment but will also work in your assay, that's the best thing that you can hope for. Of course we try to screen libraries of FDA approved compounds. But they also have much larger scale compound libraries that include up to 400,000 compounds. I wanted to just sort of wrap it up there in a since that this has really been a big endeavor for us in our laboratory, not only understanding the basic mechanisms by which genes are regulated in Schwann cell development. I've been fortunate to have a lot of really great graduate students and technicians work with me on the project. I'll just highlight a few of them. Erin Jones and -- have done a lot of work that I've talked about in this talk as well. I also wanted to, whenever I give a talk like this, I often think about people that have affected me one way or another. I wanted to kind of dedicate this to a person known as Arthur Goldberger. 

He was actually on faculty here at UW in the department of economics. He unfortunately passed away in 2009. He became my wife's coffee shop buddy. He used to hang out in a coffee shop on University Avenue and really became a friend to my wife who brought in our small kids. He's one of the first persons I met who had CMT. He was really a wonderful person to get to know. He had the fortune or misfortune, depending on the way you look at it, of having a Nobel Prize awarded for work that he did together with his advisor. Unfortunately, his advisor got the Nobel Prize for his work in economics and his name is on the model that he developed, but he did not actually get the Nobel Prize. Later on, he was asked, was that disappointing that that work-- You know, that you didn't get the Nobel Prize? He said, well, it's okay. It wasn't my best work. 

[laughter] So anyway, he was one of the first persons I got to know that had CMT. He really was a giant in the field of economics, and also a very nice person that befriended my wife as well. With that, I'd be happy to take any questions, and I appreciate your time. [applause]