9UNP0246SD_Jeff_Johnson 

- I'm here to introduce our speaker Dr. Jeff Johnson who's really bringing us the very latest information about delaying the progression of Alzheimer's and similar diseases. Dr. Johnson received his PhD from the University of Wisconsin in 1992, did a postdoctoral fellowship at the University of Washington in 1995, accepted an appointment as assistant professor at the University of Kansas Medical Center and then was recruited back to Wisconsin in 1999. Since that time he's established himself as a leader in the field of neuroprotection and neurodegeneration and has garnered significant state and federal grant support for his work. The research emerging from his lab in the areas of Alzheimer's, Parkinson's, Huntington's and Lou Gehrig's disease ALS has led to patents, the startup of a pharmaceutical company named Mithridion and several state awards. Please help me welcome Dr. Jeff Johnson, and I think his wife Delinda and two daughters are also here this evening, Emma and Taylor. So welcome Dr. Johnson. 

(applause)

- So first I want to just make a little few announcements before I get started. The first one is I want to thank the academy. I always wanted to say that. 

(laughter)

 I'm waiting for the little bald-headed statue. 

(laughter)

 I kind of feel like I'm in the Da Vinci Code, Robert Langdon in the Da Vinci Code up here doing this stuff. But anyway I also need to acknowledge, for everybody in the audience that doesn't know it, the Edgewood Campus School 8th grade Smart Team members are here over there. 

(applause)

 I'm actually serving as the Smart Team mentor for this year's Smart Team and I did it two years ago and it's actually a very rewarding situation. I have to specifically acknowledge Mr. Toomey who is the science teacher at Edgewood who really makes it a great experience for the kids. 

(applause)

 Of course the only reason they all showed up is because he's giving them 50 extra points to come here. 

(laughter)

 And he promised me there was a definite no iPod rule while I'm talking. Because if kids are whipping out their iPods while I'm talking, I'll walk out in the audience and grab them. Just kidding. The other person that's here who I want to acknowledge was my Ph. D. mentor Frank Siegel. Dr. Frank Siegel right there. 

(applause)

 Frank taught me a lot of things not only how to be a good scientist but also to be a good person. And so what I'd like to do tonight is I would really like to focus, and I'm going to walk around if I get away too far from the mic just holler at me. What I'd like to do is start with what my goal is. And I think the goal of this talk tonight is basically to make you as excited about the research that we're doing in my laboratory as I am. And I hope I can do this by the end of this 45 minutes or the half hour talk that I'm going to give or in the question and answer. So that's my goal so if we start with that. Let's move on to the presentation. I need to grab my pointer. So we're going to talk about a general concept of neurodegenerative diseases and neurological diseases and how we can hopefully intervene in the pathologic progression of these diseases. And I'm just going to start with some general introduction slides. People probably hear a lot of things about oxidative stress and how you should go to Willy Street Co-Op and buy all the antioxidants and take them all because antioxidants are good for you and so on and so forth. And there is some foundation in that in that oxidative stress, if you think about it, is actually contributed or at least has been associated with virtually all of the neurodegenerative diseases that we've listed up there so that would be Parkinson's disease, Alzheimer's disease, ALS with Huntington's. And in all those cases they have found multiple markers of oxidative stress. 

And oxidative stress is usually characterized by this reactive oxygen species and so basically the normal oxygen that we breathe in can be converted into toxic chemicals that will modify proteins, lipids and a variety of other things, then cause or contribute to the development or the progression of these diseases. Mitochondria, I'll talk a little bit more about mitochondria. Mitochondria is an organelle in your cell that's kind of the energy factory of your cell and generates ATP which we all need to survive. And this mitochondria is also one of the major generators of reactive oxygen species in the body. And so this dysfunction in mitochondria can significantly contribute to the pathologic progression, and it's been shown to be involved in all of these diseases as well. So it's very clear that oxidative stress and reactive oxygen species and mitochondria dysfunction are really intimately involved in the normal cell death in neurological diseases especially the chronic neurodegenerative diseases. And so what we've tried to do or what we're trying to do is understand ways to control that generation of reactive oxygen species and how we can attenuate that or mitigate that so that we can have some -- into slowing the progression of these diseases down. So the mitochondria shown here, and like I said, mitochondria in normal conditions will generate reactive oxygen species. No engine is 100% efficient, that I know of. And so what happens is you get the spewing of a little bit of a reactive oxygen species just under all normal conditions. 

And so these reactive intermediates then, what we've done as a human being and as a cell is we've developed significant cellular defenses. And I'm not going to go through a list all these enzymes or all this stuff. But needless to say if we wouldn't have developed these cellular defenses we all wouldn't be here now. We would all be dead. So what that does is these cellular defenses then actually divert or shift in equilibrium towards breaking down or preventing these reactive oxygen species from causing damage and cellular damage which then leads to presumably DNA mutations and a variety of other things that could cause cells to die. Now I'm going to try to sell you on this point. So Alzheimer's disease, Parkinson's disease, ALS and Huntington's diseases are extremely different diseases, right? I'm sure people have seen people with Alzheimer's and Parkinson's, for sure. ALS is less common. Huntington's a less common. 

But if you look at these three or four diseases what you'll find is they're different in that they're initiated from different proteins, let's say. Because usually a protein or mutation is involved in this. They affect different neural populations in the brain. And there's a very brain region specific effect of each of these diseases. And the outcomes, if you watch the outcomes, and by the outcomes I mean the phenotype, the changes in posture and gate and the tremors, those are all different for the different diseases. But the one thing, as I pointed in the previous slide, is that all of these diseases funnel into what is a common mechanism presumably that leads to the chronic progression of these diseases. And I'm going to argue that that is focused around the mitochondria. Now I could be arguing with a lot of people about this for a long time. There are certain people that believe mitochondrial theories and essential people that don't. But I think from a standpoint of understanding what I'm trying to talk about tonight we're going to focus on the mitochondria. 

And so what happens then is when you have these diseases they target the mitochondria or during the processes of these diseases the mitochondria gets targeted and so what happens is you end up damaging the mitochondria and in doing so they become far less efficient in what they're suppose to do and spew out a lot more reactive oxygen species which actually overwhelms the cellular defense mechanisms so those shrink and then the cellular damage actually increases. And so with this accumulation of cellular damage that's how you get the progression of these diseases. So what are the targets? Well, one of the targets obviously would be to identify how we can block the reactive oxygen species at this point during the disease process. The other target would be to actually see in we can plug the funnel. So if we could plug the funnel with some kind of a drug or some kind of a pathway that we can activate, we would not let the toxic metabolites or toxic proteins actually get to the point where they screw up the mitochondria. That's really scientific language, screw up. 

(laughter)

 That's what happens when you have two teenage daughters. If I start rapping then we're really in trouble. 

Sorry girls. So anyway, if we can target this and this then we could have at least we can plug it, what leaks through then we may be can stop over here. So it's kind of a bi-functional attack if you want to think of it that way. So one of the things that we can do is we can take that cellular stress or cellular defense and we can crank it up. If we crank up that cellular defense, then we shift the equilibrium away from this pathway into this pathway. Here we can basically try to block the mitochondria from being affected. So the way I like to affectionately call this, we need mitochondria on steroids. And if we can get mitochondria to be stronger and more resistant to these stimulations that are causing them to have problems, then we could prevent this whole process at that level. And, fortunately, there seems to be a protein, one of my favorite proteins, called Nrf2 that has the ability to increase cellular defenses and also put mitochondria on steroids. And so what I'm going to talk about is I'm going to talk about how we worked with this protein to try to mitigate these affects and develop, well we haven't developed drugs yet but we're trying, but tried to develop an understanding of the mechanism and how the cells in the brain can be manipulated to actually modulate this pathogenesis. 

So what is this transcription factor? It's a transcription factor so that's a big word. What this thing does is it binds to DNA in the nucleus in the cell and once it binds to the nucleus in the cell it increases over 115 genes simultaneously, a majority of which are involved in detoxification and neuroprotection. And so it's maintained, it's held in the cytoplasm of the cell by this protein called keep 1. And upon stimulation with stress or with chemicals or by certain enzymes, this enzyme gets released, moves to the nucleus and then activates the transcription of a lot of these genes. Some of these are involved in glutathione synthesis, thioredoxin, reductase. There's a lot of proteins involved in protection. And I have a summary slide to just show you to give you some idea of what we've done and that will be in a little bit. So what we wanted to do is we really wanted to look at how this pathway is regulated in vitro and in vivo. So I'm going to show you a brief bit of in vitro data. 

Now when I say in vitro we're talking about a system where we take the brain out of an animal, we dissociate the cells, we put them on a plate and we look at them in a dish. So it's very different than looking at the brain in vivo, in a live animal. But it can give us hints as to how we should maybe go after systems in the animal to try to modulate this. So one of the things that we've done to sent up tools for this is to make animal models that we can use to actually follow this. And this mouse, this hPAP reporter mouse, this mouse is actually an in vivo sensor of Nrf2 activation. So if we activate this protein that I like, Nrf2, what happens is that binds here and you see this gene go way up. And what this allows us to do is it gives us the ability to look both in vivo and in vitro at the cells and the regions of the brain that are responding to either the pathologic event or to any kind of therapeutic intervention. So this has been a really valuable tool in establishing the pathways that we've been able to do it and I'm proud to say that mouse was developed by my wife. Good job, Delinda. And then the other mouse that we have is a mouse where they have this favorite protein of mine, Nrf2, is actually deleted from the mouse. 

So now we have a mouse that doesn't have the ability to actually activate this protein. And if you can't activate the protein what does that mean then to the mouse? Well, mice are resilient, as are humans. So this mouse actually lives, breeds, generates pups, walks around like nothing's going on. But if you live in the environment that these mice live in, which is better than most of our houses probably, you could probably walk around without this protein as well. So they're very well taken care of animals. What I want to talk about, and I just want to get everybody on the same page, is that the brain is not a simple organism. The brain is made up of multiple cell types, and the focus tonight, at least from my perspective, is going to be primarily, this is the neuron. So everybody knows about the neurons. The neurons die in Parkinson's disease. 

The neurons die in Alzheimer's disease. The neurons die in ALS. The neurons die in Huntington's disease. If I look in the audience now and I say everybody wiggle my big toe, everybody wiggles their big toe. You get a signal from your brain that goes down a neuron to your big toe and it wiggles. Now, what people don't appreciate, a lot of people don't appreciate, is that there's another cell type in the brain called an astrocyte. The astrocyte, in your brains, the astrocyte actually makes up 60% to 70% of the cells in the brain. The neuron is the minor population relative to the astrocytes in the brain. And these astrocytes play a critical role in the maintenance of the neuron. And we've been looking at how this pathway, this Nrf2 pathway actually integrates with this communication between the neuron and astrocyte. 

So I guess you could call me an astrocentric speaker because I really think that these guys die if these guys get sick. And if these guys never get sick, then these guys never die. Okay? So keep that in your mind as we go through the talk. So astrocyte is the cell that supports the neurons and the neurons are the ones that die in the end that cause the outcomes that you see, the phenotypes that you see. So I love doing this. I'm summarizing five years of work in three bullets. 

(laughter)

 So Delinda's work is actually just one bullet which is unfortunate but that's the way it goes with time limit. And then Andrew Craft was another grad student in the lab that did some work. 

And what we've been doing, and this work is in vitro, so what we've been able to do is make these cultures in dishes where we have both astrocytes and neurons in the dish and we can see how they kind of talk to each other. And what we have found at least in vitro in those systems is that this Nrf2 pathway is primarily in the astrocyte and not in the neuron. So the question then becomes, well, here you have a protein that's activated in the astrocyte, what does that do to the neurons in the culture? And what we've been able to show at least in vitro is that this Nrf2 activation can protect against a bunch of oxidative stress induced killing molecules. Hydrogen peroxide and glutamine are a few. But if you put on, melt chemicals on to these cells to kill them what you can do is you can treat the cultures with an activator of Nrf2 and you can protect them in the wild type cultures but in the knock out cultures where they don't have Nrf2 there is no protection. So clearly the protective effect is dependent on Nrf2. The other thing that we've developed in the lab is viruses. So we can selectively transduce astrocytes versus neurons with genes that we want. And so in this situation we were able to selectively transduce astrocytes with Nrf2 and show that the Nrf2 astrocyte, even though the neurons in that culture are negative for Nrf2, they don't have any Nrf2 in them, the astrocytes over expressing this protein actually completely protected the neurons that were negative. 

So it looks like a pretty powerful system. And just to give you some idea from a picture standpoint because pictures are always nice. So in this culture what you see is the red cells are neurons and then the blue stain is nuclei. And what you treat with this molecule hydrogen peroxide, which I'm sure everybody has in their cabinet, at home in their medicine cabinet, you basically obliterate all the neurons in the culture. Now with the viruses, what we have in the viruses, you know that Nobel Prize that was just given out for that green fluorescent protein thing that they talked about on the radio all that time, that's what it is. The green is the green fluorescent protein. So we have a virus that has this green fluorescent protein in it that we can follow what cells get transduced with this virus. And so what you're seeing here is a virus that we just put in GFP, the green fluorescent protein and not Nrf2 and you treat with a hydrogen peroxide and boom, all the cells are gone. In this situation, however, we put in a virus that not only has the green fluorescent protein but also has Nrf2, and in that case we found that the neurons all survive. So what we found in a lot, and we did a lot of gene chip micro array kind of experiments to try to follow this pathway, what we found is a lot of these Nrf2 dependent genes are involved in maintaining redox. 

Now this molecule is an extremely important molecule in our cell, it's called glutathione. And glutathione maintains our cells in a reduced state so it's really critical because if we oxidize, we die. And so this is critical. And what you can see is all the genes, and I'm not going to go through any of the genes, but all the genes that are kind of highlighted here are all the genes that increase the production of glutathione or genes that actually use glutathione to detoxify bad stuff in our cells. In this situation, this is all reactive oxygen species or free radicals. And all the genes in metabolizing and clearing free radicals are also controlled through this Nrf2 pathway. And then one of the other genes that really critical is this cofactor called NADPH that's generated by a series of proteins but then is utilized as a cofactor by a whole host of proteins that are involved in detoxification and metabolic inactivation of these toxic intermediates. The other thing that we've found in these experiments is that here's your neuron and here's your astrocyte and not only do the neurons survive in these cultures because of the astrocyte but there seems to be and there is clearly a critical metabolic coupling between the neuron and the astrocyte and that the astrocyte provides a lot of the nutrients and a lot of the reducing agents that the neurons need to use to survive. And so you can imagine in a situation is that if this guy gets sick and can't provide all of these things to the neuron, the neuron can't do its function and not only does the neuron get sick but the neuron dies. The astrocyte won't die, it has, it's a much stronger cell more resilient to this kind of thing. 

But if it's sick it can secondarily cause these neurons to die. So we have this really dramatic protection and all this stuff that we've talked about in vitro. Cells in a dish. But what about in vivo? In fact, if try to qualify the data or quantify the data, a single Nrf2 over-expressing astrocyte in a culture like this can protect over 700 naive neurons from oxidative stress induced toxicity. If we can translate that in any way, shape or fashion into the in vivo situation, that would be very good. Now remember, your brain has 70% astrocytes. If we could find a way to turn on those astrocytes with this pathway, we can have significant impact on a lot of these diseases. And so that's where we're going to try to go tonight. I'm going to talk about two, this data is unpublished, hopefully will be published soon. 

They're supposed to be submitted. But I'm going to talk specifically about ALS or Lou Gehrig's disease and I'll have another little section on Parkinson's disease. And I want to show you the outcomes of these experiments from the in vivo standpoint. Lou Gehrig's disease, does anybody know somebody who's had ALS or Lou Gehrig's disease? Lou Gehrig's disease is definitely a serious disease. It's a much rarer disease than Alzheimer's or Parkinson's. But what it results in is motor neuron degeneration and muscle atrophy. So you lose the function; you become completely paralyzed basically. And it's common in people over the age of 40. It affects a relatively small population. 

There's 30,000 people have it on an annual basis. The thing about this is the life expectancy is only two to five years. It's a very rapid degenerative disease. Now because the life expectancy is only two to five years it's also, pharmaceutical companies don't touch it because there's no profit margin. You can can't, in a drug that's long you just don't have the means, you're not going to invest billions of dollars to develop a drug that's going to treat somebody for two to three years with that low number of people. So a lot of the work that I've talked about that I'm going to talk about has been funded by the ALS Association which is great association for ALS research. The Packard Center for ALS Research at Johns Hopkins has also funded us and NIH so most of the stuff is coming, all my funding comes from foundations or from NIH, nothing from drug companies. But anyway 90% to 95% of these incidents of ALS is unknown cause. Nobody knows why it's caused. And the other 5% to 10%, it's inherited. 

It's familial. And you'll see these same kinds of numbers when we get to Parkinson's disease. And now within that familial set there is one gene that's very interesting and that's the gene called superoxide dismutase. And what we do as scientists is we identify these genes, and this was done which Mark Gurney. He identified this mutation in the human gene in this SOD gene that caused this ALS pathology in humans. So what we do then is we take these human genes and we stick them into mice. That's not quite that easy. 

(laughter)

 But we do it that way. I don't want to go into the details of how we do that. 

But anyway what we can get then is we get a stable integration of human genes in a mouse that's mutated that's produced as a mutated protein in the mouse and the question is does that human gene result in anything that looks anything like what we see in a human. And the reason I want to talk about this is because this one is the really the only one that's really given us a complete picture of what happens in the human. So this ALS mouse model actually has, it's the SOD mutation and it's expressed in the brain and spinal cord. You see oxidative stress. Of course oxidative stress is something we're very interested in. And what happens is this mice has about 120 to 135 day life span. And you're at a presymptomatic stage. All the sudden you start seeing symptoms. Now these are mostly in the animal; you can't really physically see these symptoms. But then you have this age of disease onset and then you get paralysis. 

And so what you're looking at in the movie is these are litter mates and this is a normal mouse that doesn't have the mutation and this guy is the mouse that has the mutation at 110 days, so some place in here. And what you'll see is see how the mouse is so low to the ground and how its legs are splaying out. What's happening is the mouse is becoming paralyzed. 15 days from now this mouse will be dragging both back legs and in about another ten days from that he will have been completely paralyzed and, of course we can't let them do this and initial experiments show that they die from asphyxiation, they drown in their own fluids. And that's what happens to ALS patients. So we have a mouse that we can look at and to study these things and this has been a really critical mouse to try to identify drugs and other things that can help prevent this pathology. So in this field the sick astrocyte, this is the one field, this is why I love this field, everybody in this field over the past five years as become extremely astrocentric. The neurons don't even matter. It's all the astrocyte. So the question is, Marcelo Vargas who is now a postdoc in the lab, published a paper in 2006 and this was followed up by two other papers in Nature Neuroscience showing that the astrocyte, if the astrocyte has the mutated gene and the neuron doesn't, the astrocyte still kills the neuron. 

So what that's implying is that this mutated gene that's driving ALS, at least in vitro, it doesn't have to be in the neuron, it can just be in the astrocyte. And that the astrocyte under the stress of this gene is actually secreting factors that are toxic to the neuron and it kills them. And so the question is, and in this model which is a very clean, it's a very aggressive model. So the data has to be interpreted carefully. But it's also nicely clean model to look at. So the big question that we're trying to answer is in this model and all the model is can Nrf2 fix sick astrocytes. If we can fix the sick astrocytes, then the neurons might just come along for the ride. So the way that we decided to do this is, obviously, make a mouse. So in this situation what we've been able to do, and Marcelo's characterized this, is we've been able to make a mouse that's selectively over-expresses Nrf2 in the astrocyte in vivo. So this GFAP is a protein that's expressed in astrocytes and so we kind of replaced, we took the normal gene and we put it, so normally what happens is when genes are expressed you get these different proteins in your brain or in your cells, there's specific promoters that cause the expression in the specific cell. 

Now what you can do it take those specific promoters and put any gene you want to in front of them and then make this transgenic mouse and what you have now is a mouse that expressing a protein that you want selectively in the cell type you want. So in this case we were looking for astrocytes. We wanted to see what is the role of Nrf2 in the astrocyte. And so we've made this mouse and of course we have to characterize it so what we did is we took that mouse and we crossed it on to Delinda's mouse which is the reporter mouse that's going to tell us if this pathway is being activated in the brain. And what we see here is a bunch of different brain regions and we have this is the basil activity and this is when it's crossing that mouse. Now I have never seen anything like this before. In the cerebellum of the mouse there is a 12,000-fold induction of the reporter gene. Okay? I mean, that's just huge. You don't need statistics on that. 

It's 12,000-fold. So the other thing that's selective about this that you see is that other tissues in the body don't have the activation. Which means that we've been able to control this mouse through this promoter that we've been using so that the astrocytes are over-expressing Nrf2 and the peripheral tissues, other tissues are not. So we can really focus on what's going on in the brain. And Marcelo has taken some beautiful pictures, as well, looking at increased green here means there's increased activity. So you see this big increase in activity and this is the protein, this is actually the protein that stains the astrocytes and then when we overlay it we see that actually indeed what we are is we're seeing this increase in these positive cells which means that the promoter's actually working right. So all of this is a characterization to make sure that the mouse we generated is doing what we think it is. So now the big question is of course, what happens when we've made this mouse, we spent, I don't want to tell you how much money, to get to this point. It's going to work, right? Is it going to work? 

Yes. So what Marcelo was able to do was to do a series of experiments where we actually looked at onset of disease and survival in these G93A mice that do not have the Nrf2 and that do have the Nrf2. And what we're seeing is around a 20-day extension of lifespan. Now you're saying 20 days, that's wimpy. But you have to remember that these mice die in 120 days so that's a 20% extension of lifespan. The only drug that actually FDA approved to treat ALS gave a ten-day extension of lifespan. So we're not talking about peanuts here, right? This is a pretty significant finding. What's interesting is that we didn't see any delay in the onset to death but what it looked like it was doing it was making the animal stronger so it could last longer until the disease started. Once the disease starts in this aggressive model it just goes, they usually go the same rate. 

So what we've had is we had this. So we try to figure out what's going on, and these are just pretty pictures you don't need to understand them. This is bad. We don't see the bad stuff here, right? So that's good. So this is the double transgenic. This is what we see in our Nrf2 mouse when we should be seeing this but we've protected the G93A mouse from all that bad stuff. Okay? And again pretty pictures. So what this is, this is actually a muscle fiber, right? 

In this disease we lose the innervation of the muscle. So we want to not only do we want to look in the spinal cord but we want to look at the muscle at the amount of innervation of the muscle. So what you're looking at here is a single neuromuscular junction on the muscle fiber, and if it stains both red and green, so it turns yellow like this, that means it's innervated. If it stains just red like this, that means it's denervated and then you have this kind of partial intermediate denervation here where you have both the red and the yellow colors in there. And what you see is what Marcelo just quantified, the number of these, this is not an easy process, by the way. He's got to count all this stuff and take all, it's hard to process these tissues. But what you'll see if you look at this pair of data and you'll see that the double transgenic you're increasing the amount of innervation and you're decreasing the amount of denervation. So not only are we sustaining the cells in the spinal cord, and this is what's really cool about this right, is we're preventing motor neurons from dying in the spinal cord, but we're also preventing denervation in the peripheral muscle tissue by sustaining the Nrf2 in the astrocytes in the spinal cord. That's pretty cool. So what we think and what Marcelo thinks is that we, and we have some data to support this, is that it looks like in these GFP Nrf2 mice in the spinal cords they have higher levels of glutathione that we've talked about before, remember it's the major antioxidant, and that level of glutathione is sustained in this model so that might be one of the major mechanisms, at least in this model, in the ALS model, that the Nrf2 protein is affording, or how it's affording this protective response. 

So I want to briefly talk about Parkinson's disease because we have some new data on this too that's not published. And Parkinson's disease I'm sure, how many people have known someone with Parkinson's disease. So you can tell the incidence correlates with the people that raised their hands. A lot more people know people with PD than with ALS. This is a progressive disease that has a mean age of onset of 55. Again 95% of it's sporadic. We have no idea what causes it. There are a number of familial mutations in this situation. Now however in this case there have been tons of money spent on making mice with these different Parkinson's genes mutations and none of them have actually recapitulated what we see in Parkinson's disease in humans. They all have a little bit of this and a little bit of that. 

But we don't have a really, really nice model of PD. A genetic model of PD. We do however have a chemical model of PD and that's what I'll talk about. The symptoms are -- resting tremor, rigidity and gate disturbance. And here are some pictures. The arm is tremoring and I'm sure people must have seen these kinds of postural issues. And if you actually have heard that if you talk to Parkinson's patients the tremor is not the part that bothers them, it's the inability to start. So what they do is they want to walk and they think step but it's delayed and so that's why you see this kind of leaning forward and posture instability. And I think that's what they find the most irritating. The tremors, that's not it but it's this inability to start that once they start they're fine. 

It's just that initial starting. And so really all treatments in this disease, as well as any of the other diseases, are palliative. In other words, all they treat is the symptoms. So, for example, in Alzheimer's disease the FDA approved drugs they increase acetylcholine in the brain by knocking down the enzymes or activating receptors of acetylcholine. But that's not the pathology. So as you're getting treated with aricept basically in the background the disease is still going full blast. There is no way that these drugs have any affect on the progression of the disease at all. In fact, in some countries they don't even have them on the drug registry because they just don't work. So we do not have a disease, the label that we want on a pharmaceutical is disease modifying drug. And we do not have that for these diseases. 

So we need to find some way to do this. And I want to point out at this point too is that one of the problems, or not a problem but one of the other major areas of research that needs to work in these fields is the diagnostic part of this. And the reason that that is is that, and this is just a diagram of Parkinson's patients I'll talk about in a little bit, but the main reason of that is that in this situation of PD you need to lose 60% to 80% of the cells that have to die before you can actually even diagnose the disease. So intervening at a point where you only have 20% of your cells left is going to be high risk. It's going to be hard to fix it. It's going to be hard to treat it. Now, I mean what we're doing we think we may be able to have some affect even in those patients who are at that far stage. But we haven't tested those theories yet and we are in the process of doing that. But what you end up with Parkinson's disease is there's this place called the substantia nigra that has neurons that send axons up into the caudate and the putamen in the human. And what happens is that as you age, or if you have a gene that causes Parkinson's disease, is these neurons in this area die. 

And they die as we normally age. These neurons are dying as we age. So if there was no other disease in the world and we all lived to the ripe age of getting Parkinson's disease eventually and so what you lose is you lose these innervations. In losing these innervations or losing these axons that's what manifests and physically is what you see. The tremoring and things like that. There's also inclusion bodies and as with most of these diseases there seems to be some kind mess up of proteins in the cell. And they tend to aggregate and form clumps. So in Alzheimer's they're outside the cell. In Parkinson's they're inside the cell. And ALS they're inside the cell. 

And Huntington's they're inside the cell. And what this is there's some gummed up machinery cell they start to precipitate out and form these aggregates and it's the formation of these aggregates at the early stages that people think are actually toxic to the cells. And there's a huge amount of work going into trying to understand what forms these proteins might actually be causing the oxidative stress that leads to this kind of pathology and cell death. So I guess not fortunately, but unfortunately for drug addicts in California there was an accidental byproduct identified in synthetic heroin. So Bill Langston was at a clinic, he's a chief in neurology, I forget the clinic name right now, and they had two patients come in, two young patients, 25 years old or 30, that were completely rigid, like stiff. Couldn't move. And Bill said, gosh, that kind of looks like what I see in end stage Parkinson's disease. And it turns out that around hospitals in that same area all of the sudden a number of people started showing up completely stiff. Turns out that that's what it was. They had taken, injected themselves with synthetic heroin that had this byproduct in it and they presented with these symptoms like a full blown Parkinson's symptoms. 

Would you want to isolate this molecule? They went around and they got a hold of this heroin batch and they actually were able to isolate the molecule that caused Parkinsonism directly in humans by a chemical and that chemical is MPTP and that's an acronym so I'm not going to try to say the whole name, it's too long. But they identified that MPTP was the cause and they used animal models both in primates and in rodents to identify that this was causing, selectively targeting those same neurons, those dopamine neurons in the substantia nigra -- that we're losing as we age and that contribute to Parkinson's disease. And so what this, if you put MPTP and you go to PubMed, put in MPTP you're going to get 35,000 papers or something like that. It's used all over the world as a model for Parkinson's disease to see if we get rid of these neurons how can we rescue them, how can we treat them, how can we modulate the sensitivity of these neurons to MPTP as a surrogate for having any good animal models. And the other justification for MPTP is we know it works in humans. It does the same thing. So we have that molecule. And what this does is it targets these dopamine neurons that are lost in Parkinson's disease, and this is lots of time quantified by this enzyme called thyrosine hydroxylase and you'll see some stuff I'll probably say TH staining. And this is an enzyme, remember we talked about how GFAP is selectively expressed in the astrocyte and we can use GFAP to drive Nrf2 in astrocytes. 

In this situation this enzyme is selective to these dopamine neurons. So this is what makes that dopamine. And so we can use this as a marker to track those neurons to see if they're surviving, if they're dying, whatever they're doing. And there are a number of different models that have been developed for this and we're using a subchronic model in the lab to look at this right now and moving into chronic models. So again I want to remind you that we have this GFAP Nrf2 mouse developed by Marcelo and these Nrf2 knock out mice and wild type mice. So these again are the tools that you're going to see used in this to try to show you that astrocytic Nrf2 actually works. This is a work done by -- Chen who's a postdoc in the laboratory as well. And across the top you have the wild type mice, and across the bottom you have the knock out mice. And you first thing you should notice is if you give increasing doses of MPTP, you kill these neurons which are the dopaminergic neurons. They're stained for tyrosine hydroxylase. 

But what you also should notice is that you're starting to see death at those neurons here and almost here these are virtually killed more than they are out here. So what this is telling us is that the lack of Nrf2, although it may not have an affect on the development of this process or the development of the dopaminergic neurons, it clearly is making them more sensitive to the toxin that we're giving them. And so these guys are definitely more sensitive. And what this is, this is actually measuring dopamine levels because if you're killing the dopamine neurons then the dopamine levels should also go done in the brain. Makes sense right? So in this situation what you'll see is here it the wild type animal and you are seeing loss of the dopamine neurons or the dopamine metabolites. This is dopamine and these are the metabolites but you are seeing the loss dopamine in both knock outs and wild types but it's dramatically accelerated in the knock out. So there clearly is a differential sensitivity here. Now what's nice about this is that we want to test our astrocyte hypothesis here, right? And remember what we did in culture where we cultured knock out neurons and we just put Nrf2 in the astrocyte and we saw that the astrocytes alone could protect the negative neurons. 

Well, we wanted to test that in vivo. And so what we did is we took this GFAP Nrf2 mouse, again this astrocyte specific mouse that's expressing Nrf2 in the astrocyte, and we crossed it with Nrf2 wild type and knock out mice. So the first test was well does this thing even work against Parkinson's disease or MPTP toxicity? So here is what you see is MPTP causes a loss in Tyrosine hydroxylase, as we've shown before, but in the situation where you have this Nrf2, GFP Nrf2 present in here, you completely rescue that phenotype. Now the ultimate test, if we only have astrocytes over-expressing Nrf2, can we still rescue the dopaminergic neurons that are targeted by MPTP? So just a synopsis of what we saw in the previous slide and the answer is yes. So what happens is you, here is the normal cell death or loss of TH staining associated with the giving of MPTP in the knock out mouse. And when you look at that in the wild type or the mouse where you have no cells in the whole body expressing Nrf2 except the astrocytes in the brain, you completely protect the dopaminergic neurons from toxicity. And again it's even more dramatic when you look at the metabolites. So here's the dopamine again, don't worry about the rest of them just look at dopamine. 

When you give MPTP to these mice in the knock outs the dopamine is completely wiped out. I mean, it's gone. You don't have any left. You add Nrf2 to the astrocyte, it's not even touched. So what are we doing now? Well, what you just saw is what we've been doing for the past six months. So what are we going to do? What are we going it try to do with this? Well, one thing is we already have experiments going on looking at Huntington's disease with Nrf2 and astrocytes and Alzheimer's disease with Nrf2 and astrocytes. They also look promising. 

Even though the genetic models in Parkinson's disease don't necessarily manifest in a Parkinson's like phenotype we're still going to test some of those models because we know there's some neurotoxicity in those models that we may be able to rescue. If you remember way back to the beginning when I showed this keep 1protein. So what happens is keep 1 is like the hand and Nrf2 is the fist. And this hand keep 1 holds on to Nrf2 tightly and keeps it away from activating Nrf2 genes. That was kind of like a dance. But anyway so what happens is when stress is applied, or oxidative stress, this protein keep 1 gets modified and it allows Nrf2 to go to the nucleus. So one of the techniques that we're trying to design and trying to work out is that there are ways through -- and knocking out we can therapeutically target this protein and shut it off in the cell. That leaves all of this Nrf2 free to go. And we can do this in a transient kind of system. So it's not like a transgenic mouse. 

It's almost like a drug. Well, it's not quite a drug yet but it could be close to drug if we could actually modulate that interaction. We're also doing small molecule screening trying to identify drugs that would actually activate this pathway and astrocytes in the brain. The problem with the brain, of course, is the brain has the blood/brain barrier. So a lot of drugs that work on peripheral systems don't have access to the brain so you really have to work hard to try to identify drugs that actually will cross that blood/brain barrier, get into the brain and have the effect that you want. But at least what we've been able to do with this GFAP Nrf2 mouse is we've been able to show proof of principle that if we can get something into the brain that activates to this equivalent level that we see Nrf2 and GFAP Nrf2 mice that we should be able to mitigate and slow down the progression of these diseases. The other thing that we're really interested in is astrocytes. Now the astrocytes, remember that we said they're in the co-culture in the system. The astrocytes are touching the neurons but we also know that if we neutralize Nrf2 selectively in the astrocyte, knock it out in the astrocyte only, we completely lose the protective effect for neurons. So what that's telling us is there's something that's Nrf2 responsive that's probably being secreted from the astrocyte that's changing the neuron to be more resistant to toxicity. 

So we already have data indicating that there are creative factors and now we're trying to identify those. Because those would be clearly new drug targets for the treatment of these neurological diseases. Again we're looking at mitochondrial function as a mechanism as we talk about earlier. And glutathione we also have a number of glutathione mice that we can manipulate glutathione levels in these mice. So we can see to what affect does the glutathione levels in the mice, we said that glutathione is a good thing. If we take glutathione down a lot how does that affect the pathologic process in these different models that we're setting. And of course I don't do anything except come and talk to you guys which I enjoy immensely. And I do write grants and get money. But the lab as it exists is here, Delinda and I talked principally about Marcelo and -- Chen but we have a number of other postdocs and graduate students and a couple of clinical faculty that are working in clinical translational research with regard to oxidative stress. Some past members of the lab whose data I mentioned. 

A number of collaborations at the university. -- is actually been a very supportive individual. He's the one that generated the Nrf2 knock out mice. Sam Sisodia at University of Chicago, we worked with him on the Alzheimer's work. Tim Dalton is providing the glutathione mice. And as I mentioned before, really we can't do anything without the funding. And I've been very, very fortunate over the past years to have an immense amount of support from a lot of different places that you see listed there that I very, very much appreciate. And with that we can have discussion and questions. 

(applause)

- Just form your questions and all I want to do is remind you to speak loudly and Dr. Johnson will try to repeat the questions so that not only the audience here can hear but those that might hear this recorded at a later time. So thank you so much Dr. Johnson and it's open for questions. 

- Yes? 

- If I understand correctly, in the beginning you were talking about mitochondria essentially producing bad stuff. 

- Right. 

- And you indicated also I believe that those four diseases essentially funnel in products or messages that further exacerbate that. 

- Yes, so let me just kind of repeat. The mitochondria have been the focus of that what has been shown in these diseases the mitochondria seems to be a common kind of switching point. 

So decide whether a cell dies or lives we're at that kind of potion. That seems to be the switch. 

- Now if you plug that funnel so that you don't get the stimuli coming from those four, the mitochondria are still going to be doing that to some extent because, as you characterize it, no machine is perfect. 

- Right, right. 

- Okay. 

- So no machine is perfect but what the problem is with these diseases and why we don't want that funnel unplugged is because we have a limited capability to deal with these things. And even among this audience if I sample all your blood, found your whole genome pattern, what we would see is we would see 10, 20-fold variations in the expression levels of these gene cluster which I refer to as cellular defense. So cellular defense is dependent on a multitude of factors. One can't control is genetics. The other one we can control is our environment and diet and if you're a smoker you're probably not going to get Parkinson's disease or Alzheimer's disease because smoking actually prevents these diseases. 

Now I'm not encouraging smoking. 

(laughter)

 So don't go light up, okay? That is not what I meant. Just go get the nicotine patch. But I'm just saying there are a number of factors that modulate mitochondria's ability to be more efficient or less efficient, as well as the cell's ability for deal with the less efficient mitochondria. So we need to understand that. But we also need to know how effective we can be across the spectrum. Because there is going to be variations. And one of the things we're really interested in doing and we're talking about doing this with a group at Harvard is trying to identify serum signatures from people like you that would be related to Nrf2 to give us some idea of who might have the lowest Nrf2 in the population, who might have the highest, and based on that we can suggest diet modifications to change those ratios so that the lowers ones aren't the ones that are going to get Parkinson's and Alzheimer's and all the other associations with that. 

- Everyone is interested in nutrition. 

(inaudible)

- Go eat your broccoli, lots of broccoli. Broccoli sprouts are the best actually. I prefer red wine because resveratrol in red wine induces this pathway dramatically. I'm going to go home tonight and have some red wine. But the cruciferous vegetables, and there are a number of molecules in the cruciferous vegetables that actually cause the activation of this pathway. And a lot of the studies have been done using products that have been isolated from broccoli sprouts and from broccoli that are dramatically chemo preventive. They prevent cancer formation and the way they do that is principally through activating this Nrf2 pathway. 

God knows we have tried to test these things to get in the brain. And we just have had very, very little success at delivering these things orally and actually having any activation of this pathway in the brain. We've tried. A lot. And so what we're trying to do is to use these mice that these -- reporter mice because all we have to do is feed the animal and then just take the brains out of these animals and be able to see if this pathway is being activated very easily, but we haven't been able to do that with any good drugs. You should go eat as much of those as you possibly can because there are a lot of good things in those vegetables that activate this pathway and prevent cancer formation. And there are a lot of companies that are trying to, a nutraceutical kind of approach to treating these kinds of diseases. Almost every antioxidant therapy that's been tried with Parkinson's, Alzheimer's has failed if it's an antioxidant itself. So it's not just that things, just because an antioxidant's going to work in your brain, that's not necessarily true. So it's difficult to get something there that's actually going to do what we want it to do. 

Yes? 

- The diet associated with resveratrol has that been tried or demonstrated to have any affect on disease in humans instead of mice? 

- Calorically restricting humans, in Wisconsin I think that's a contradiction in terms. 

(laughter)

 I'm speaking from my own experience here. But there have been caloric restriction studies done in mice and one of the things that I can point out is that if you look at the changes in the brain with caloric restriction, one of the, there are a number of these Nrf2 genes, these Nrf2 proteins that we are really interested in that change. And so what we're doing right now is Mark Matson at NIA, National Institute of Aging, actually has Delinda's mice and is calorically restricting them to see to what extent caloric restriction will actually have the ability to activate this pathway in the brain. And based on that, we can determine if we want to proceed into these other kind of large based animal studies in these transgenic models to see if caloric restriction affects them. So that's a really good question actually. We should all caloric restrict. 

Eat broccoli. Skip the brats on Badger day. Yes? 

- (inaudible)

 Antioxidants but you don't know if they work. Do you know any oxidants that actually reduce this type of problem. 
(inaudible)

- You mean as far as drugs that would. 

- Extended factors. 

- There are a lot of astringent products that activate this pathway but the problem with them is we haven't been able to show that they get into the brain. These things are good for you, you should eat these things and take these things because they are really good for you, for your body. And if your body maintains health, your whole body maintains health that also can be reflected in your brain. I think one of our friends, Dr. Charlie --, always says treat your brain like you would your heart. Because if you keep your heart healthy, your brain will stay healthy. Now the one thing that has been shown to have significant impact, outside of smoking, is do the New York Times crossword puzzle every day. That will have a significantly attenuating effect on the development of Alzheimer's disease. That's been shown in human studies. 

So it's an activity issue as well. So exercise to keep your heart healthy, exercise your brain to keep your brain healthy. And those effects have been shown. We do not have, and I hesitate because what we've done is we've taken, for example, a molecule that we've had synthesized that we treat ourselves with and it activates. It's a natural product, let's just say. I'm not going to say which one. But it's just a natural product. Then we'll go to the health food stores and we'll get the tablets of this same natural product and we know that we activate 10, 20, 30-fold this Nrf2 pathway in our culture systems. We'll go get these tablets, your pulverize them up, we'll throw them in ourselves, nothing. So almost everything that we've tested that way has failed to activate unless we actually buy the pure stuff from a chemical company that's making the molecule. 

So you really have to be careful I think with the real food, the real nutrition is important but the supplementation if it's not working doesn't mean it's not working because they drug's not working. It may not be working because they actually never are functional in the first place once they're packaged. So you've got to be careful. 

- With exercise has been an indication that the insulin-like growth factor has been increased and it impacts the brain. What might that have on your molecule? 

- The insulin-like growth factor pathway, for those of you that don't know it, that pathway in neurons, like in the dish ,have been shown to be extremely protective. There are a series of things that are activated when you treat with insulin-like growth factors and insulin itself that in neurons that really have a dramatic protective effect. We have, needless to say, we've done that. And the insulin-like growth factors and insulin at least in these neurocells that we've been doing do not activate this pathway. So it looks like it's something different than that. 

We have been working on other signaling pathways to try to figure out if there's cross talk and which pathways might be involved but, unfortunately, we can't just throw insulin on our neurocells and get activation. We haven't done that in vivo. We can actually take molecules like that and directly inject them into the brain. And that's why I say you got to be careful. This sulfurophane, which is a product in broccoli, is a really good, you eat it, it's really good at blocking cancer and activating this. If you take that molecule and you directly inject that into the brain, you die in an hour. The mice die in an hour. It's nasty toxic. So clearly what's getting out of your gut and into your body hopefully is not the same thing that causes your brain to completely stroke out and die. Because we thought we could bypass the blood/brain barrier and see if it would work and it didn't. 

God knows we only tried this experiment and few times. Go ahead. 

- Some years ago there was, maybe not a robust association, but some studies indicating that there was a Parkinson's patient in Canada overlap with high pesticide and I wondered whether there's a feeling that environmental toxins have an impact on the development of these diseases? 
(inaudible)

- I'm having flashbacks to my thesis defense. 
(laughter)

 Yes and no. That's what I said in my thesis defense too. No, actually in my thesis defense I just said I don't know, you're so smart, I just want to pass. 
(laughter)

 Actually one of the drugs that's actually used to induce a Parkinson's-like type phenotype in rodents, although it's not, well it's reproducible in certain labs but it's been hard to do, is -- which is a pesticide. And I think there is, if you look at studies, and I teach lectures in toxicology about this stuff, if you look at studies on Parkinson's and environmental components, there are probably just as many studies that say no as say yes. And the problem, I think, with these studies is they're retrospective studies. They're looking back. They're basing a lot on what a population or what a spouse or a sibling or a child would have said. And the cohort of individuals that they have in these studies, if you're lucky you might hit the two cohorts that give you the right situation. And if you're unlucky you just don't have enough control population versus the Parkinson's population to show that. I think everybody believes just in a concept-wise that the environment, at least in Parkinson's disease, the environment is a major contributing factor to the development of the pathology. And that it's probably what we talked about earlier. 

Each of us has our own baseline, Nrf2 or baseline ability to deal with oxidative stress. And those with lower baselines, and add to that an environmental component, those people may actually be at a much greater risk. But to date I don't think we've been able to define that cohort as populations. And that's why we wanted to look back to see if we could identify the human populations that had low versus high and then retrospectively look back at say families because that would probably be a genetic, well it could be diet too, but we have to go back and look at those and see if there's an association with risk. I thought I saw another hand go up. Yeah? 

- So I'm just trying to relate this to, my father had Parkinson's and you mentioned environment as a factor. 
(inaudible)

 Running a dairy farm he exposed himself to -- 
(inaudible)

- Yeah, filling up the fertilizers. 

- With that said, I've always looked at myself and my kids and my brothers is it genetics? Is it not? I always thought it was environment. I really thought it came on, in his case, because of his --. 

- I think people would, there have been a lot of studies looking at farming association with Parkinson's disease and -- was known as one of these farming pesticides that was used routinely. So I do definitely think that if you're putting yourself in a high risk environment and exposing yourself to these different things it's definitely, it's probably a contributing factor. What component is the contributing factor we don't know. But again I think what I'm talking about from, when I'm talking genetic component I'm talking about a mutated gene that's been found in a familial association with Parkinson's disease. 

I may have misspoke. I didn't mean, Nrf2 is a gene but it's not that the gene is mutated or changed what it is is that it's just expressed at different levels. So if your family, for example, is a low Nrf2 expresser, I'm just trying to make this an analogy, if your family is a low Nrf2 expresser and a hard-core farmer using all these things, then your risk probably goes up dramatically. Whereas, if you are a high Nrf2 expresser and you're a farmer you may not see anything. And so I think that's why you have 10 studies saying that there's a farmer association with Parkinson's and 10 studies saying no. So there's two multifactorial diseases to try to, we have to find a way to find out what cohorts are the best representation to make the conclusions based on and I just don't think we have that technology. We just don't know yet. It's really hard to identify that. Sorry about your father. 

- I have a question about glutathione. 

I understand that it's a very powerful antioxidant. It decreases with age. It's difficult to introduce into your body in a pill form. Could you tell me a little bit about it? 

- Glutathione is basically a three amino acid peptide. And actually if you give precursors to glutathione or glutathione itself it actually can be toxic to the GI tract and stuff like that. So they've tried that. There are precursors to glutathione that are tolerated. That do have affects on increasing glutathione in organs and things like that. And we can do that on some of the mice. 

Again we've tried to do a lot of work with glutathione administration chronically, glutathione precursor administration, chronically in mice and we have not been able to get any significant changes in glutathione level in the brain but we can in the systemic outside of the brain. In the livers and the kidney and stuff like that. And this is I think maybe one of the approaches that we think take antioxidant, take antioxidant, take antioxidant for these diseases. And I think that's what makes this such an interesting target, I mean Nrf2. If you can target Nrf2 the glutathione will go up because the genes that are responsible for making glutathione are some of the key genes that are changed. So glutathione levels, for example, in the GFAP Nrf2 mice, those mice we made that extended the life span of those ALS mice, the glutathione concentration in the spinal cord of those mice are increased only 20% to 30%. I mean, that's not very much. But that much of a change with have a dramatically significant impact on pathogenesis. But we can't even get that much to be able to see in the brain in these systems. So approaching it from the way that we're doing it is using glutathione is generating increasing glutathione but we're doing it from a completely different approach than taking antioxidants. 

We're activating the pathways that result in increased intracellular synthesis of the product itself. And that may turn out to be one of the major outcomes of this work is that we can actually activate those pathways in vivo using this system and get the benefits of the glutathione in the regions that we need to. 

- What options are you exploring to try to get things past the blood/brain barrier? 

- All kinds of stuff. We can do chemical modifications. We can put adapter molecules on these. So if we know a molecule activates Nrf2, what we can do is we can possibly take that Nrf2 activating molecule and stick things on it that might allow it to be actually taken up by the brain. There are lots of things. Glucose, a lot of sugar. Stuff's got to get into the brain, right? 

We need this stuff. So there's a lot of stuff that's transported in the brain. So if you want to think of it as a hijacking is probably not the right word. Taking advantage of an existing endogenous system that we know accumulates something in the brain and then being able to attach that to molecules. Kind of like making bifunctional molecules. You can put them in an animal, they get in the blood stream and because they get taken up through these systems that we've kind of pirated through we can get them into the brain. And so those are one of the ways we're doing it. The other way we're trying to do is you can do antibody you can tag these things with specific antibodies that are also taken up. And the other thing is you can permealize the blood/brain barrier if you want to. Although that's not necessarily a good thing to do. 

The other thing that I should mention too is that in a lot of neurological diseases once the brain becomes stressed the way it is in the pathogenetic process, the blood/brain barrier actually gets looser. So drugs that may not get into the brain when we're testing them in an animal or drugs that may not get into the brain of a patient of just you or me, well presumably we're okay, in these patients they may have access to the brain. Just because the blood/brain barrier is not as tight or intact as it was when you were healthy. And this is more chemists' field. We collaborate with other groups on campus to try to do these things, to try to find out how we can get things across. And some molecules will just go across. A lot of the drugs like LSD and those things they just go right across. 
(laughter)

 So maybe we can find out ways to transport stuff in, you know? Using these, there's some cool pharmaceuticals out there that have a lot of affects in the brain and if we can figure out how they work we can maybe hijack that technology. 

And it may just be a side chain or something that's on this molecule that we don't even know yet that that's how it's going. Maybe just passes through. So trying to identify drugs that are lipophilic and go through cell walls and that kind of stuff. So there's a lot of ways to do that. It's just it has to be a very designed approach to try. Taking that kind of, I got a thousand things I want to do and getting it to an animal where you can actually physically test it is ungodly expensive to try to screen. So we have to develop prescreening methods so we can take a thousand molecules that we know activate in vitro, find ways to test secondarily test them so you get down to maybe 10 that maybe kind of candidates and then those you push forward into the animal models that we have with the -- hPAP model. And we can look at that. And that's exactly what we're doing. And we're trying to design the system so that we can start with large number and as we develop or as we go we can whittle down and by the time we get to the in vivo system, so we have it all set up. 

We can screen in vitro. We can screen in primary cultures. We can take the hits from primary cultures, go right into the -- reporter mouse. Then we can take those right into MPTP models and then we can take those successful ones in the animal model that are MPTP, take those right to primate models and then we can go to Michael J. Fox, they write us a check and they'll move into clinical models. And we've done this already with one drug, an FDA approved drug that's hopefully going to go to clinical trials based on findings that we've had here in the primate center that will move into clinical trials with Parkinson's disease. So that kind of proves that we can push this through. We just need to find drugs that may get the brain more efficiently and activate where we want them to. That's the other thing. The brain is not a brain. 

It's not just one big hunk. There's all kinds of different regional distributions that you have to deal with as well. I saw another question. You had a question? Yeah? 

- Well, I'm just thinking of timeframes you partial answered the question. What kind of a timeframe are you thinking about? 

- Timeframe? 

- (inaudible)

- Tomorrow? 
(laughter)

 You mean for development in this? 

- Yes. 

- If there's the right investment and I have to step back a little bit too. This kind of research is really not the kind of research that we do in my lab. We do lab on mechanistic stuff, identifying targets and things like that. But where you basically try to churn through this whole stuff that requires partnering with a company, forming a company. Somehow you need to have a huge amount of money to be able to set up and run these studies and base that in a company format because I went to my graduate students and say I have 10 molecules I want you to give this thing five doses each to the animals over the next three years they're going to look at me like where's my hypothesis. 

We've been very careful to not overlap that because that's a problem with having thesis driven research and hypothesis driven research is just churning through a bunch of stuff. So we need to do something in a partnership. And we have done some of that stuff with -- and GNF in screening molecules. We screened a 1. 2 million molecule library for Nrf2 activators with GNF. And so we're trying to get people to pick up those molecules in a company standpoint and begin to try to develop them and we have the tools to offer them to do that. And if somebody doesn't do it pretty soon then I'll just start another company myself. 

- Does this work have application. 
(inaudible)

- Yes, I would say yes. It depends on, the astrocyte stuff, no. Because astrocytes are not in the periphery, obviously. But there may be ways that we can do that. And we've never tested that. And one of the things that one of the postdocs wants to do is actually look at spinal cord injury and spinal cord crush to see how this pathway may be involved in that. And Delinda is actually looking at Multiple Sclerosis models and looking at how this pathway can modulate inflammatory responses associated with auto immune disease. And so we have a bunch of kind of irons in the fire trying to test to see how broad this application could be. But I think there could be help with periphery. Maybe not getting the feelings back but at least slowing down the progression. 

Regeneration is a whole other question. Any other questions? Yeah? 

- I just want to know what did they do with the people that had that synthetic heroin, how did they treat it? What did they do? 

- My guess is that most of them, I'd have to look back at the history. I don't actually know for sure if they lived. I'm assuming most of them lived but I think they probably have severe, they may all be dead by now, they probably have severe neurological effects that were persistent for a very long time. And the theory was that these may have been the worst cases. It's the addicts that didn't take as much dosing as these guys, that these initial people that they found did, that they're concerned that there's going to be a rash of, if the addicts had lived there may be a rash of Parkinson's disease in that population over the next 10 years because of this contamination. 

I saw another question in back. 

- You said that you get most of your funding from private foundations? 

- NIH and the government and from private associations. 

- Do you think your research would be affected if you got your funding from a pharmaceutical company? 

- That is one of the reasons I tend not to deal with pharmaceutical companies just because I don't want anybody to think that the research is biased. There can be great funding from research companies that give you free reign and there are other companies that may not give you the same kind of free reign. We do get, for example, we're testing, -- and I, are testing another molecule that's an Eli Lilly molecule. But we have no restrictions on that. They just provided the drug; they didn't provide any money. The money actually came from Michael J. 

Fox to test this whether it would work. So I've really worked hard to make sure that the research is not in any way biased in that way at all. And it never would be the way that we work. But I even try to avoid the perception at all. 

- Could you tell me a little bit more about Lewy body? 

- Lewy bodies are the inclusions or the proteins aggregates that form in the dopaminergic neurons in Parkinson's disease. So what they are is it's a protein and usually alpha synuclein is one of the common ones. So there's a number of mutations in this protein called alpha synuclein that will facilitate this aggregation. And that's one of the genetic forms of Parkinson's disease. So what they are, say what? 

- Is it just in the frontal lobe. 

- No, these guys are actually in the substantia nigra. The Lewy body formation. You'll get Lewy bodies in a lot of other diseases. So for example actually all of us are probably getting Lewy bodies right now as we sit here and protein aggregates in our cells. There's a very famous study actually done in Wisconsin with nuns and so they looked at as nuns died they looked at demented nuns and undemented nuns, and my wife's Catholic so I'm not even going to go there. 
(laughter)

 But what they found is that Alzheimer's patients have plaques and tangles. But what they found is a lot of these undemented nuns had just as many plaques as the demented nuns who were diagnosed with Alzheimer's disease. And so this is a natural process, again it probably goes back to is a Lewy body going to be a bad thing or a good thing. 

There are people that argue that aggregate formation in cells actually prevents them from being toxic. So why, when we look at Parkinson's brains, do we see all the cells we see they're still there, have Lewy bodies in them and the ones that are gone did they ever have Lewy bodies? And the way they think it works is that there's a certain machinery in the cell that degrades these proteins. And they think that somehow that mechanism, normal mechanism of degradation may get messed up by these mutated proteins and when that gets plugged, let's say your garbage disposal gets plugged, things start backing up and when things start backing up you get enough of this stuff in association with each other that it begins to form these aggregates and fall out of solution or fall out in clumps in the cells. Did that answer your question? 

- I think so. 
(inaudible)

- I think we're toward the end here. Help me thank again Dr. Johnson. 
(applause)

(inaudible)

I know Dr. Johnson will be here for another couple minutes if there are any other questions. I'd like you invite you to all come back for a special treat November 18th and 19th, two back-to-back academy evenings on the celebration of the 10 year birthday of stem cell research. We'll be back in the capitol theatre. There'll be several stem cell displays. Dr. 

Jamie Thompson will be speaking the first night and Michael West the second night and an added bonus is there will be two stem cell documentaries earlier in the day starting at 3:00 and both will be having their Wisconsin screening preview and there are a lot of university professors here. These are films that were made nationally. One that was shown at the republican and democratic national conventions on stem cell research. So we hope you'll come and join us and share in this community of sharing knowledge and, again, please turn in your little cards on your way out. If you haven't filled one out there's some out on the table and we are very pleased to have you part of the Wisconsin academy community. Thank you. 

(applause)