1997 LECTURE SERIES

The Secret Life of an Aging Neuron: Successful Brain Aging vs. Alzheimer's Disease

Dr. Carl W. Cotman
Institute for Brain, Aging and Dementia
University of California, Irvine
April 9, 1997

What I want to share with you tonight is some of the impact that aging and Alzheimer's Disease are having on us as a society and why we really have to address this problem. I'm going to try to take you to the neuron level and take you through how the neurons see this and what they're trying to do about it, and what we can do to help them.

Last year in Time Magazine, they ran a series of really healthy distinguished elderly people. For example, Sidney Amber works in Los Angeles and is actually the maitre d' at a restaurant he founded decades ago. He still works there, helping to seat people, greeting old friends and so on. He's 109, which his truly remarkable! So people can, in fact, age successfully. We need to find out why this is and what makes that happen -- and then what steals away that important edge of successful aging.

Sometimes, people think that the enviable consequence of aging is to lose cognitive function and, you know, to go "down hill" so to speak. There's a recent study that was done in Berlin called the Berlin Aging Study, where they looked at the loss of intellectual function with aging up to 100. Their big hypothesis was that maybe some of this is due to losses of hearing and vision, and so when they corrected for that, they in fact noticed that this line is now no longer going down, but going flat. That's extremely important fundamentally, because it means that you can get glasses, you can get hearing aides, but you can't get a memory machine.

Over 65 Population is Increasing
By the year 2020, one out of five Americans will be over the age of 65. That means that when you're off in Fashion Island or whichever shopping area, one out of five -- maybe even in Orange County one out of four -- will be over 65. That will make 39 million people in America over 65, conservatively, in the year 2020. Of those, 12% will come down with Alzheimer's Disease. And startlingly, it goes to over 45% for those over 85. It is the fourth leading cause of death in adults. So it's an enormous problem. Put it another way, it's an epidemic that's sitting there looming unless we do something about it now.

As Paul Boltis said when he finished the Berlin Study, the bad news is the Alzheimer's Disease, the good news is that if biomedical research can do something about this, the potential is really there. Now, the cost is enormous. Nationally, it's actually $100 billion annually, and when I first heard that number I didn't believe it, but you can actually recalculate that number. In Orange County, there's roughly a billion dollars spent, according to a recent survey taken by the Alzheimer's Association of Orange County. A corporation with 500 people spends $225,000 annually for employees giving care to the elderly. That's missed work time, excuses of having to go out to take care of somebody during the day, etc. So it's a huge expense.

What is Alzheimer's Disease?
Now what is Alzheimer's Disease? Sometimes it's really best to go back to the original source, which was E. Louis Alzheimer in 1907, who first described this disorder and he actually wrote about a two-and-a-half page paper that is certainly become a landmark for this century. And I quote out of his paper, "...One of the first disease symptoms of this 50-year-old woman was a rapidly increasing memory impairment. She could not find her way about her home. She was then institutionalized and during this, her gestures showed a complete helplessness. She was disoriented as to time and place and she would state that she did not understand, that she felt confused and totally lost."

This is really quite an insightful, very brief description of the disorder. It starts with kind of a memory loss of which there are several sub types. It then becomes one of confusion, disorientation and abnormal behavior, including aggressive behavior in some cases, and what it does is it begins in one area of the brain. It increases the severity in that area, but then it spreads through the brain, essentially taking away some of the higher processes that are basically so important to us for learning, thinking, and emotional stable behavior. In order to address this problem, the real hope for us is research, and in order to do this kind of research, you need a multi-level approach.

In the Alzheimer's Disease (AD) area, we were fortunate to be awarded one of the several Alzheimer's Disease research centers, both from the State of California and also the federal government. What we have put together is a clinical program that does diagnostic and treatment work. We have an imaging component that does research on trying to pull out modern methods for detecting the disease. We have an integrated basic research program including animal models, training and outreach program, and we have a parallel thing run by Ira Lot here on Down Syndrome, and a number of external collaborations both within centers and so on.

What are the Neurons Trying to Tell Us?
What I'm going to talk to you about tonight from the neuron's point of view is what our neurons are trying to tell us, and what they are telling us in the AD brain. You have two basic hallmarks of the disease. One is that the neurons that are dark are staining with a particular subs292 that's abnormal to them and they shouldn't be showing this at all. A normal control case would have none of this staining. The second thing that happens in the brain is there's this build-up of this deposit called beta amaloid and it accumulates outside the cells in the extra cellular space of the brain and these actually were described initially by Alzheimer.

Okay, so what are the neurons trying to tell us here? Amaoloid has been thought to be one of the central molecules in the disease. It's found in all of these plaques. It's derived from a large pre-cursor protein which I'm proud to say was actually defined by, out of Irvine, Dennis Cunningham and Bill Van Nostrom in the medical school. It's known that beta amaloid will assemble into essentially what's called beta pleated sheet or it folds into an insoluble structure that's called beta amaloid. And so, this is the large precursor protein and then amaloid is a little 42 amino acid chain that has the ability and the information in it to self-assemble and then become insoluble and non-degradable. It's basically as insoluble as hair. That's the beta structure that you've got in your skin and your hair and it forms within the brain.

Neurons start to grow into it and start to degenerate and as I was looking at this kind of image several years ago when I first started some of this work at Irvine, I kept looking at these and people just thought that they were just degenerating. But not really -- the amaloid was inactive, metabolically inert. And so we asked the questions, "Is this really true?" And frankly, for three decades, amaloid was believed to be biologically inactive and you could read it in paper after paper.

Well, one of the exciting things about science is that you can test these hypotheses and do an experiment and so the first thing was you had to get the amaloid some how and make it basically synthetically. So initially I got it from a colleague of mine and then I couldn't get anymore out of it so I went up to Charlie Glade, who is a very distinguished biochemist in the biological sciences division, and convinced him to make me one batch of it. And he said, 'You know, Carl, okay I'll make you one batch of it, but this is kind of, you know, this isn't really very interesting. It's a little long because we're only used to making 10 amino acids in a row, not 42, but you know, just this once.'

And so Charlie made it once and Charlie is now one of the leaders in the study of amaloid and its assembly in the neurons and found the whole area totally fascinating. First, when the amaloid is made, it's all soluble, but you just leave it in the test tube for a few days and it forms these little plaques. It actually does this spontaneously in a test tube. So in other words, the molecule has its own mind and its own information that drives it into forming these amaloid-like deposits. And that was a really big discovery.

So it's sort of a love/hate relationship. The amaloid grows there. It likes it, but then it actually kills it in the process and it finally kills the nerve cells. The actual biological activity of amaloid, this subs292 that accumulates in the brain of elderly patients that come down with Alzheimer's Disease, has two activities. If it's an insoluble form, it actually stimulates and acts as a weak factor to keep cells healthy, but as soon as it assembles, it becomes toxic and will kill the cell so the survival goes way down. In other words, when the amaloid ages and forms this way, it changes its biological activity. So the same molecule can do two different things. This assay actually is now being used in the pharmaceutical business for screening many new compounds and identifying new structural leads to study drug candidates to treat the disorder -- so that's a real accomplishment.

Well at the time, everybody was telling me that that's really nice information Carl, but that this really doesn't apply to Alzheimer's Disease because we know that the amount of amaloid doesn't really correlate with cognitive function, and previous papers had not shown that correlation. So Brian Cummings in my group, together with our clinical staff, decided to challenge that assumption, using our own patient population where we took the time from the last assessment to autopsy as being approximately a year, and we used computer-based methods to do the analysis and not qualitative accounting methods. And, we were able to show that yes, indeed, there is a correlation.

As the amaloid "accumulates," cognitive function drops. So this means that this is an important target and we need to find out more of what the mechanism is. There's many people working on that now.

The Degenerating Neuron
Let me show you where we are with this now. It's an exciting and new concept. In the life of a neuron from the beginning of birth to death, there is only two ways that the cell will finally degenerate. One is called program cell death or apatosis, and you can say, well I learned a new word tonight. Apatosis, it's Greek for falling leaves, it's a natural form of cell death. And it's characterized by a number of different things, like the membrane actually bubbling apart. It's gene dependent. It's actually driven by the genes expressing killer type products and it's associated with degrading the DNA so that the DNA can't be passed on at all. It's actually used as a mechanism that never even generates an inflammatory response or a local infection because it just takes the cell away and the next cell isn't even affected by it. In contrast, the other way that cells die is by accidental cell death or necrosis. That's just basically swelling up and breaking. It's like you drop the egg on the floor and so it's an accidental form and people previously thought the major form of cell death in the nervous system was this.

We decided to test that and see whether amaloid was killing neurons by apatosis or program cell death. In other words, are they programming their own death? That's a significant piece of information because if you can learn what the program is, you can control the program. Just as if you can fix your computer when the program doesn't work correctly, this is what happens when you add amaloid to the cultures. They essentially become covered by it and they start to bubble apart. These are the healthy cells. These are the cells that are present with amaloid and here's the bubbling that actually takes place. The cell is literally taking itself apart bubble by bubble. And as it does this, the DNA is degrading and the DNA breaks down into a series of forms that actually generate a ladder of breakdown products, which is exactly characteristic of the program. The program runs to do a particular kind of breakdown and you can use that to determine that it indeed is a program cell death or apatosis. That can also be picked up histochemically in culture. We were very interested to find out what it looked like so we could then test it later on in the human brain and find out whether this kind of mechanism is actually operative and find out what we can do about it. And this is showing the DNA breakage, you know, looking at essentially the cultured neurons. You can pick this up now by a new histochemical method and the nucleus actually falls apart. The nucleus breaks into fragments and the DNA is degraded by this type of method.

Now when we finish this work and publish the first couple of papers -- we were actually the first group to demonstrate this for central neurons and particularly for the model for Alzheimer's Disease -- we realized that many of the conditions that accumulate in the human brain in Alzheimer's Disease are stimuli that will induce hepatosis in neurons and this includes the beta amaloid I talked about, and it also includes oxidated damage. It includes low growth factor or neurotrophic factor support and BD&F for example drops down, low energy, and then combinations of these conditions. All of these, in fact if you had to list all of the risk factors for hepatosis they are all of the same factors that occur in a human brain in Alzheimer's Disease.

Okay, here's for example what happens if you expose neurons to oxidation. See, as soon as you hear about oxidation, you say 'ah maybe there's a way you can control it because this is what happens if you put neurons in the presence of a small amount of an oxidating agent.' Hydrogen peroxide, actually the same stuff you use when you have a cut and you want to clean out the infection, and you just pour it on and it bubbles away? That's basically oxidizing the material, and it will then kill the cells around there, but the healthy cells away from the injury will survive. Well it turns out that you can control this by various, naturally occurring anti-oxidants, including vitamin E. Trollix is just simply a soluble form of vitamin E. So, that's very significant, because that predicts that vitamins out there already today may have a protective effect against Alzheimer's Disease.

So again, program cell death, the thing runs the program but there are substances already which can protect it. It turns out against amaloid mediated program cell death, the anti-oxidants are only partially protective. It will protect some of the lipid oxidation, but they do not completely protect the cells forever, and that means that newer therapeutics need to evolve that will be effective for all forms of this. I also want to mention genetic risk factors. This is a very actively growing field and there are two forms of Alzheimer's Disease. The early onset occurs in the late 40s and early 50s. It runs in the family and it, in our impression at least in our population, is male dominated. Actually that's never been reported in the literature, curiously enough, and it largely runs in a couple of genes that were discovered last year called presinelle one and two. A cholesterol transport protein called Apoe 4 is a particular risk factor and has been in many of the newspapers. It's a risk factor for the early onset.

For the late onset disease, Apoe 4 still is but gender also is. And females are at a greater risk for coming down with Alzheimer's disease than men, and that's going to give you another clue as to how we could begin to think about this disorder. It's about a 5% additional risk factor and it doesn't seem to be that women live longer. All those things have been controlled.

Interestingly enough, while we knew some of these early genes provide risk for dementia, it turns out they also are conditions that will make the cells vulnerable to apatosis or program cell death. In the presence of a cell where you've over expressed this presille gene, the cells are more vulnerable to this oxidative insult, hydrogen peroxide in this case. So that means that we should be taking these early onset cases and start to look at whether we can't do something with again, anti-oxidant type protection and multiple types of therapies. That has actually not even been started yet to date. So that's sort of the culture story: There are all these risk factors that we know damage neurons in culture, damage them by program cell death, and these are the natural things that occur in the human brain.

Now let's go into the human brain and let the human brain neurons teach us what is happening and see what their secret is. And they'd be here, running away, trying to get away from all of this program cell death and apatosis. I mentioned that there is a histochemical method for looking at DNA damage in the human brain. This is a control brain. A healthy individual that had no signs of dementia and no signs of DNA damage.

This is a moderate stage Alzheimer's brain, and notice all of these neurons that have this dark brown deposit which is indicative of DNA damage. When we first saw this, we were so worried about it because we said maybe we've got an artifact here, and it's something to do with the way we were handling the tissue or the precondition of the patient and to make that story really short, that is not the answer. The answer is that there is more DNA damage that's physiologically occurring in the Alzheimer's Disease brain and we've had many, many arguments at science meetings on this and there is yet nobody to show that that particular conclusion is wrong. Furthermore, I mentioned that the nuclei actually fragment and you can say DNA damage, by itself, indicates the cell is in trouble, on its way and in a program of some sort, but it doesn't really show you it's in the terminal stages of apatosis. And this is showing two cells in actually a very mild Alzheimer's Disease case where here there is very clear evidence that the nucleus is coming apart and the DNA and the nucleus is being damaged.

Previously I mentioned the classic hallmarks of neurons in stress. In the AD brain it is the classic tangle, and so let's see now if it's tangles or apatosis that comes first. So does DNA damage precede tangle formation and who is the winner, is it 1997 or is it 1907? So Joseph Su in the group did some experiments looking at very early cases and also looking at brain regions that were passed the primary areas where tangle formation occurred but that are on the way to tangle formation to see which comes first and the answer was very clear. Here is the normal brain showing all these blue tangles like I showed before. This is a very mild case. Here's even before that. DNA damage two tangles and all of this DNA damage, including indications of apatosis neurons, but the curious thing is that there's many, many neurons that have DNA damage. I mean it's almost like a wave of DNA damage has hit the human brain and it's only the regions that are vulnerable and then it spreads. So it means that these cells are under some kind of massive attack, but what is it because it seems like it's too much damage? Why so many neurons? And what I haven't told you so far is that once the program starts, in all other cells it happens real quick. Once the neuron starts the program, it runs it just like it's trashing the thing and somebody put the program onto erase and it's just out.

So if the mechanism was classic apatosis, the neurons would degenerate in a few days, which is the normal time of degeneration by this mechanism, yet the DNA damage in neurons continues and you can see it through the disease. Why? The hypothesis was maybe the brain is fighting the damage. You know, neurons are non-dividing cells and basically you have what you have after you're about seven or eight years old. There may be few stem cells which can do a little bit of replacement, but you can't replace the bulk of the brain. The neurons do not divide again. So we tested that.

BCL2
There are various genes that are known to actually protect against program cell death, and particularly one called BCL2. Its only known function in a cell is to protect the cell against dying. That's its whole job and it become active when the cell dies. Curiously, it was originally discovered in cancer, because some of the tumors actually developed too much BCL2 and so they couldn't die. This is actually what's called a proto anka gene and then it turned out then it wasn't unique to tumor cells and all cells have low levels of this. There's a whole family of these. So what's the state of this in the AD brain?

We have had an argument about this in the lab. You know, a lot of times we like to do this. We say how many people think BCL2 goes up, how many thinks it goes down, how many don't want to vote. So we were pretty split down the middle. We didn't know. We let the neurons teach us, and the surprise was that they have induced this protective gene. We thought they might be fighting the DNA damage, but here's the thing. It's almost a one-to-one correspondent. Almost all the cells that have induced DNA damage have also induced this protective gene. In other words, we would argue that the neurons are in a battle between life and between death and that they're struggling with it. It goes further than that, too, because not only are they inducing that will protect against program cell death, but they've also got genes to protect against to repair the DNA and there is several DNA damage repair enzymes. Recently, we found that indeed some of these appear to be unregulated and so not only is the neuron active in terms of preventing it but it's also in the repair path. It went to the garage and it's getting in there to get a tune-up and get repaired, and that's certainly good news.

Now what is it, what is the condition that seems to be going along with all these early changes that actually haven't been described before in the human brain? I should say also if this was on terminal apatosis as you would see it in culture, this activity would be down regulated, not up regulated. Okay, so what is it? This is a study that shows that the cells that have DNA damage have also extra oxidated damage as seen by the number of proteins that are oxidized. The brown is oxidized proteins and the cells that are light do not have that whereas the cells that are damaged have more oxidation. So we suspect that there's some oxidative effect in part that is driving this to put the cells under stress and to start to cause the DNA and some of the other factors to be damaged. And you know, we can go into that more but I'd like to keep moving on the story and sort of show you what we have as the bottom line on the whole sequel.

We think there's an apatosis decision cascade. In other words, the neurons are trying to make a decision and what they do is they're seeing all these cell death signals and they're taking and saying okay, program, what are we doing, are we on or are we off? And there's a death check point. So the cell is trying to make a decision. If it passes this checkpoint, then it goes onto another one. And in the process of this, it also can go backwards and repair. So it's not really over until it's really over. And at the last stage is then terminal apatosis which is probably rapid. When you're seeing those multiple nuclear bodies in the cell and we're at the terminal stages. You can think of this like a computer program when you're trying to erase your file and you dump it in the trash and say okay, I'm going to throw this out and then the computer says, "are you sure you really want to get rid of that?" and you can even throw it in the trash and you can then go ahead and pull it back out of the trash and save it -- well the neurons are doing the same thing.

Can We Help the Aging Neuron?
What can we do to help aging neurons? Obviously, finding out what the problem is important to addressing the solution, but we all want solutions. And I mentioned the $100 billion problem, the 20% rate constancy, if we can slow it down, so what do we do about that? Well, let's look at it from the point of view of the neuron. Just like people, maybe neurons like to be active and they want to be healthy. So why not ask the question whether neurons like to exercise and fire away and do good, healthy things that neurons do. You know, they want to go to the gym at night and stay healthy. You know, it's been a long day and I need to do something to get my genes back in order and relax and so on. So, we did a simple experiment in the lab and tested this hypothesis that maybe activity is actually protective to neurons.

This slide shows that, in fact, if you would just stimulate neurons here by essentially depolarizing them which makes them fire more, that instead of being affected by amaloids here that they're actually protected from the amaloid for quite a period of time. This has to be preinduced. It's dependent on on-going protein synthesis. It occurs by program cell death when it occurs and so what it's telling you is that the neuron level, there's something about stain active that's healthy to neurons. But let's go now to the next level and say what can you do to test these ideas in a whole animal. I was interested in the idea that had come out of some MacArthur Foundation studies that I was honored to be apart of that showed that strenuous activity, education, and a measure called self-advocacy predicted successful aging. Let me say that again. A measure called self-advocacy, that is how sure you are that you can control your situations. That doesn't mean you have to do everything, it just means you have to sort of be in control of them. The second one was education. So you may be actually helping your neurons listening and hopefully you're listening. And the third thing was strenuous activity.

So there have been no animal experiments and no rational basis for that, so we decided to do one. So we had this hooked up with a computer to essentially test the revolutions and could then evaluate whether this worked or not. There is a particular class of molecules that we tested called growth factors. These are sort of neurotrophic factors and growth factors. They're small proteins that aid in neuron survival and health such as nerve growth factor. And so what we're doing is looking at running and the animals are self-selecting at running, and so the only behavioral change that they're really participating in, you could argue, is really exercise. It turns out that it's increased with just a few nights of running, and some of these animals actually run a fair distance at night. It does seem to be linear related to a degree, to the amount of running until too much running is not actually beneficial.

When we first started this, I think our expectation was that gosh, if we get a change in the motor and sensory cortex, we'll be really happy, because at least some of the brain is benefiting, but the huge surprise was that it occurred in the hipacampus, which is a brain area that is vulnerable to Alzheimer's disease and involved in learning and memory. Why in the heck is it that running, simple running, goes ahead and induces these factors in the hipacampus? Well, actually there's two reasons. One is that it's a natural behavior to keep the brain area healthy, and secondly, it may also be involved in learning, memory, and decision making.

So we asked the next question. Does learning itself maybe add something to just running? So if you're on the treadmill in the morning and you're volunteering rather than being forced and you're reading the newspaper, you know, depending on what you're reading of course, but if you're studying and learning something, maybe you're actually doing better for your BD&F than if you're just out there running. So we did this again in our rat model, and we put a rat in a little swimming pool. So we used what's called a Morris water maze, to essentially see what would happen if the animals got in the swimming pool and had to find a little platform, which in this case was a life pres296 in case they were having trouble, but there actually would be a platform underneath that they would escape to. And so what we asked then is does BD&F actually increase? And what they showed is that if you did the swimming controls, just controls at 100% here and then put the animals into swim, there was an increasing in the swimming in the BD&F in both the cortex and in the hipacampus, which would be consistent with our previous data from the exercise. But the exciting finding was that learning actually added to that and further increased BD&F expression.

So what this suggests is that there is an additional benefit from continued physical and mental stimulation but now instead of being qualitative and at the structural level, we can actually put it to the gene level. BD&F has been shown to actually protect cells against program cell death or apatosis and it is one of the factors that is actually lower in the Alzheimer brain.

Okay, what else about interventions do we know? Some of what I've argued is that DNA damage is essentially one of the central pathways and one of the earliest changes that has yet been reported that's clearly pathological in the AD brain. It can be driven by oxidated damage. It can be driven by beta amaloid and it can be driven by low energy. It is also the case in inflammation here, it can essentially drive some of the amaloid and will essentially put these cells under additional stress. If there is too much prolonged stress, I've argued that apatosis will occur and the neurons are unhappy. Several things can actually protect. I mentioned previously that vitamin E will and there's been indications in literature that also estrogen will. And I want to spend a minute on these two.

Currently, there's work from U.S.C., John Hopkins, and other places that estrogen may be neuro protective or rather protective against some of the symptoms of Alzheimer's Disease. Ruth Mulnard at the Institute is now heading a national clinical study involving approximately 30 centers across the U.S. to actually test this in a patient population of very well characterized elderly women with Alzheimer's Disease. There's also a study where vitamin E has been evaluated in Alzheimer's patients. The data has been completely analyzed and the paper will be coming out shortly. I'm actually not allowed to pre-release the data, but I'm saying the paper is coming out shortly, and journals don't generally publish negative data. So I didn't tell you what happened. But it should be out any time now and it's very exciting because if it is as I'm hinting, it means that there is something you can buy for $8 for 500 pills that may actually be a benefit to the human brain, and to the course of Alzheimer's Disease.

So it's a very exciting potential finding, as is the estrogen finding. You know, this is probably due to the fact that many post-menopausal women who are no longer on estrogen and that may be needed in order to keep the cells and the nervous system healthy. Also, recently it's been shown that Ibuprofen, but not aspirin, is essentially able to be protective against dementia. This is a study that came out two months ago in the Journal of Neurology. So, you know, this is something that is as simple, compound and one can take in various forms and it looks like it's beneficial. So again, the list is growing. We don't understand all about what this is actually doing yet, so this may be just a prototype compound that allows us to look in more depth at other mechanisms and why aspirin doesn't work and this is not clear.

I've also argued that exercise and learning and other activities in the elderly population are important, and it's worth noting that many of the individuals that have Alzheimer's Disease will become inactive in the home and sometimes even isolated. This may actually be counterproductive to the health and the vitality of their nervous system.

So in conclusion then, what we've shown is that neurons degenerate by apatopic mechanisms. They are very vulnerable to apatosis. The AD brain accumulates these conditions. Beta amaloid, oxidative damage, etc., low energy, low growth factors. I've shown you evidence that it exists as one pathway in the AD brain. I then said that there's an apatosis decision cascade that may very well be operative and this is actually new for the whole field of apatosis. I just wrote a review on this for a book that is going to talk about this for the whole field, and it seems like it's a new concept.

Neurons seem to develop their own protective mechanisms. Being non-dividing cells, and probably the smartest cells that we have, their secret is that they know how to protect it and as we learn more about their protection strategies, we can buy into this and this is certainly suggesting new interventions and new prototype compounds, even based on some common vitamins and other compounds.

So future directions is that in the Institute we're pursuing studies on apatosis and early gene changes. We're looking for faster and more efficient data management methods through informatics because as you accumulate all this data and on these patients, Alzheimer's Disease actually turns out not to be a single, but it's got multiple sub types that present in different ways. And we suspect that they're slightly different pathways initially and maybe final. It looks like apatosis is common, but that some of the features are going to be different and so we need to be able to correlate these patient qualifications and sub types to the basic biology. And to that, we basically need things where we can capture the cell data. We're using machine vision techniques and also object-oriented databases to organize and manage the data in collaboration with several people in computer sciences and electrical engineering. And we need early detection methods. Recently, the administration has given the final approval to buy a high field MRI which will allow us to have the most avant garde research machine on the campus that can do human studies on dementia patients. We believe that using functional MR and others, we will be able to follow the disease early in its course and actually test circuits to see how the circuits are working and to look to see if these interventions are working even at a sub behavioral level and we're still searching for bio markers.

Then, finally, we need to do something that's kind of unconventional and test combine therapeutics. With that, the system had been tipped to apatosis and through work in the institute and research, we believe we can tip the call back again and put more life into years.

It's been a real pleasure to talk to you tonight and share some of the excitement and advances. Thank you.