Cause and Patho - Genesis of Alzheimer's Desease and Aging of the Brain
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Transcript: English(auto-generated)
00:22
I thank you, colleagues and fellow students. I am going to present an update over my lecture here three years ago in which I explained that we were just
00:41
then at the beginning of recognizing that it might be better to look at Alzheimer's disease as well as normal aging of the brain as basically a cerebral amyloidosis. The word amyloid was first presented to the world
01:04
here in Germany, but as a misconception in that it was named after the prefix amyl. Amyl was referring to carbohydrates. And it was later discovered that these insoluble precipitates
01:23
inside of cells and outside of cells clogging the intracellular and extracellular physiological functions was actually not a polysaccharide, but a polypeptide, in some cases glycosylated.
01:43
And therefore, it had staining properties with PAS, such that it appeared to the early German morphologists to be a deposit of a starch-like material, which really was not.
02:01
Now we have known that with the aging brain, we lose cells. The curve of the loss of 50 or more grams of brain gray matter with each decayed after the 70s, such that the mean of normal brains, not brain damaged
02:23
by chronic brain disease, is a good 200 grams less in people in their 80s and 90s than in people in their 40s and 50s. This curve of slow loss of gray matter has bothered people.
02:43
And the optimists among us, all of us hoping to live to our centennial, would rather not confront this problem. And therefore, it has always been easier to mysteriously claim that this loss of gray matter is interstitial substance, not neurons.
03:02
It happens to be neurons. It happens to be neurons and their connections. But more than just finding some atrophy which we can measure today by CAT scan quantitatively, the aging brain shows three other findings.
03:23
It shows amyloid deposits inside of cells in the form of neurofibrillary tangles. These neurofibrillary tangles are found in a full 30% of people who are 65 to 70 years of age. Not many, but they're there. And they are never found in a healthy person's brain
03:44
under 60. By the time you're 75 to 85, they're there in more than half the brains. And what we didn't know three years ago, although we had an intimation of this, was that if one looks at the brains of the most
04:01
healthy persons one can find who die in their ninth decade, nonogenarians, these are still chess-playing, Mozart-playing, jogging 90-year-olds who die of a stroke or a heart attack while jogging.
04:21
In such brains, we're finding these changes of neurofibrillary tangles a full 95% of the brains. So the curve is getting pretty close to 100%. Now, in addition to this intracellular, intranuronal deposit of amyloid, and we call it amyloid simply because it's insoluble.
04:43
It's a precipitate. It's in this particular format called a neurofibrillary tangle. And it's birefringent once it's stained with Congo red, which all amyloid stains with. And that's all there is to the definition. It was defined and so named long before its biochemistry was understood.
05:00
And now we've learned that such structures, whether they're in the spleen, liver, brain, or elsewhere, there are many kinds of amyloid, are actually beta-pleated sheets that are formed into fibers with a 10.6 and a 4.7 angstrom crystallographic lattice diffraction pattern
05:22
on X-ray crystallography. And these beta-pleated sheet structures are a confirmation of a polypeptide chain, which can occur spontaneously, even in an old wallet or an old ski boot, or on a slide in vitro, from what was previously
05:42
a soluble globular protein, simply with its hydrophobic amyloid genic area falling into a lower energy state of secondary structure. But in that formation, it is out of metabolic cycle.
06:00
It does not go into aqueous solution. Its solubility properties are totally changed, and it usually has obtained enormous resistance to proteases which previously cleaved it easily, even though there has been no change in polypeptide amino acid sequence. Well, that's the basic philosophy of what we're talking about.
06:21
Now, what about the brain again? Besides these hallmark of aging, we have amyloid there in two other formations. One is a amorphous deposit of amyloid scattered punctate around vessel walls. I'll show you pictures of this in a moment. That's called amyloidosis of cerebral vessels,
06:43
or vascular amyloidosis of the brain, or congophilic angiopathy, a big word for simply saying Congo staining of the wall, Congo red staining of the wall of a vessel that's birefringent thereafter. That's amyloid too.
07:02
And finally, you have neuritic plaques with the neurites decaying end terminals of dendrites, microglial cells around the core, which is called a neuritic plaque core. It's very insoluble. You can't get it digested by trypsin, or pepsin,
07:23
or protease K. And it often is associated with a surrounding deposit of amyloid, making it into a large amyloid plaque. These amyloid plaques are extracellular. And they're formed by electron microscopy, one can see, of fibers, single-stranded fibers,
07:44
which in high resolution have two or four subfibrils. And they're totally different in appearance, morphologically, from the double-stranded fibers that make up the neurofibrillary tangle inside a neuron.
08:01
Those double-stranded fibers are called paired helical filaments. And there are fully 1,000-some papers published in the last two years on the immunology and structure of those kind of fibers in the brain, for good reason. Because we're all going to get them in our brain
08:21
if we live into our 90s. And they are the hallmark of Alzheimer's disease, a disease which affects one in five of our aged population in the United States, producing about two million patients at present. It's a total misconception that the normal aged play golf,
08:44
Mozart and chess, and jog in their 80s and 90s, fully 20% of them aren't there to be on television or at the meetings. They are being taken care of with their bowel and bladder functions and need escort. And that is 20% of us who now healthy
09:03
live into our 80s or 90s. It's only the lucky 80% who might not have that. And then as I've told you, if we are the lucky or unlucky ones to be nonogenarians, we all have these findings. May I have the slides now?
09:20
Now, why I went into that slowly is that it was the recognition that these findings of amyloid deposit are all made of the same 42 amino acid polypeptide, a basic building block that builds them all that allowed us to realize
09:41
that this phenomenon of laying down amyloid in different structures, which had led the immunologists and the morphologists, brain pathologists, to conclude they were all different processes, different matters. They stained different. They had different epitopes exposed. And therefore, we have this vast literature
10:01
right until this last few months, claiming they are made of different materials. They are not. We've slowly learned that a 42 amino acid polypeptide builds them all. And a key laboratory in this discovery was the Max Planck Institute for Guineetique with Konrad Beyereuter, who's recently
10:21
moved to Heidelberg. And in more recent years, Benno-Müller-Hill, also in Köln, and the Köln Institute with Beyereuter and Benno-Müller-Hill have been a big collaborating laboratory in some of the work I'll tell you about. And they helped with Colin Masters from my laboratory, who
10:42
is an Australian from Perth, to establish this matter clear and loud that all amyloid of the brain in normal aging and Alzheimer's disease is built of the same building block. If we weren't aware of that, we would not have gone on to the next exciting aspect of the story, to look for the precursor protein that
11:04
led to that amyloid. That precursor being the precursor which is going to break down in all of our brains as healthy individuals we age, and if we're unlucky, break down in a galloping senescence
11:21
in the form of Alzheimer's disease. But we also know clinically one other thing of great importance, and that is there are many other diseases that lay down these structures, like progressive supranuclear palsy, like Pick's disease, like all 20 or 30-year-old people in the ring,
11:42
fighting with boxing, who have too unlucky a history of too much head trauma. Those who are knocked out too often all get pugilistic encephalopathy with dementia, like our world champion Muhammad Ali now shows with Parkinsonism. And those people lay down amyloid in the form of plaques
12:03
as well, all of them, even at a young age. So trauma alone will cause it. And since all unlucky boxers get it, we can dismiss the thesis as wrong, that it requires an abnormal precursor protein or gene. Since all of us, if we live into our 90s, get these
12:22
changes, we can dismiss as wrong the concept that there's an abnormal gene producing an abnormal precursor that leads to these changes, since everybody gets them. So now may I have the slides? Can we dim the lights? I don't need lights here.
12:41
In the 20th of February issue of Science, the whole issue was devoted to a series of papers, starting with the paper by Dimitri Golgarber and Mike Lerman in my laboratory, which announced the isolation,
13:01
characterization, cloning of the gene for the precursor of amyloid in the brain of Alzheimer's disease, normal aging, and Down syndrome, with its chromosomal localization as well. And a series of laboratories that quickly confirmed
13:21
this discovery of two Jewish refuseniks trained in Moscow, who have left the Soviet Union and been working with us in my laboratory for the last seven years, Dimitri Golgarber and Michael Lerman, and a team of others in my laboratory, Bill McBride and Ritrich Inigahara,
13:41
you'll see their names, managed to get this out last October. We knew it was important, but we had no idea how important it really was. We were looking for the gene of the precursor of the only structure we see in the aging brain or Alzheimer's disease. Having found it, it could just as well have been
14:02
on chromosome two, four, six, or seven, or be a complex protein with its message on several chromosomes. That was immaterial. We knew that post-translational modification from environmental or genetic-determined causes in familial Alzheimer's, for instance,
14:21
could easily modify that precursor into amyloid, giving the post-translational changes we were looking for as the cause of Alzheimer's disease and aging brain. But to our amazement, it mapped onto chromosome 21. When my team told me this only four days
14:42
after we had the clone, I told them, oh, get to work and find it fast. I thought they were joking. Now, many of you won't understand why I would think they were joking with me when they told me chromosome 21. That is because all of us working in this field know
15:01
that Down's syndrome is chromosome 21 trisomy. It is a duplication of chromosome 21, and children with Down's syndrome, formerly mislabeled Mongolian idiocy, all get these changes of aging, not some of them,
15:21
all of them, if they reach the age of 40. And already in their 30s, many have Alzheimer's-like degenerative pathology and clinical picture. But over 37, every brain in the world ever yet looked at in Down's syndrome has the full changes of Alzheimer's disease, with no exception.
15:42
Therefore, this pediatric problem, which has now become a problem of premature senility, as though these poor children didn't have enough of a problem with their trisomy 21, their mental defect in utero, their facial and cutaneous defects, they also have the inevitable degeneration
16:04
into Down's syndrome in their 30s and 40s. All right, that amyloid is the same as well, and we know that's on chromosome 21. But what we didn't realize was that when we map, as Gazella and his group have been mapping in Boston,
16:21
large families of familial type of Alzheimer's with genetic autosomal dominant inheritance, they also are mapping onto chromosome 21. So when Dmitri Golgava, Bill McBride, from Maxime Singer's laboratory, told me it's on 21, I thought they were joking.
16:41
It is on 21, and it is exactly where the Down's syndrome defect is, and that's what I'm going to show you in detail. Next. I have no pointer here that I see. Well, the upper neuron is simply a neuron with a neurofibrillary tangle in it. Those of you who've had neuropathology
17:01
can recognize it very easily. Does it light? Yes. Will you respond to my asking for the next slide with this button, unless I'm, all right. This is a purification by cell fractionation of those neurofibrillary tangles using SELCO's method from Boston. We've developed a method we call
17:21
the Collin Masters method. We have another method from Bachman Yar, a Iranian doctor in my laboratory, and these three independent ways of purifying these neurofibrillary tangles give us electron microscopic fields that are pretty pure. They have fibers in them. If we look at them here, one sees very little debris,
17:41
and these are the tangles, which are birefringent, having been spun out of cells, and most of the mitochondria, the nuclear membranes, the nuclei and the nucleoli themselves are totally removed, and they can be cleaned up very much because these preparations can be treated with trypsin and protease K and nucleases
18:01
without touching this insoluble material. In fact, when we go to electron microscopy, we see the telltale paired helical filaments, which are the amyloid double-stranded fibers that make up the intracellular deposits of amyloid. It's because of their 80-nanometer spacing of thinning and thickening and their two-stranded nature
18:23
that then massive literature is accumulated assuming they are totally different matters than the amyloid in these neuritic plaques, extracellular, which often gets to be masses of amyloid of considerable magnitude. We have some brains of aged people, healthy,
18:43
and others with Alzheimer's disease where the entire field is filled with these plaques so that it occupies more than two-thirds of the area of the field for many centimeters of hippocampal tissue. So the quantity of this amyloid is grams percent, not a few lambda percent.
19:03
Well, this amyloid, the extracellular plaque, can also be spun down by cell fractionation using a cell coat technique or one that Colin Masters developed or one that Bachmanyar developed, and we get very pure amyloid plaque cores with no neurofibrillary tangles visible in them at all.
19:22
It's a separation of the two structures. And from the point of view of contamination, that is easily controlled, at least contamination, with soluble components, not only by the detergent treatment, but by the fact these are so damn insoluble, you can treat them with proteases and SDS,
19:42
you can then wash them with Protease K, you can then treat them with nucleases, you remove everything that's soluble. You don't find many people writing their PhD thesis on the molecular biology and the Western blotting and amino acid sequencing of insoluble materials that can't get into their gels. And it's that reason that we've waited
20:01
until this late time in history to have the story in more detail. Colin Masters found what anyone could have found in the Ustiks Libiks Laboratory in the last century, that insoluble proteins like this usually go into solution in formic acid. But most of my and other laboratories,
20:21
PhDs in molecular biology, have ceased reading the old literature long ago, or they never read it, and they did not realize that this was the trick that was needed. Solubility in formic acid has permitted us to get back into aqueous media, put these things into gels, get them out with a little detergent, and then amino acid sequence them.
20:41
That's been the whole trick, and without that, we would not have been where we are, and for the last 50 years of study, we weren't there because of lack of solution. Now, these materials are made of the single-stranded amyloid fibers. The immunology is very different. We have antibodies that light these up and don't light the other ones up, or vice versa,
21:03
and that has led to a decision that they are completely different. With practically no one allowing for the possibility, we were looking at the paint, the stucco, the aluminum siding over the same basic fiber structure, all made of a known protein.
21:22
This is the sequence that Colin Masters and Konrad Byroider first got of neurofibrillary tangle amyloid from Alzheimer's disease neurofibrillary tangles, and the same sequence was obtained doing the same thing over on a young person who died at 35 with Down's syndrome.
21:42
The polypeptide was 42 amino acids long, and here is the N-terminal, and it had N-terminal raggedness with one, three, seven amino acids missing, but all these polypeptides continuing the same way to position 42.
22:01
Interestingly, the amyloid plaques, amyloid plaque core of both diseases gave the same polypeptide with less N-terminal raggedness, and to their amazement, and in this slide, there's an error, this should be up here. This means congophilic angiopathy amyloid,
22:23
amyloid of vascular walls. This is not from Konrad and Kern and our laboratory. This is the published sequence that was already in Biochemica and Biophysical Research Communications three years ago, published by Glenner and Wong for vascular amyloid,
22:44
and almost no one in the profession either read it or paid any attention to it, and to our amazement, in Bethesda, Maryland, in Kern, and in Perth, we find material that gives us the same peptide, the Glenner-Wong peptide. Glenner and Wong have had trouble
23:02
on these two of a glutamic acid or a glutamine. It turns out that the GLU is correct in all of them. It's an error. It is not really a difference. Now, this vascular amyloid was obtained not only in cerebral cortex, but by stripping off the meninges, and the coating around the vessel wall
23:21
continues into the meningeal vessels. Such preparations have no possibility of being contaminated with intracellular amyloid from the tangles or plaques, extracellular, since they are not made from cerebral cortical tissue.
23:41
Now, this was Colin Masters' attempt to summarize what he thought this led us to believe, that we have vascularization of amyloid, that we have amyloid in plaques, and intracellular amyloid in tangles, which is the hallmark of aging of our brain as well as identical morphological biochemical events,
24:04
but speeded up in Alzheimer's disease. The guess that it was all coming from a precursor in the serum and moving by retrograde axonal transport into the pericarean sounded to all of us to be highly unphysiological. Killed neurons, whether from an abscess,
24:23
surgical injury, shrapnel, a sword, rapidly form, leave with their substance going out in the urine, and large brain cysts and large cerebral atrophy to the tune of hundreds of grams is known to all clinicians and all x-ray people
24:41
who do pneumoencephalograms and now easily evaluated by CAT scan. The root for dead neurons is from the neuron through the interstitial spaces out to the vessel and to the kidney. It's not the other way. Now, from Abramson and Kirchner, I've taken a picture showing the 4.7 and the 10.6 peaks
25:04
for both the neurofibrillary tangle paired helical filaments and the straight filaments of Alzheimer's disease. And here is where that spacing comes from. It is the twisting of the amino acid polypeptide to form a 4.7 crystallographic spacing in the sheets
25:24
and the beta pleated sheet structure refers to the piling up of these sheets with a fiber axis in that direction. It's a crystallographic problem of how fibers can be made. This simply is showing you the congophilic angiopathy around vessel walls.
25:41
I didn't have another picture to show it. It's the same amyloid. Now, we were in trouble. The established neuropathologists of the world were outraged to have these thousands of papers called into question in the last few years that indicate clearly these three different types of amyloid are different.
26:01
And since they insisted that we must be contaminating our preparations, we knew we weren't because the yield of this polypeptide accounts for better than 80%, in some preparations, better than 90% of the amino nitrogen in the purified preparation. So we know that this polypeptide is the building block and whatever the immunology is is the paint
26:23
or siding or stucco on the top of the fiber. But there's one place in the world that people make these intracellular neurofibrillary tangles in enormous quantity without ever having any vascular amyloid or extracellular amyloid
26:42
in the form of plaques. And that disease is a group of diseases called amyotrophic lateral sclerosis and Parkinson dementia that occur in high incidence foci in the Western Pacific. One of them is in Guam, another is in Japan. And in these populations, these diseases of post-encephalitic Parkinsonism
27:02
like dementia Parkinsonism or motor neuron disease of Charcot and in addition, early appearance of Alzheimer's neurofibrillary tangles occur in the whole population by 30 years of age. And the diseases occur between 40 and 70 to be the first cause of death.
27:22
This is the Western Pacific form of epidemic of these diseases in isolated populations. The one I'm going to mention, will you change the slides? Is that we have in West New Guinea villages where every single house has a person dying this way every year of classical Charcot's disease.
27:42
And we have the Parkinson dementia instead of the preservation of the higher cortical functions, this husband of the last patient is demented and stiff with Parkinson dementia. And all of these patients have in their brain vast enlargement of the ventricles
28:01
and enormous, please to the next slide, enormous atrophy. You're not responding to the slide. And the other aspect of these foci is the dramatic matter that these incredibly classical diseases that are problems in Berlin and Zurich,
28:21
just as they are in New York, are in these populations, particularly West New Guinea, occurring in people who've never in their life seen white man before this incidence got to be high, including classical neurofibrillary tangles. They have no cattle, no sheep, no agriculture. They never heard of metal or potsards or fabric.
28:44
And when we first encountered these diseases, they were still living in the trees, unaware of the existence of coffee and tea and petrochemicals and anything manufactured on Earth. Imagine if one could throw away all of that epidemiology in cancer or multiple sclerosis and say it was meaningless.
29:03
Those materials account for a few hundred million dollars a year environmental investigation for our major idiopathic diseases. Well, here where this occurs, only in the 10,000 people in this area, and none of it in the big rivers.
29:21
We now know what's happening, and I'm not going to lecture on that. It was simply that it was the brains from this area that we went to use because they're loaded with these tangles intracellularly without any extracellular amyloid. There's no other conditions like this. And therefore, in situations like this,
29:40
what we've done is we've purified these tangles. Here's the paired helical filaments. Here they are. They sometimes occur in the form of triplets. And we now know that we can get these triplets to form even in vitro when we form fibers from the synthetic precursor polypeptide. And when we make a Western blot,
30:00
here is the Guamanian material. Here is the Alzheimer's material at 4.1 kilodaltons. And here's the synthetic polypeptide made in the organic chemical laboratory. And these are marker proteins on Western blots. And the, I'm sorry, you're not responding to the, this is the Parkinson dementia complex from Guam.
30:21
It turns out the polypeptide is the same. It was only this demonstration since the brain had been looked at by many of our critics before we did the preparation and was declared absolutely free of extracellular amyloid that made it absolutely clear that all these structures are made of the same
30:42
Masters Biroiter Glenner-Wong polypeptide. Well, this is the paper, and these are the two authors I told you about. Here is Bill McBride from Maxim Singer's Laboratory. And that's what we have just published first in October of last year. Next slide, please.
31:00
For those of you in molecular work, I'm not going into a molecular biology lecture, but Dimitri and Mike Lerman's probe was rather sophisticated. You usually go after a cDNA library these days if you know the product you're after with an antibody. But we had found that our antibodies to the synthetic polypeptide were reacting
31:22
with dozens of dots on our electrophoretic focusing of brain gamish, meaning that hundreds of proteins, dozens of them actually, were reacting. And that told us we would not know which gene we were cloning if we used an antibody.
31:40
As a result, we abandoned the antibody technique and ran to do a probe. Usually you can succeed with probes based on 20 amino nucleotides, six, seven, or eight amino acids. Here we decided we couldn't because we made a computer search of the entire protein library at Oak Ridge and in the Cancer Institute,
32:02
from Drosophila yeast to man, and found enormous redundancy until we got to 20 amino acids. So we had to settle on a 20 amino acid probe aiming at 60 oligonucleotides. We synthesized that, used diazaxionecine for every third codon, and where there was redundancy, we made both probes.
32:22
Well, with that type of a probe, we pulled out four clones from the human brain cDNA library, one of them 1.1 kilobases, the others all one kilobase, and the sequence strategy gave us, to our excitement, the Glenner-Wong sequence of our amyloid of brain.
32:42
There it is. The probe started at term three, and at 18, we knew we had the right probe, and we celebrated on October 16th with a special amyloid fest, which started at 3 a.m. and went until morning. We then finished the sequencing and found an end tag codon
33:00
only at position 157 amino acids after the cleavage site. Now, next slide, please. Here is the entire one kilobase cDNA, and the reading frame ends with that tag codon, and here are two glycosylation sites. One is proline, and it's therefore not a likely site
33:21
for two glycosylation, but you notice the cleavage that makes the amino acid of the amyloid deposits has no glycosylation site within it. When one looks at this protein for its, where it, well, before we had the sequence, we decided to chromosome localize it. This is using hybrid cell lines
33:41
of one, two, or three human chromosomes with hamster cells, 57 varieties. Each cell hybrid contains a fragment of a chromosome or a whole chromosome, but never more than three. Then we used 30-odd mouse-human hybrids, and together it's easy to figure out, as we did,
34:00
that it had to be on chromosome 21. More about that later. The hydrophobic hydrophilicity diagram, hydrophobic, hydrophilic, shows that the cleavage site of 42, 43 is right in the middle of what is probably a transmembrane region. The major hydrophobic site. This is a quantum mechanical calculation
34:21
showing that here is the major area for beta-pleated conformation potential. This was published in the March 13th issue of Science from my laboratory together with Cine's laboratory in Paris, Delabarre, from Cine's lab, came over to our laboratory, carrying with him
34:42
material I had not known about a few months earlier. When I reported this in Zurich at Alzheimer IV in January, Cine's group told me about micro-duplicated Down syndrome. As a pediatrician, I must admit, I didn't know about that. When they told me that there were Down syndrome patients
35:02
that had no trisomy 21, I was confused when I heard of non-karyotypic Down syndrome. It turns out that the French have several such patients, eight in all, that other people in Europe have dozens and so in America. And these children not only have the brain defect
35:21
and the hyperteolarism and the small little finger and the dermatoglyphic signs, they have total Down syndrome without the karyotypic change. This means that all our pediatric literature is wrong when it tells us that it requires duplication of the long arm of chromosome 21. What is here instead is a micro-duplication
35:42
that cannot be seen by optical karyotyping. And three enzymes are known to be in the duplicated area giving three copies per person in the diploid person, namely superoxide dismutase, proto-oncogene ETS2 and phosphofructokinase. No other genes have been found to be duplicated.
36:03
And in that type of material, we use that to look for the gene dosage of genomic DNA on southern blots looked at by the laser interferometer for gene dosage. And here we found three gene doses in all the Down syndrome, three gene doses in a combining of 11,
36:23
here are six Down syndrome and here are the non-karyotypic Downs and here are three Alzheimer's. Here is our normal patients, five of them controlled and here we are using anonymous gene markers outside of the micro-duplication area on the long arm of chromosome 21
36:41
and on the opposite side of the centromere. So we see that Alzheimer's patients, like Downs patients are carrying three copies of this gene for Alzheimer amyloid. What actually turned out was that they are not only carrying three copies of our amyloid gene but they're carrying three copies of proto-oncogene ETS2.
37:03
We thought they were not carrying three copies of STS of the superoxide dismutase. That is a partial error. We have now found using with Mary Sanders up in Boston University where she has the cis area of this gene in a probe
37:21
that the cis portion of the reading frame is duplicated in all sporadic Alzheimer's disease, not the whole gene. So we know exactly where the micro-duplication starts and it's in the middle of a reading frame. And these anonymous DNA loci on chromosome 21 are not duplicated.
37:40
What this means is we can use the probe now to localize quantitatively messenger RNA in the brain. We found already using the whole probe that the message is 3.4 kilobases, that the whole message is much larger than our first probes. Using cDNA from early autopsied humans
38:01
with Rose's laboratory at Duke, we found 75 probes that are 3.1 to 3.4 kilobases long. And using a computerized in situ hybridization with Joe Martin's group and Sina Bachmanyar from my laboratory out at La Jolla at the Scripps Institute,
38:20
we find that in layer three of the human cerebral cortex and layer five, we have massive intracellular neuronal material. What we're doing here is in the hippocampus making sections here. This is the antisense probe for messenger RNA, the sense probe. No hybridization with the sense probe, massive hybridization with the antisense probe.
38:43
Looking at it with a color, this is the layer five with six and four minimum message. This is neuroanatomically the same thing here as the cornus ammonus, one area of the hippocampus most affected in aging and Alzheimer's disease. This is Alzheimer's, this is normal.
39:02
Many cells are lost, but the message is in the normal as well as the sick. The message per cell is higher in the Alzheimer's patient, but he's lost over three quarters of his cells in his cornus ammonus as a result of his disease process. The final story is then that all neurons are loaded with this message.
39:22
It is also present in lymphocytes, microglia, vascular endothelial cells and macrophages, but not present in liver and epithelial tissues except that 100th to 1,000th the dose. All neurons have the message, but many neurons have fully 100 fold more than others.
39:41
And those with a high message are the neurons that get tangled. They are the neurons that drop out with age like the large pyramidal cells of the cortex. There they are lighted up with a, and here is the type of hybridization one gets by the direct probe. It's very convincing. Now these papers will be out in science and nature.
40:02
Next slide, please. Now I will finish by simply contrasting what you've now heard, that it looks as though the Alzheimer's story is simply a normal gene that has in a hot spot been in prezygotic immunogenesis duplicated, giving us three copies.
40:21
Not only are those first three sporadic Alzheimer's proved, but other laboratories and our own have now produced a list of over 40 Alzheimer's so looked at, all have three copies. Obviously, 10% of us in this audience and myself included should have three copies since at least 10% of us are doomed
40:40
to have Alzheimer's disease, maybe as high as 20%. I don't know if anybody would be so dumb as to want to know in advance. I am sure your faculty supervisors would like to know to consider this in awarding tenure. I'm sure your insurance company would like to know or the airlines or army in giving you a pilot license,
41:03
but your friends aren't going to want to know and if you really want to know, I would wonder whether you weren't starting with Alzheimer's disease already. At present, we have no way of preventing this change and we do not know what causes are causing it. It is nice to realize that that 42, 43 amino acid
41:23
intra-membrane cleavage site is a specific cleavage site for elastase beta, which we've learned through the biochemistry department with Mary Singer at Rutgers and that amino peptidase B cleaves even our synthetic polypeptide at exactly
41:42
that amino acid raggedness N-terminal. Now, there is another kind of cerebral amyloidosis, namely the cerebral amyloidosis of Kuru-Kreuzvalyakov disease and Scrapy, which produces plaques, which are just like the plaques of aging and Alzheimer's disease. These are from Kreuzvalyakov in a mouse
42:01
and these are from Scrapy in a mouse and those plaques, when the amyloid is extracted, are the same amyloid that forms the virus-like Scrapy associated fiber of Pat Mertz, which is inextricably associated with the infectivity of these unconventional viruses. This is a totally different amyloid.
42:21
It comes from a precursor, which is these fibers, which we think may be the infectious material, are pure amyloid. They are 27,000 daltons and in prisoner's hands, this is his prion protein. None in the normal Scrapy animal in the Scrapy. 27 to 30 kilodaltons, much larger
42:42
and the precursor for this protein can be eluded from those gels and it forms fibers in vitro called, these are Stan Prusiner's prion rods. And in many laboratories, including nearby, Weissman Laboratory in Zurich, can I have the next slide?
43:01
One has the whole gene from hamster, one has it for man and one has it for mouse at present. And there is the distribution of this transmissible dementia all over the world, a very rare disease indeed, causing what we thought was a good model for aging. But it isn't a good model for aging,
43:21
it's a different amyloid. It's the only additional amyloid we know of in the brain and it's this amyloid which we've transmitted in our contaminated human growth hormone to cause four human cases of Creutzfeldt-Jakob disease just by our neo-cannibalism of injecting children
43:41
who are sexually immature or doomed to be dwarfs with growth hormone made from pooled pituitary glands. The process of purifying that hormone has failed to remove the amyloid material that is transmissible to a sufficient degree to prevent infection.
44:01
And first four came down in our first reports and then Paul Brown has summarized three more. We now have seven down, actually we have eight. And these represent many batches of hormone. The blue show the possibilities that the batches these children have received were shared. But the whites are not shared batches
44:20
and the case in New Zealand and the case in England shared nothing with the American cases. And now we have an eighth patient that got hormone only in 1983 and four. And so there's no sharing there. So there's been massive contamination of many batches. Those children who develop as young adults Creutzfeldt amyloidosis have the prion protein
44:41
in their brain tissue. They have the SAFs and these are from those patients. And when we purify their SAFs, here they are from one of those children with a control from a hamster gold beaded with beaded antibody made against a synthetic polypeptide of the prion protein, not of Alzheimer's.
45:01
So it's here in New Guinea that we've got one lead and it was here in New Guinea we got the other lead on amyloidosis. We had realized already 30 years ago that Kuru was a galloping senescence of the juvenile because 80% of Kuru are loaded with amyloid plaques. We thought it was a good model for aging
45:21
and we got the virus out and we found that the plaques occur in the brain of Creutzfeldt-Jakob disease as well as Kuru. We thought these plaques were the plaques of aging and then we discovered it's a different protein and with Bayreuter's work here, with Derringer's work in Berlin, with Prusiner's work in San Francisco's Weismann's at Zurich
45:41
we found that this transmissible amyloidosis is on chromosome 20 with the precursor different and the amyloid glycosylated. The story of Kuru you've heard here before I'm not going to repeat it just go through these last few slides fast. There's still six cases of Kuru this year in New Guinea
46:01
but this is the disease that gave us the first concept that massive amyloidosis of the brain was a degenerative disease that could be infectious. It was following this through and pursuing it to a precursor on chromosome 20 which is truncated by proteolysis
46:20
and finally deposited in massive plaques that led us to realize that the model of these plaques in animals was not a good one for aging and instead we decided to do the whole thing over again for non-transmissible dementia, namely the amyloid of aging and Alzheimer's disease.
46:40
But here is the disappearance of Kuru with the cessation of ritual cannibalism so we have almost none left and all that we have left are an aged people with 30 years incubation and here is the disappearance of Kuru in three-year cohorts or four-year cohorts
47:01
by age under 10, under 20, the 20-year-olds, 30-year-olds with first disappearance of the child disease and now all cases now are in people over 35. The loss of the disease in children, the loss of it in adolescents, the loss of it in adults
47:21
and this transmissible amyloidosis is different but since we can control it in the laboratory, since we can make this precursor which is conserved between Drosophila lobster and man, convert into amyloid plaques at will in the mouse or the hamster, the biochemical elucidation
47:41
of the post-translational modification of the transmembrane protein will probably be a good model for us to pursue to find out a little more about how a similar process is happening in aging and Alzheimer's since we have no model in the laboratory and our transgenic mice given two and four doses
48:02
of the human amyloid gene are still not developing lesions and so we are not likely to biopsy your brains or anyone else's serially to see how the post-translational processes arrived at and we have a model in a more complicated situation of Scrapi and Kuru in the laboratory.
48:22
We know it's not a perfect model and it's on a different chromosome but it's easy to manipulate and so we're back to using the unconventional viruses as models and it was pursuing that model that led us to the discovery that our aging brain is a phenomenon
48:40
of a protein on chromosome 21 even in the mouse where Down syndrome is on chromosome 16. This is on chromosome 16 and we're looking with Seymour Benzer to find where it is in Drosophila because Kurt Stern from the old Kaiser Wilhelm Anstauten spent the 1940s,
49:01
the warriors working on microduplication of hot spots on the genome of Drosophila and if we find that the superoxide dismutase, ETS2 and Alzheimer beta protein gene are all together there, we will have pretty much the answer for what's going on and perhaps some way to get at the environmental matters
49:22
which cause the microduplication in gametogenesis. I thank you.