Glutamine Repeats and Inherited Neurodegenerative Diseases
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Lindau Nobel Laureate Meetings239 / 340
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Transcript: English(auto-generated)
00:22
Well, thank you for your warm reception, and I should like to thank, like other speakers, Countess Sonia Bernadotte and the Curatorium for inviting me, but also the ladies of the staff, Fra Shielin, who had to write me at least a dozen letters to organize
00:42
it all, and the very kind ladies who look after us here with motherly care and make sure that every wish, every little need of ours is immediately fulfilled. I think they contribute very much to the success of this meeting, so very many thanks.
01:04
Now, in 1949, Linus Pauling, Itano and Wells published a sensational paper in Science. The title was Sickle Cell Anemia or Molecular Disease. The paper was about
01:31
inherited blood disease, which afflicts mainly blacks, sickle cell anemia, and they showed
01:43
that this was caused by a change in the electric charge of the hemoglobin molecule. Sicklers, sufferers from the disease, had a hemoglobin which contained two fewer negative charges
02:06
than normal hemoglobin. They established this by a new technique, which Arne Tiselius had invented in Uppsala, electrophoresis, and for which he had gained a Nobel Prize.
02:22
They didn't have such a machine. In order to investigate the problem, Pauling got his collaborators to build the first Tiselius machine in Pasadena, and with that, they made the discovery. Now, in Cambridge, I was working on hemoglobin. I was very excited
02:44
for this discovery, and then was then joined by a young biochemist, Vernon Ingram, who decided to have a go at this problem and see what the change in electric charge is really due to. And after a few years' work, he discovered that it was due to the
03:08
replacement of a single pair of the 574 amino acids in the hemoglobin molecule, but a different pair, a single pair of glutamic acids. A single pair of, yes, a single
03:25
pair of glutamic acids was replaced by a pair of valines, and that meant, you have to realize, that meant the replacement of two oxygen atoms by, yes, two carbon atoms led to a
03:45
lethal disease, an extraordinary discovery at that time. Imagine this enormous molecule containing 10,000 atoms, and you replace two of these atoms, and that leads to a fatal disease, fatal because this change made the hemoglobin crystallize inside the red
04:06
cells, and made the red cells so rigid that they get stuck in the capillaries and clogged up the circulation. Now, with Vernon Ingram, this was the first time that
04:21
the cause of a genetic mutation was discovered. You see, we had no idea before what a genetic mutation is. We then realized the genetic mutation causes the replacement of an amino acid, and, of course, this posed in acute form the question of the genetic code, which
04:44
was still unknown then, and was solved a few years later by Crick and Brenner. So the discovery gave rise to a huge, enormous amount of research, and since then, hundreds if not thousands of different amino acid replacements in proteins have been found,
05:06
which may or may not give rise to diseases, and, of course, they are all due to faults in the DNA, which occur in replication. Now, at that time, Ingram had to analyze the
05:24
amino acid sequence of the protein, but now, thanks to the recombinant DNA technology, this is no longer necessary, and you just sequence the DNA of the gene, and that's
05:41
much simpler, and you discover the cause of genetic mutations much faster, but there was one severe inherited disease which resisted all attempts to find its cause, and that is Huntington's disease, Sankfeitstanz of Deutsch, and a severe, dominantly inherited
06:06
neurodegenerative late onset disease, which first manifests itself by uncontrolled movements, mood disturbances, then leads to dementia, and finally to death. It's a terrible disease,
06:27
which afflicts people in middle age. Its frequency is about 4 in 100,000 among European populations, and a few years ago, its cause was totally unknown. Then there was a tremendous
06:44
collaborative effort of 61 molecular biologists, biochemists, geneticists, medical people by 61 scientists in eight different universities in the United States and Britain. The gene
07:04
was discovered and was published in a great paper in Cell, signed by the Huntington Disease Collaborative Research Group, and they found that the gene stretched over a tremendous
07:24
length of DNA. It covered 67 exons, about which Gilbert talked yesterday. Sixty-seven exons spread over 180 kilobases of DNA, and the gene codes for one of the
07:45
largest known proteins, a protein of over 3,140 amino acids in a single chain. So about, what is it, six times larger than the number of amino acids in a single chain.
08:04
Than hemoglobin. Now, so they found the gene. So what was the difference between the gene of normal people, the gene of the patients of Huntington's disease? It was in the
08:26
lengths of a repetition of codons, in the length of a repetition of codons, in the normal people, starting at the codon number 18, there was, is a long repeat of only CAG.
08:45
So it goes CAG, CAG, CAG, CAG, but anything from about 10 to 35 of these, and in the sickle cell, in the Huntington's disease patients, this was extended. So here you
09:10
see a slide of the base sequence of the DNA, and written underneath the amino acid
09:24
sequence in the protein, in the single letter code, which you may not all be familiar with, but never mind. So there are 17 mixed amino acids, and then here the amino acid is 18,
09:42
you see QQQQ, and Q stands for glutamine. So there's a long stretch of only glutamines coded for by this repetition of CAG, CAG, CAG, and then there's a sequence of prolines, and then the amino acid sequence continues in that large protein, the sequence shows
10:06
no homology with any known protein. So it gives you no clue whatever, what its function might be. But the amazing discovery was that the only difference between the gene
10:24
in the normal people and those in the patients consisted in the lengths of the CAG repeat up to about 37 repeats. People remain healthy, and with more than 40, they get the disease.
10:42
And the longer the repeat, the earlier the disease sets in, and the more severe the disease. So you see here we had a completely new cause of a genetic disease, not an amino
11:02
acid replacement or, as often happens in recessive diseases, the deletion of a gene or the putting out of action of a gene, but an elongation of a repeat of a single amino
11:20
acid. Now, you know, what does it mean? You know, I am known as the hemoglobin man, and yes, I haven't said that this paper, excuse me, this paper appeared in Cell in March 1993. So I am known as the hemoglobin man, and a little before that, I worked on
11:48
an abstruse hemoglobin, the hemoglobin of a parasitic worm, Ascaris, which interested me because it has a very high oxygen affinity. A group in Antwerp determined its
12:03
amino acid sequence and found it is made up of eight subunits, and each of these subunits, there was a peculiar sequence of a kind that I hadn't seen before, which meant that
12:21
along a strand, a straight strand of polypeptide chain, there would be an alternation of positive and negative charges. So on one side, it would be plus minus, plus minus, plus minus, and on the other side, also plus minus, plus minus, and I realized this
12:41
was a polar zipper. So then I wondered what other, there must be some other proteins that contain polar zipper, and because of that, I came across some proteins in Drosophila, in the fruit flower, which Vishal will speak later today, and there I found several proteins
13:08
which had long repeats of only glutamine, and so I wondered, what does this mean? And just out of idle curiosity, I built an atomic model of a polyglutamine chain,
13:25
and I brought this along for you. I'm afraid it will be hard to see from the back, but people at the front at least will see it. You see, what I've got here is two polypeptide chains marked out by these white strands, and here on the right and left are the
13:48
glutamine side chains, and what I found was that if you have two chains on the glutamine sides, you can tie them together by hydrogen bonds between the main chain amides, which
14:02
are along here, and also on each side by the side chain amides, so that such a chain of only glutamines also acts as a polar zipper, and I wrote a little paper about polar zipper, and polar zippers sent this in, and this was impressive. I thought that
14:27
was the end of the story, just the curiosity, you know, and then I read this paper in Cell, and it suddenly occurred to me that my observation of my model might possibly
14:43
provide a clue to the molecular mechanism of the disease, and I got very excited about this. I mean, nobody would pay any attention if I just say I built a molecular model and look, this is what it would be like. We had to do some experiments, so I asked
15:05
a chemist in our laboratory to make me a synthetic polyglutamine, so he made a chain of 15 glutamines in a row, but this would have been insoluble, so he put two aspartic acid
15:26
residues, one end and two lysines at the other end. They are electrically charged, so they made this soluble, and then we looked at a solution of this 15, with a spectroscopic
15:47
method, circular dichroism, which actually tells you what kind of form such a chain would take, whether it forms straight chains linked together like this, or whether it
16:01
forms helical chains, depending on the shape of the chain, you get different spectra, and they've all been characterized and are well known. Now, when we looked at the spectra of this solution, we found that indeed the chains have the form that my model predicted,
16:24
it forms straight chains, which tend to tie together like that, and then we found that in acid solution at pH 2, when neutralization at pH 7, this polymer slowly precipitated
16:45
in the form of tiny little worms, and an x-ray picture of these worms showed that indeed the protein has this structure, but the chains don't run along the lengths
17:00
of the worm, they're wrapped around the lengths of it like that. Right. Now, we have a look at the next slide. Here, then, I have an atomic model of this model, sorry, a computer
17:22
drawing of this model, which will help those of you who are at the back and couldn't see this very well, so you see here you have the, yes, here you have these straight chains, you see four of four such chains of glutamines, and the dotted lines show
17:46
the hydrogen bonds which hold them together, so here would be CO on this side and NH on the other side, and the CO combines with the NH to form a hydrogen bond, which has
18:01
a strength of between three and five kilocalories, and these bonds, you see, hold the chain together, and then you must imagine sticking out from the screen on one side would be these side chains, and they also form hydrogen bonds, which you see there, and going back
18:24
into the screen would be another lot, and they again form hydrogen bonds like that. So that was the predicted structure, and the chains you see written here are 4.8 Angstroms
18:41
apart, which is important because that's the signature of this structure, and now, having got so far, I decided I could stick my neck out and published a paper in the Proceedings of the National Academy suggesting, making this suggestion, extension of the glutamine
19:06
repeats may cause the affected proteins to agglomerate and precipitate in neurons, symptoms may set in when these precipitates have reached a critical size or have resulted
19:22
in a critical number of neural blocks. Well, I made this suggestion, but there was not the slightest evidence in support because people, of course, had cut-scene sections of the brains of the patients who died from the disease and had examined them with immunostaining,
19:46
so they made antibodies against the hunting proteins, and they stained these sections with the antibodies, and they found that the protein was in isolated dots in the
20:05
evidence of any aggregates whatsoever. So I thought maybe this is all nonsense, but then in August 1997, suddenly there was a complete turnaround. Gillian Bates at Guy's Hospital
20:30
in London had succeeded in producing the disease in mice. She had made transgenic mice. She introduced a fraction of the human gene into the fertilized eggs of mice, and she introduced
20:56
two kinds of genes. That is, first of all, let me explain. She didn't introduce the
21:04
whole gene, but only the first axon, and the first axon codes for the glutamine repeat, for the protein repeat, and for some of the adjoining acids, amino acids. So she introduced this gene, and she introduced it with 18 CAGs and with about a hundred
21:25
and fifty CAGs, and she managed to breed these mice, and to her astonishment, the mice, transgenic mice with the hundred and fifty glutamines, began to show the symptoms
21:44
of the human disease. They first showed the uncontrolled movements and seizures and the general loss of weight. They didn't develop as well as normal mice, and they died prematurely.
22:06
On the other hand, the transgenic mice with the protein with only 18 glutamines remained healthy and showed no abnormal symptoms, whatever. So this was a terrific discovery
22:21
because this was the first time that the human disease had been reproduced in an animal which gave one hope that one might possibly find the treatment. Now, she handed these mice to a lecturer in anatomy at University College London, Stephen Davies, and he cut
22:43
thin sections through the brains of these mice, and one morning, he came up with a question. He came to see me, and he burst into my room, and he was so excited that he began to tell me his results before he had even shut the door because he had
23:06
found in the brains of these mice, the next slide, please, there we are, yes, he found that in the cell nuclei of these mice, he found aggregates of protein which stained
23:36
with an antibody against this N-terminal peptide of the hunting protein. So clearly
23:45
they're composed of it, and it consisted of fibers and little granules. So he found what he called the intranuclear inclusions in the neurons of the cortex and the striatum
24:06
in the brain of these mice, and he also found stains in the nuclear pores of the neurons, and in neurons, these are sort of little extension of the nerve fibers,
24:26
which extend in different directions. So he was terribly excited about this as he may well have been, because this suggested that maybe there was something
24:42
in this prediction that I'd made. Now, his discovery stimulated people at the Harvard Medical School, Marion Defilia and Neil Aronin, to look again at the sections of the human
25:00
patients, and they found that these nuclear inclusions were there and that they had been overlooked. In fact, a paper appeared in Advances in Neurology in 1979, so many years earlier, by another group of Americans who showed that in the cell nuclei of hunting
25:29
patients, there was some protein which they could stain with urinal acetate, and they found that this sort of structure, but they didn't have any immunology, of course
25:44
the gene wasn't known, the protein wasn't known, they didn't know what the protein was made of, but the paper was forgotten, and because, you know, it was a discovery before its proper time, and of course afterwards the people remembered that it had been there.
26:11
So now, yes, the next slide shows you this human nuclear inclusion, which Defilia
26:24
and her associates had discovered, and again, you see, if you look closely, you find that it's a mixture of granules and little fibers. Now, since that time, seven other
26:43
neurodegenerative diseases have been discovered, all due to extension of glutamine repeats in different proteins. They give rise to neurodegeneration of different neurons.
27:01
For instance, there's one Kennedy disease which is due to an extension of glutamine repeat in the receptor, in the androgen receptor, and which affects motor neurons. Well, Huntington disease affects neurons in the cerebral cortex and the cerebral striatum.
27:32
These proteins have nothing in common, but there's one feature, an astonishing feature, which they all have in common, in all but one of the cases, repeats with fewer
27:47
than 37 glutamines are harmless, the people remain healthy, repeats with more than 40 glutamines produce disease. So this means that the extension of the glutamine repeats
28:02
must be associated, must cause a change in structure of this repeat, and that change of structure starts the process, which finally leads to aggregation. Now, what seems to happen is that the first stage seems to be that it makes the protein
28:28
susceptible to proteolytic attack. Defilia also used immunological methods to find out what's there, and she tested this with an antibody against the first 17 amino acids
28:48
of the Huntington protein, and this aggregate was stained with it. And then she tried an antibody against a peptide about a sixth of the way along, around an amino acid 5
29:07
hundred, and it failed to stain it, suggesting, as later confirmed, that only a fragment of this large protein gets into the nucleus, and the fragment is associated with ubiquitin,
29:27
which is a signaling protein that attaches itself to proteins that are unstable, that are in the process of unfolding, and prepares them for digestions where other enzymes in
29:47
the cell, which then split it into individual amino acids. So this protein, also the mouse protein, stains with antibodies against ubiquitin. It also stains with antibodies
30:02
against chaperones and the proteasomes, so complexes that digest proteins, and probably various other proteins may be associated with it. Meanwhile, Jillian Bates stimulated a group at the Max Planck Institute for Molecular
30:31
Problem in a different way. Erich Wanker and Scherzinger and their colleagues introduced
30:42
this same exome into colibacterium, and they actually manufactured this protein in colibacterium by recombinant DNA technology. Now, they, in fact, developed this protein
31:01
and made it with a varying number of CAGs, so a varying number of glutamines, and with 20, 30, 51, 83, and 122 glutamines. And what did they find? They found that the
31:27
protein with 20 and 30 glutamines remains soluble, but the protein with longer glutamines repeats aggregated and formed precipitate states. So there is a precise correlation
31:46
between the lengths of the glutamine repeat that causes the disease, and the lengths of glutamine repeat that causes the protein to precipitate when you make it in vitro.
32:02
But now other questions arose. The Detlof at the University of Alabama asked himself if any protein with long glutamine repeats would produce neurological symptoms. So he had the ingenious idea of taking a protein, which normally occurs in brain, hypoxanthine
32:34
phosphoribosyltransferase, a terrible mouseful, but it's not such a big protein.
32:43
So to this protein, he attached a long glutamine repeat, a repeat of 146 glutamine repeat, and then he made mice transgenic for this protein, and indeed, the mice developed seizures,
33:07
behaved abnormally, had shortened life spans, and when he sectioned their brains, he found in the pyramidal layer of the cerebral cortex about a third of the cells developed
33:26
the same kind of nuclear inclusions. So showing, you see, really that it doesn't matter what sort of a protein. Any protein that is expressed in brain with a long glutamine
33:42
repeat will produce these symptoms, produce neurodegeneration, and these aggregates. Nancy Bonini at the University of Pennsylvania in Philadelphia had another ingenious idea. Why not introduce such a protein into the geneticist's favorite pet, into drosophila,
34:08
the fruit fly? So she did this. She took, not hunting disease, but another one of these spinocerebellar taxa proteins and introduced a fragment of this into drosophila,
34:25
and the great thing about the drosophila is that so much is known that you can actually target this gene for a particular organ. So she targeted it to the eyes, and promptly
34:41
the eyes began to show malformations, nuclear inclusions, late onset degeneration, and cell loss. Well, this is not only interesting, so that you sort of ask yourself, so what,
35:05
but of course it's marvelous because the drosophila would be the ideal animal for testing possible therapies of the disease. Yes, what I mean by that is that there
35:24
are, what about possible therapies? The various approaches have been sort of, but you might be interested that the group in Berlin at the Max Planck Institute for Molecular Genetics,
35:43
Wanker persuaded Merck to put at his disposal their entire library of 160,000 organic compounds, and he built a robotic machine with which he can test whether any of these compounds
36:03
inhibit the aggregation of the protein made in cola bacteria, so that he can test the hundreds if not thousands of such compounds very quickly within a few days. And when
36:21
I saw him in April in Berlin, he had actually got one compound which did show such an inhibitory function, so that there's hope that something might be found which prevents this kind
36:40
of aggregation. There's one other very interesting and exciting feature about the discovery, and that it has brought a remarkable unity into neurodegenerative diseases because
37:03
Alzheimer's disease is due to proteins precipitating in the form of neurofibrillar tangents, not actually within the neurons but between the neurons, and Parkinson's disease is due to aggregation of another protein, synuclein, into what are called levibodies
37:30
after the German medical man who discovered them earlier in the century. So they are little
37:40
balls of this protein forming in this nuclei. And then, you know, you heard a lot about prion proteins, Crotchfeld-Jakob disease and bovine spongiform encephalitis, BSE, recently,
38:00
and they are due to the aggregation of prion proteins in a mysterious manner which we do not understand. So, you see, each of these diseases is due to precipitation of proteins which makes me think that most, if possibly, all neurodegenerative diseases
38:24
are due to protein precipitation, as indeed are many other diseases. You know, the one sickle cell disease with which I started this talk is due to precipitation of hemoglobin in the red cells. So the next, the great problem which faces us in the next century
38:50
is to find ways and means of preventing this aggregation. And I think this is one
39:01
of the great challenges to you, the young people who are attending this meeting. Thank you very much.