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So welcome Maria Grazia. It's a pleasure and a personal honor to have you on the podcast. I would like to start by asking you to give us [00:01:00] a historical perspective of how you, and of course, your team came to the discovery that alpha synuclein is present in Lewy bodies.
[00:01:08] Dr. Maria Grazia Spillantini: Okay. Yeah, that is a bit of history. So in the early 1990s, we were working with Michel Goedert and try to identify the post translational modification of tau protein in the neurofibrillary tangles of Alzheimer's disease. And one approach was to raise antibodies against brain extract from Alzheimer's patients and, so in controls.
But at some point we found that one antibody that we had raised did not recognize only tau, but also two smaller bands of a different molecular weight and so we became interested in trying to determine what this small band that were about 15 kilodalton, 14 kilodalton, what they were. And so this started as a side project in the lab with Ross Jakes that was a [00:02:00] technician in Michel's lab.
And at that time I was doing my PhD at the very beginning. Then I was a postdoc and finally I was a lecturer at the university during the study of alpha synuclein, I would say. And what we found that these two bands were homologous. And when we sequence them, we decide also to produce antibody that recognized their amino terminal region, the carboxy terminal region, the middle region, and then also that they could distinguish between the two.
And then Michel went to a meeting in Japan and Saito presenting the cDNA sequence of a protein that he had called plaque component. And this protein was identical to one of the two proteins that we had found from the strata from human brain. So we were surprised because we never saw staining of amyloid plaques and we had all these antibodies. So [00:03:00] just, the other day while I was tidying up my office, I found a fax that I had exchanged with Saito to say that we didn't find this protein in plaques. And so he sent me his antibody to test and he was right. His antibody was staining plaques, but not our antibodies.
So this work we continue and then Michel saw that this sequence of these two protein was homologous to one described in Torpedo California by Marto, and Shela in 1988, and they had called this protein synuclein because in the fish it was distributed both in the nucleus and in the synapses.
And so seeing the homologies between these two proteins, we decide to call them alpha and beta synuclein. And while the one that identifies as alpha synuclein was the same plaque [00:04:00] component of Seito. The other was similar to the phosphoneuro protein 14 extracted from bovine brain. So it had never been looked at in a human brain.
And so the name alpha beta synuclein started from that point. And at that point, I also determined the chromosomal localization of the genes encoding these two proteins. And I remember that we sent to genomics. We sent the data and the editor asked us to give a name to the two genes. And so we called them SNCA and SNCB to distinguish the two. And so when we continue the work, but as I said, was a site project, although after Seito published the plaque component we became much more interested and we found that alpha synuclein was really really very abundant in the brain as described, and it was mainly localized both [00:05:00] beta and alpha in synaptic terminal.
And so that is how we end up to identify alpha beta synuclein to name them and then also the genes encoding for them. And we had many antibodies. And we knew quite a lot about this protein, so much that one of them alpha synuclein was very, very good in every experiment. immunoprecipitation also immunogenicity to this antibody.
And so Ross Jakes decide to call the protein that then we name alpha synuclein, perfecting, because any experiment you are doing seemed to work. And the other was, imperfect, because beta synuclein wasn't that good. And so that is how the alpha beta came up and how we knew the, chromosomal localization.
And then in 1996, the linkage was found of the first family with Hereditary Parkinson's disease to exactly the location where the alpha synuclein [00:06:00] was.
I tried to find some DNA, just out of a curiosity, but we were not geneticist. We had no DNA, we had no connections, and so we had to wait. And then the following year we had that a mutation actually had been found in the alpha synuclein gene. So we had all the tools. We had the antibody, we had the knowledge of the protein, we knew how they look like in the brain, but we were mainly working Alzheimer and the front temporal dementia and so on.
And so I went to the brain bank in Cambridge and I asked for some Parkinson brains. And I also was looking for Dementia with Lewy body brains to see if this lewy body that were characteristic of Parkinson's disease, neuro pathologically, if they contain this protein. But these were not familial cases.
They were all sporadic Parkinson's disease cases. And so I used the antibody that I had and initially stained in some Parkinson's brain. And it was [00:07:00] wonderful the first time I saw lewy body in the microscope, late in the night and then actually it is the one that is also in the paper because I went and I called everybody that was around to show to them in the microscope because it is really impressive to look at this structure in the microscope. The problem was that we did not have any dementia with lewy body in th e brain bank here. They didn't have it, and so Michel was collaborating with John Trojanowski and Tao. And he asked them for some dementia with lewy body tissue.
And so John sent us some sections of four cases of dementia with lewy, and I still have the box because I never threw away anything with this writing and saying the case. And so we found that actually there was a lot of synuclein stain in these cases and both from the sections that John sent with dementia with lewy Body and the one that had Parkinson that we had here.
And so from that moment it was beautiful to see that we found Lewy body. [00:08:00] What we now know is a lewy body in Alzheimer brain. This big blob that I thought maybe was golgi, maybe it was endoplasmic reticulum. So I showed to several neuropathologies. Asking, what is this? Because I've never seen a lewy everybody in Alzheimer.
They're not round, nicely shaped like in the substantial nigra. And so that was the first encounter of synuclein with lewy body. But then we had really to show that it had any relevance because there are many protein that are stuck in the Lewy body.
And so we had to extract the filament and we extract from dementia with Lewy body patient brain, and then also from a multiple system atrophy because they are similar in type of inclusions, although they were not lewy body but glial cells. And then we tried to reconstruct the filament use recombinant alpha synuclein. And so just by shaking like many people do now, this recombinant protein and we [00:09:00] could see that it was by nanogold labeling identical to the one that we instructed from the filament.
And that indicated that really the alpha synuclein was the component of these filaments in the lewy body. And so that is what we published earlier.
[00:09:16] Dr. Tiago Outeiro: That's history. Wow, that's really interesting. It shows how it's important to be attentive to details and, how you and Michel connected all the information that you are reading about. So that's really interesting. And I want to pick up on something you just mentioned.
Usually we read this in all papers that alpha synuclein is the major component of Lewy bodies. Do we know what percentage does alpha synuclein representing Lewy bodies, when you did your extractions? Could you estimate we know there's a lot of other proteins, but what is the percentage of synuclein in Lewy body?
[00:09:49] Dr. Maria Grazia Spillantini: Well, we have never really measured the percentage per se, because you have to dissect the lewy body to know that, and the fact is there is an evolution in the lewy body formation. So you have pale [00:10:00] bodies that are much looser. They have less filaments, they have more organelles around, and then they become more and more compact.
There are some reports saying that about 90% of the content of lewy body is alpha synuclein. Others say that the tissue can vary between nanograms to microgram and so I don't know exactly because it depends on the area of the brain, the number of lewy body, and then really dissecting them. But what I would like to say is that I don't think that major is really related to content.
I think to amount. I would say that is perhaps better to say main because what we meant when we say this is because the filament that formed the synuclein are the main component that form the skeleton of the lewy body. And then when you have this structure in the cytoplasm can be beside the filament. Also, granular alpha synuclein, oligomers, and so on, [00:11:00] many other protein remain trapped on it.
And so you have a lot of chaperone protein, a lot of enzymes. You have a cytoskeletal protein. Some people say that there are hundred and fifty to hundred protein identified as attached or that come out if you struck the lewy body. But if there are not the filaments of alpha synuclein, you don't have this sort of compact aggregation of other protein that remains stuck there.
And so I think that the meaning is mainly main component because without the filaments of synuclein, you don't have the lewy body. And then on the other end, there is also something else. The lewy body are one aspect of it, because there are the small synaptic aggregates that in my view, they're really very important.
So the lewy body is something that disrupt the soma of the neuron. Disrupted transport, but the synaptic aggregates that are not as big as the lewy body, they [00:12:00] really disrupt the function of the synapse and therefore the dysfunction in dopamine release and so on. And so, yeah, I think one has to look at that in the context, the lewy body are important. But there are also these other aggregates of synuclein.
[00:12:15] Dr. Tiago Outeiro: You're leaving me a good lead here because I wanted to go after this and I wanted to ask you we read so many things these days. There are so many opinions and people are actually questioned whether Lewy body pathology is relevant and whether Lewy body are the culprits or even whether the toxicity that we seem to believe is taking place in the presence of alpha synuclein is because of the aggregates, or is it because its function is lost when alpha synuclein aggregates in Lewy bodies or in those smaller aggregates that you mentioned. What do you think is more relevant?
[00:12:51] Dr. Maria Grazia Spillantini: I think that, for example, this is my personal view, but based also on the results we have had in mice, the synaptic aggregates [00:13:00] are the one that disrupt the function into the initial dopamine release dysfunction and so on. And there's the beginning of the aggregation and the synuclein also start to aggregate in the soma.
We don't know if it is protective or not at the beginning, but when you have a big lewy body disrupt completely the transport, the trafficking in the soma and certainly will become toxic, but I don't know if it is a loss of function at the synapse, that is due to this aggregation. So you have the SINAPS, again, of toxic function due to the aggregates, but also you have a normal loss of function because synuclein is involved in movement of the vesicles containing the neurotransmitters. And so, for example, in experiments that we have done in our transgenic mice where we have aggregation of alphas synuclein, both in the cell body and in the synapse, what we find is that the dysfunction at the synapse seems to proceed the dysfunction in the cell body. [00:14:00] So the loss of dopamine release in the striatum comes earlier than the neuronal death of dopaminergic cells in the cell body. And when we use oligo modifier or chaperone protein to disrupt these small aggregates that we know colocalize with nerve proteins and therefore, is a full disruption of the system in the presynaptic terminals and could be postsynaptic.
We have studied many presynaptic. Then we find that there is a huge increase of monomeric synuclein, and that is the question, is there are less aggregates. The aggregates become smaller, and there is also this increase of monomeric alpha synuclein in the same place. So what is relevant. Is the increase in the monomeric synuclein or it is the decrease of the aggregate.
And I think this is an important question that we have to try to answer to understand. Because for example, many people, they are trying to inhibit [00:15:00] alpha synuclein expression, reduce the alpha synuclein to avoid the formation of aggregates. But what if the aggregates continue to form and you just reduce the normal synuclein that could still be functional? And so I don't know if at this stage we know for sure that in this condition, because reducing alpha synuclein in normal brain, nothing happens. There are a lot of mice that do not have alpha synuclein and knockout or spontaneous mutation, but in the context of the aggregation will be very important to find out.
I'm sure that probably the aggregates are the culprit. They are the toxic gain of function. But we shouldn't forget also the monomeric synuclein.
[00:15:47] Dr. Tiago Outeiro: Absolutely. You raised important question, so is there any other major open question in the field you think it's important to highlight? Because maybe we have more junior researchers [00:16:00] that are entering the field and maybe they get from you some ideas for what they should really focus on.
[00:16:05] Dr. Maria Grazia Spillantini: Yeah, I think there are so many things to do yet, it's curiosity that drives, so like this for example, what is that makes the synuclein start to aggregate. Well, what is the trigger because it could be the synapse like we think because we see the dysfunction there, but you have also aggregates of synuclein, oligodendrocytes, where it comes from.
And then now the cryo EM work that has been done has shown that clearly there is a difference in the shape of the aggregates coming from Parkinson and dementia with lewy body and multiple system atrophy. And we know that they are in different cell types, so you can have the same aggregates in different cell types.
So what is the triggers? Because you probably need to have a common denominator that trigger the aggregation, but then there are factors in the different cell type that induce a different [00:17:00] susceptibility. And so I think that these are really important question because you should know. The other important question, what does synuclein do?
If you read the literature now, there is everything. You can find any sort of function for synuclein, but in the brain, besides being involved in binding synaptic vesicles or lipid membranes, in particular, or for example, in plasticity, we know that is involved in plasticity through the work that has been done in birds.
And then there was this report of parrots that they had aggregates of alpha synuclein. But what is important is what is the real function? Why do we have so much? Because it is almost 1% of the total protein content of the brain.
And that I think will be really important to find out. But also what induced the aggregation that is the two things that I think would be important to focus on.
[00:17:56] Dr. Tiago Outeiro: Great. Well, Maria Grazia, thank you so much. It has been [00:18:00] an honor having you on the podcast and discussing all of these ideas and hearing from you firsthand how you made that initial discovery that started whole field. So thank you so much for your time. It was a really a pleasure. And I look forward to talking to you in some other occasion about some of your exciting work that you're doing.
[00:18:20] Dr. Maria Grazia Spillantini: Thank you very much Tiago. Thank you for having me. Thank you.
[00:18:24] Dr. Tiago Outeiro: Thank you. So we have just interviewed Dr. Maria Grazia Spillantini for the History of Movement Disorder podcast series. Thank you all for listening and join us in our upcoming podcasts. Bye.