Podcast – Molecular Basis of Alzheimer’s Disease

Voice Over:

Welcome to the Dementia Researcher podcast, brought to you by dementiaresearcher.nihr.ac.uk, a network for early career researchers.

Megan Calvert-O’Hare:

Hello and welcome to another podcast brought to you by the NIHR Dementia Researcher website. My name is Megan O’Hare, and today I’m delighted to be joined by two biochemistry scientists from the University of Sussex, who have travelled up from Brighton today to discuss their research into some of the fundamental sciences behind the causes of Alzheimer’s disease. Before we start, we know a lot of people listen to our podcast, but don’t necessarily use our website. It is a very good website, so I really would encourage you to go and have a look at it. You can sign up and then you get a short weekly news roundup email every Friday. And we add new content every day from blogs discussing people’s research and their careers, a full dementia and research events planner, details of all upcoming funding opportunities and lots more. So head on over there after you listen to this podcast and register.

Back to today’s topic, I would like to welcome Louise Serpell, Professor of Biochemistry and Director of Science, Sussex Neuroscience. And Karen Marshall, research scientist at the University of Sussex. Hello.

Dr Louise Serpell:

Hello.

Dr Karen Marshall:

Hello.

Megan Calvert-O’Hare:

And a warm welcome to both of you. As I mentioned in the intro, you have both travelled up from Brighton today and before the podcast, I was saying we recently recorded another podcast down in Brighton, and I made a joke about seagulls and it was a really good joke. Anyway, preparing for this podcast this weekend, I was trying to entertain my three year old son while his pasta cooked. And all I could think to entertain him was pictures of seagulls. So I’ve just had them on the brain since then. Now seeing you guys has made me think of seagulls again. Anyway, so should we start with a quick round table and you can introduce yourselves, your job titles, bit about your background and also who funds you. Karen?

Dr Karen Marshall:

Hi. Yes. My name is Karen Marshall. I’m a postdoc in Louise’s lab and I’ve worked on this subject of protein misfolding since I started my PhD in 2006. I was doing more of a biophysical project then, looking at the structure of amyloid fibrils. I then went to the U.S., where I worked on prion diseases like mad cow disease and CJD. Worked more with cell culture model systems, and then came back to the UK to work with Louise again in 2013, because we work well together.

Megan Calvert-O’Hare:

You work well together and also I believe you sing well together. Is that right?

Dr Karen Marshall:

Yes.

Dr Louise Serpell:

We sing beautifully.

Megan Calvert-O’Hare:

There’s a love of karaoke, a shared love of karaoke.

Dr Karen Marshall:

Yeah. Yeah. It’s quite a lovely thing. Yes. Then I was working more on Alzheimer’s focused projects, trying to understand mechanisms of misfolded protein induced cell death, and I’m still working on that.

Megan Calvert-O’Hare:

Great. And Louise?

Dr Louise Serpell:

So I’m a professor of biochemistry, but sort of part of neuroscience now. And I started off really trying to understand the structure of amyloid fibrils. And so proteins that misfold and form amyloid can be lots of different proteins, not necessarily just amyloid beta. And then gradually throughout my career, I’ve looked more towards the causes of Alzheimer’s disease, so more focused on Alzheimer’s disease. And so my lab now work on all the proteins that are associated with Alzheimer’s. So amyloid beta, tau and APOE. So we use structural biology and we use cell biology to try and understand what the causes of Alzheimer’s are.

Megan Calvert-O’Hare:

Okay. And you mentioned amyloid fibrils, and I think a lot of our listeners are basic scientists, but we also have social care scientists. We have a kind of mix. Maybe we could talk a little bit about some definitions of some terms that come up in biochemistry a lot and come up in Alzheimer’s disease. But maybe people don’t know the exact definition. So what is an amyloid fibril, when you use this term?

Dr Louise Serpell:

So I always define amyloid as a misfolded aggregate of normally folded proteins. So amyloid beta is well known for forming amyloid fibres and it forms in amyloid plaques in Alzheimer’s disease. But actually, there are at least 30 or 40 different proteins that can all form amyloid. One example of it is a prion that’s found in CGD and mad cow disease. Other examples are things like in diabetes type two, there’s a protein called islet amyloid polypeptide, and that will form amyloid fibrils that deposit in the pancreas. And so this is actually quite a reasonably common feature of proteins, that they can misfold, aggregate and cause a disease, or at least be associated with a disease.

Megan Calvert-O’Hare:

So you said there are a number of proteins that do this. Do they share any sequence in common? Could you predict a protein would eventually form amyloid?

Dr Louise Serpell:

Not really. The sequences are really different from one another. The proteins themselves have very different structures. There are some algorithms that can help you sort of to predict whether something’s likely to form amyloid or not. But some people have suggested that it’s a sort of fundamental property of proteins that they can unfold and then refold into amyloid fibres. They’re really, really stable so it’s sort of energetically favourable that they would form an amyloid fibril. And in a way, it’s more interesting to think about why they don’t do that and why we have so many mechanisms that can prevent protein misfolding. Most of the time proteins don’t misfold. Most of the time, we don’t get these diseases.

Megan Calvert-O’Hare:

Okay. And Karen, you said you worked on prion disease and then Louise mentioned that amyloid occurs in prion disease as well. Were you working on that, was that in the States, did you say?

Dr Karen Marshall:

Yes, yes. So we were using a cell line to look at whether amyloid forms of the prion protein were infectious. So prion diseases are the only diseases in which amyloid forms that have actually shown to be infectious, as we all know, from people eating infected cow meat. Then some people went on to develop variant CJD.

Megan Calvert-O’Hare:

When you say infectious in that instance, you mean it’s been passed on to you?

Dr Karen Marshall:

Yeah and crossed a species barrier as well in that instance as well. So the whole issue of infectivity in other diseases is quite controversial maybe, and not really very well defined. But with prion diseases, there is a very strong infectious component. So in those studies, we were more interested in what was different about those to other diseases where amyloid forms. So we still see the same protein structures, amyloid. But in Alzheimer’s, they’re formed from amyloid beta. In prion diseases, they’re formed from the prion protein, they’re still called amyloid fibres. But why was there this infectivity? So that’s kind of what I was focused on there.

Megan Calvert-O’Hare:

You mentioned the structure. Maybe we could talk a little bit, quite basically about actually, what is the structure of amyloid when you talk about it?

Dr Louise Serpell:

So amyloid fibres are actually really very well-defined. They have, what’s known as a cross beta structure. So they’re a very repetitive structure, which is composed of beta strands. So when I tell students about them, I sort of liken it to the rungs of a ladder where the protein is formed the rungs of the ladder and then aggregated so that all, each one, of those molecules is added on top of one another to form a very, very long ladder. And what’s nice about that structure, in terms of its simplicity, is because it’s so repetitive, it means that you can take all sorts of different lengths of protein and you can access that structure because it’s so simple. And it explains, in a way, why proteins can misfold to form these structures.

Megan Calvert-O’Hare:

This is quite a basic question, but these proteins are intracellular and then the amyloid is intracellular as well, or extracellular.

Dr Louise Serpell:

So amyloid beta plaques are found extracellularly and most amyloid is extracellular.

Megan Calvert-O’Hare:

Okay.

Dr Louise Serpell:

But tau also forms amyloid, and it’s intracellular. There are other examples like alpha-synuclein and Lewy bodies in Parkinson’s disease. And that’s also intracellular. So it seems that these sorts of structures can form both inside cells and also outside cells.

Megan Calvert-O’Hare:

You’ve got several diseases that are associated with amyloid and then you’ve got different proteins. So this obviously leads to a different set of symptoms. Is that correct?

Dr Louise Serpell:

Yes, exactly. So even the same protein can cause lots of different diseases. So for example, tau is associated with Alzheimer’s disease, but it also is involved with tauopathies like PSP and CBD and various other tauopathies. And it seems to aggregate and misfold in different ways that cause the different symptoms of those diseases. So it’s quite complicated. And often, where the protein is misfolded is really important. So for example, like I mentioned in diabetes type two, you have islet amyloid polypeptide forms in the pancreas. And so presumably it’s this region of where it actually is deposited that’s causing that disease.

Megan Calvert-O’Hare:

But that protein is expressed elsewhere in the body. It’s just not forming amyloid anywhere, but in the pancreas is it?

Dr Louise Serpell:

That particular protein is mainly made in the pancreas, along with insulin. So all of these diseases, well they’re often associated with where they are expressed. So there are loads of neurodegenerative diseases like Parkinson’s and Alzheimer’s disease, and so the aggregation happens in the brain. But then there are other diseases where the aggregation might happen in the heart or in the kidneys or other organs of the body.

Megan Calvert-O’Hare:

And obviously you say the brain, but there are many different parts of the brain.

Dr Louise Serpell:

Yes, yes. So depending on which part of the brain, then that causes the different symptoms.

Megan Calvert-O’Hare:

Yeah. I wonder, this might be a really silly question, but once the amyloid is formed from the different proteins, obviously the proteins had a function before, do they retain any residual function within this structure?

Dr Louise Serpell:

That is actually a really good question. Protein misfolding can be associated with a loss of function or a gain of function. But we think that amyloid diseases tend to be associated with a gain of function. So the proteins probably do lose their function, but the main thing that’s associated with this disease tends to be the fact that you’ve got these aggregates and they cause some sort of toxic effects. That’s what we believe.

Megan Calvert-O’Hare:

Toxic effect, not just because you’ve got an accumulation of protein that you can’t get rid of. Toxic because you’ve then got an over-expression essentially or a gain of function. Is that what you mean?

Dr Louise Serpell:

So it’s partly because of the accumulation and then just to add more complication to the whole thing, it seems to be the process of aggregation that may lead to the toxic effects. So for example, in the case of amyloid beta, people talk about the toxic oligomers. So, when the protein starts to self-assemble, it forms these soluble species, which people believe are toxic, before you even see accumulation of amyloid fibrils that are deposited. So just to confuse everybody even more.

Megan Calvert-O’Hare:

Okay. And Karen, what is your actual project within Louise’s lab at the moment?

Dr Karen Marshall:

Okay. So the project I’ve worked on most quite recently is looking at these amyloid beta oligomers. So we’re able to make them synthetically. You can extract them from brains of people with Alzheimer’s disease as well, but they’re much less well defined. So we use a synthetic form that we make in the lab and we’ve characterised those really, really well. So I mean, we can look at them with an electron microscope. We can assess how big they are and use a very kind of controlled population. So we can make amyloid fibrils, but prior to the fibrils forming, they form these small oligomers. And we’ve done lots of different experiments, looking at the toxicity, as have many other groups, of oligomers compared to fibrils. And we see, again as do many other people, the oligomers in cell culture are the most toxic species.

Megan Calvert-O’Hare:

So this is pre-forming the massive aggregates?

Dr Karen Marshall:

Yes. The cells seem to be able to cope in vitro anyway, cope quite well with the fibrils. So the next stage after that was to look at well, what’s the difference? And we noticed, or we did lots of microscopy, looking at fluorescent forms of these. And we can see that the smaller oligomers are able to be internalised into the cells. So they’re endocytosed and they traffic to, so we use rat hippocampal neurons in culture. And the reason we used the hippocampal neurons is because the hippocampus is one of the first regions in the brain to experience neurodegeneration and Alzheimer’s disease, and that’s what leads to the symptoms of memory loss. So these oligomers become internalised and they traffic to a particular compartment of the cell, called the lysosome. And this is a really critical pathway for degradation in the cell. So it’s used all the time by the cell to break down unwanted components that are there, or maybe bacteria that have been engulfed, proteins that are no longer required, and it kind of serves as this recycling unit.

But we see an accumulation of oligomers in these lysosomes and they appear to kind of become enlarged. They don’t seem to be able to really break down the oligomers. So we think that this internalization has led to a disturbance of this whole system, potentially leading to the eventual death of the cell itself.

Megan Calvert-O’Hare:

You said internalization of the oligomers. How actually are they targeted for internalization? The cell obviously recognises that it needs to degrade this. Is that how it works?

Dr Karen Marshall:

Well, Yeah. So there are lots of receptors have been proposed for specific receptors on the cell membrane that bind oligomers, LRP is one I think. Anyway, there’s been quite a few. So we’ve done the same experiments with other misfolded proteins and we see those becoming internalised as well. But there does seem to be something specific about the fact that the amyloid beta is aggregated and misfolded because when we add a similar peptide, that we’ve engineered to not aggregate, so about the same concentration, that doesn’t become internalised. So there’s something about this misfolded structure that is probably binding either to a receptor or just to the lipid bilayer that leads to its internalization. So the cell probably is trying to get rid of it, but it can’t. It’s just become overloaded.

Megan Calvert-O’Hare:

The lysosome’s job is to break down protein. But once it’s engulfed the oligomer, can it continue to form more structure? Can it override? Because obviously lysosomes are quite acidic aren’t they?

Dr Karen Marshall:

Yes.

Megan Calvert-O’Hare:

So they should be breaking down the protein.

Dr Karen Marshall:

Yeah.

Megan Calvert-O’Hare:

But if they’re unable to, is that because you said they were very stable. Is it that they’re so stable and then they can continue to?

Dr Karen Marshall:

Potentially yes. We don’t know quite what the mechanism is of this dysfunction. It could be affecting the acidification of the lysosomes, it could be causing it to burst, perhaps. That’s what we’re looking at, at the moment.

Megan Calvert-O’Hare:

There are, I understand, various assays that you can look at the level of acidification of lysosomes.

Dr Karen Marshall:

Yes. Yeah, so we have done, we’ve used a pH sensitive probe that we’ve tagged to the amyloid beta, and we can see that that still, so it will only fluoresce in acidic compartment. And we do see it is still fluorescent. So we’re thinking perhaps that’s not what’s going on. But yeah, we’re trying some other assays as well at the moment, to look more specifically at the pH of the lysosome when the amyloid beta is there.

Megan Calvert-O’Hare:

And is this sort of also to do with ageing? I ask because my PhD was on neuronal ceroid lipofuscinosis, which was a similar thing, accumulation of protein within the lysosomes. But that’s also part of general ageing, and obviously Alzheimer’s is associated with old age. So is it that the machinery itself is sort of the whole cell is aged so it can’t cope with any like extra stress?

Dr Louise Serpell:

I think that what I’ve often thought is that as we age, that we get less and less, well our cells, get less and less able to clear aggregated proteins. So presumably we have protein misfolding happening all the time, but usually we have good mechanisms to clear it. We have chaperone proteins and all sorts of other mechanisms to clear misfolded proteins. But as we age, that becomes less and less efficient and we also get more and more accumulation. So maybe the lysosomes themselves are less efficient. That might be a reason why it leads to the disease that you mentioned, which the name I’ve immediately forgotten. And there have been quite a lot of studies in model animals that have shown that there are heat shock response and chaperone system seems to decline as we age. So it might be that we’re just less and less efficient as we age, to be able to clear this.

Megan Calvert-O’Hare:

Your model system is not, it’s cell culture, isn’t it?

Dr Louise Serpell:

Yeah.

Megan Calvert-O’Hare:

So it’s not aged in any way?

Dr Louise Serpell:

No.

Megan Calvert-O’Hare:

But you’re still seeing the lysosomes aren’t able to cope with the burden?

Dr Karen Marshall:

Yeah. Potentially because we’ve loaded them with more. But we’re kind of trying to recreate a disease that takes tens of years, in two weeks or something. And we do have, like I said, this quite good control of non-aggregated protein, which doesn’t have any toxic effect. So we try and control for it like that.

Megan Calvert-O’Hare:

I was reading that amyloid fibrils also fulfil a functional role.

Dr Louise Serpell:

Oh yes.

Megan Calvert-O’Hare:

Could you talk to that?

Dr Louise Serpell:

Yes. So when I was talking about the structure of amyloid, I nearly said it then, but it’s interesting because the structure of amyloid fibrils is actually quite similar to the structure of silk, that’s made by various insects and obviously by spiders and things. And we always think of that as being incredibly strong. If you walk into a spider web, it’s stronger than you would expect it to be, considering its diameter and everything. And so there’s a whole field of amyloid research, which is looking at functional amyloid. And it turns out there are actually lots of animals that make amyloid on purpose. And I guess the difference between those functional amyloids and the ones that we see in pathology are that they’re very carefully controlled. So, for example, in spiders, they make the proteins that form the spider silk, but they’re very carefully controlled so that they only form the fibres once they’re outside the body of the spider. And similarly, bacteria have proteins called Curli, which on the outside of their membrane, and they’re used as defence and scaffolding and to form biofilms. And so those amyloids, again, their formation is very carefully controlled. So we can learn a lot about amyloid from looking at those functional amyloids, that we can then use to try and understand pathological amyloids.

Megan Calvert-O’Hare:

And do you use any of your cultures for drug discovery? That’s obviously the ultimate aim, isn’t it, is to find a cure, to find a drug. Do you do that in your lab?

Dr Louise Serpell:

Yeah, so we’re working at the moment with a company called TauRx Wistar, that are based in Aberdeen and that’s what Karen’s current project is on. And what we’ve been tasked with is to take a compound that they’ve currently got in phase three clinical trials and to try and understand how it works. So our part of the project is to take the compound and follow its effect on tau aggregation. So specifically, it’s a compound that targets tau, paired helical filament formation. So we’re looking at its effect in, in vitro. So how it affects the formation of paired helical filaments, and also how it might prevent the toxicity of the tau in a cell culture environment. So we’re looking at those aspects at the moment.

Megan Calvert-O’Hare:

Is that just for fun or if the drug works, do you need to know why?

Dr Louise Serpell:

I think, I guess that people who want a drug for Alzheimer’s disease, well everybody does, will be delighted that there is one. But I think, as scientists, it’s always important to ask why. And if we want to try and improve the drug, then it’s essential to understand how it works. So clearly the company have been interested in how it works and being able to explain what its mechanism is, does seem of real importance. So it is fun, but I think the main thing is that hopefully we will understand how it works so that we can even improve the drug even further.

Megan Calvert-O’Hare:

And that’s targeted to tau, isn’t it specifically?

Dr Louise Serpell:

Yes.

Megan Calvert-O’Hare:

But obviously Alzheimer’s diseases isn’t, we’ve talked about other proteins that are involved. So do you do anything with the other proteins, drug discovery or is it understood that the tau drug will then get rid of all your symptoms and all your problems? Do you see what I mean?

Dr Louise Serpell:

Yes. I think it’s really difficult to know exactly what the most important target is at the moment. And there have been many, many drug trials that have been tested against amyloid beta, aggregation, clearance, etc. So far they haven’t worked. And I don’t know why that might be, but one suggestion has been that those drugs are not able to be administered early enough. So the amyloid beta seems to be the first thing that starts to aggregate, tau then following up. And it seems certainly that the fact that the drug is in phase three clinical trial seems to suggest that targeting tau is a useful strategy. Whether you also need to be able to remove amyloid beta is another question. So how those two proteins interact with each other, I think is something none of us really still understand. There’s so much that we still need to understand about Alzheimer’s disease before we can really design, really carefully, rationally designed new drugs.

Megan Calvert-O’Hare:

We did a drug discovery podcast a few weeks ago, and it was all about target validation, and the elusive biomarker. But I guess in this instance, you’re using the amyloid beta as a biomarker for the eventual tau, and then you’d know to use the drug early enough in the tau journey, as it were.

Dr Louise Serpell:

Yes. It may be the case that once amyloid beta has started, I sort of imagine that it might be a trigger. And so if that’s already been triggered, that actually targeting that, maybe it’s too late, I don’t know. And unless you could treat somebody early enough for A-beta, then you want to treat the next thing in the cascade. I don’t know that that’s the answer, but it might be the answer.

Megan Calvert-O’Hare:

And so the amyloid can be trafficked into the cell and then tau itself aggregates within the cell. So would you think that that could be, is that what you’re saying, the trigger could be that the internalization of amyloid triggers the tau aggregation?

Dr Louise Serpell:

Well, we’ve done some studies looking at what happens to tau after you’ve administered A-beta and it does seem that tau relocalizes. So there’s tau in the nucleus and there’s tau in the cytoplasm. And you see a re-distribution of the tau following A-beta. So A-beta causes a sort of acute toxic effect on the cells, that then leads to all sorts of downstream changes, and one of them is on tau. So it might be that the A-beta does trigger tau, but clearly we have a whole range of diseases where there’s only tau, the tauopathies. So you don’t need A-beta to do that, but maybe something else triggers those.

Megan Calvert-O’Hare:

To come back to a point you made quite a while ago, Karen, is that you do extract amyloid from diseased brains, but that you said that it’s not defined well. So that’s why you use synthetic in your experiments. What did you mean by not defined well?

Dr Karen Marshall:

I mean that if you have a person’s brain and you homogenise it and then try and use, normally you would do some kind of immunoprecipitation to pull down the amyloid beta or amyloid beta oligomers or amyloid beta amyloid fibrils, from a brain you’re probably not going to get a very pure preparation. So you might have bits of membrane or other proteins there as well. So once you’ve extracted these different types of aggregates, it’s really hard to then look at them using various different biophysical methods because the other bits that you’ve extracted will interfere with those assays. So it’s just not very clean, really. So it’s just hard to know exactly what you’ve got in your preparation that could be potentially causing any effects that you see. Whereas the way we’ve chosen to do it most of the time, is to go the other route, say “Okay, we know exactly what we’ve got and what we’re adding to the cell.”

Megan Calvert-O’Hare:

Yeah, you can tightly control.

Dr Karen Marshall:

Yeah. And there’s obviously valid reasons to use both, but that’s kind of the way we’ve gone for most things.

Megan Calvert-O’Hare:

That’s great. It’s been really interesting. So as we’re wrapping up today, maybe do you have any top tips for ECRs working in biochemistry, wanting to move into that field, or just karaoke tips?

Dr Karen Marshall:

Well, actually for both, I would say that I think your heart really has to be in it. And I think you know, from quite early on, whether it is or isn’t. So if you do even a little bit of lab work and you’re an undergraduate and you think, “I really love this” and it excites you, then stick it out. Because even when you will have awful times, you’ll cry, you’ll be so stressed, you’ll think, “Why on earth am I doing this?” But your resilience will build so much over time. I think the first time I can remember actually getting reviewers comments from a paper and I was absolutely mortified. It was embarrassing and I thought “I’m so rubbish, I can’t do this. Why am I doing this when someone’s criticised me?” And now I think, “Huh, what do you know?” No. I think, “Oh, that’s a really useful suggestion. Thank you.” It really kind of builds your character. And the excitement I think I felt then, I still feel it just as much now. So I’d say if it’s something you really like, go for it and stick with it through the hard times.

Megan Calvert-O’Hare:

Okay.

Dr Louise Serpell:

Yeah. So I agree with that, and I would only add that the rejection of grants and papers and things can be incredibly hard. And the only way to withstand that I think is to make sure that you’ve surrounded yourself with supportive people, to make sure that you’re in a group where people will support you. And I think that’s the only thing that is really, really important, is to make sure that you’re happy in your environment.

Megan Calvert-O’Hare:

Yeah.

Dr Louise Serpell:

Yeah.

Megan Calvert-O’Hare:

Good. Well, this has been great. Thank you so much. If you have anything to add on this topic, please do post your comments in the forum, on our website, or drop us a line on Twitter using #ECRDementia. We have profiles on both Louise and Karen, and you’ll find a transcript of this podcast as well. Finally, please remember to subscribe and leave a review through iTunes, Spotify, and SoundCloud. Thank you.

Voice Over:

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