Podcast – Investigating the ubiquitin proteasome system

Voice Over:

Welcome to the NIHR Dementia Researcher podcast brought to you by dementiaresearcher.nihr.ac.uk in association with Alzheimer’s Research, UK and Alzheimer’s Society, supporting early career dementia researchers across the world.

Professor Selina Wray:

So, hello everyone and thank you for tuning into the Dementia Researcher podcast, where we discuss careers science and research into dementia. This week, we’re exploring the role of the ubiquitin proteasome system in dementia, and some of the wonderful research that’s been undertaken by our four guests today. I’m delighted to be guest hosting today’s show. If I can start by just introducing myself, my name is Selina Wray. I’m a professor of molecular neuroscience at University College London, and my own research is focused on understanding the molecular mechanisms that lead to Alzheimer’s disease and frontotemporal dementia, particularly using patient-derived stem cell models.

Professor Selina Wray:

I’m really excited about today’s topic because it’s very relevant to the research that’s going on in my own lab. So I’m delighted to be joined today by four experts in the ubiquitin proteasome system in dementia. So from Imperial College London, I’m delighted to welcome Emma Mee Hayes, Dani Pulgar Prieto and Liina Sirvio, and they are joined today by Georgie Lines, who’s from University College London. Hi everyone and welcome.

Georgie Lines:

Hi.

Dr Emma Mee Hayes:

Hi.

Professor Selina Wray:

Thank you everyone for joining us today and I thought we could maybe just start by some quick introductions. So Emma, could I hand over to you and ask you to introduce yourself?

Dr Emma Mee Hayes:

Sure. So hi everyone, everybody listening. My name is Emma Mee Hayes and I’m a postdoc at the UEA Lab in Imperial College London. My research is mainly focused on using super resolution microscopy to look at the proteasome in also induced-derived pluripotent stem cells.

Professor Selina Wray:

Fantastic, thank you. Dani, can I hand over to you?

Dani (Katiuska) Pulgar Prieto:

Yes. Hi, I a PhD students also in Dr. Yu Ye’s lab at Imperial. I started my research career at Stony Brook after I graduated from biochemistry and since then, I’ve been looking at immune pathways in cancer, like solid and hematological cancers. However, last year I had the opportunity to join Dr. Ye’s lab to also study immune pathways, but in a degenerative condition.

Professor Selina Wray:

Fantastic, we need more people to come to neurodegeneration, so it’s great that you’re bringing your skills and your talent to us. Liina, can I ask you to introduce yourself please?

Liina E Sirvio:

Yes, of course. I’m Liina Sirvio, I’m also a first year PhD students in the Ye lab at Imperial. I have been London-based since I started my undergrad. So I’ve been at King’s and Imperial for bachelor’s and master’s, I was also a technician at the Crick for a year before starting this PhD. So I also came from a somewhat similar background, of working either on unfolded protein response or kinases in the brain, so post-translational modifications.

Professor Selina Wray:

Fantastic. Thank you. Last but by certainly no means least, Georgie can I hand over to you?

Georgie Lines:

Hiya, thank you for having me on the podcast today. So, my name is Georgie Lines and I am a PhD student at UCL. I’m currently in my third year out of four, and it’s gone really, really fast so far. My PhD project is centered around investigating tau proteostasis in induced pluripotent stem cell models of frontal temporal dementia. It sounds super nerdy when I say it out loud, but I just think that the ubiquitin proteasome system is super interesting and so obviously important in neurodegenerative diseases, so I’m really excited for our chat today.

Professor Selina Wray:

Amazing, well thank you everyone. I’m really excited to hear about your individual research projects and the different techniques that you’re using, but I thought before we get into the details, it might be useful for any listeners who aren’t familiar with the ubiquitin proteasome to just do a bit of scene-setting and maybe give some very brief and basic introductions so that we’re all on the same page. So, I wonder if anybody would like to take on the challenge of giving us a very brief introduction to the ubiquitin proteasome system, what it does in the cell.

Dr Emma Mee Hayes:

I’ll take that one. It’s a pretty complicated system so I’ll try and boil it down into two, three sentences. So our cells produce proteins and eventually these proteins get old or they get misfolded so the cell has to figure out some way to get rid of them or deal with them. So the ubiquitin proteasome system will tag different proteins that are, as I said, old, damaged, misfolded, which is obviously a feature of neurodegenerative diseases, misfolded proteins, will tag them so that they can get degraded by the proteasome system. So it’s a way of thinking, I suppose, like the garbage disposal of the cell and the way they do this is they tag these proteins with a protein called ubiquitin.

Dr Emma Mee Hayes:

So if something’s got a ubiquitin tag, it should be degraded and that’ll probably be like a debate we get into if it actually is degraded, then in neurodegenerative diseases, et cetera, et cetera, et cetera. But that’s essentially what it should do in a normal cell. So I hope that explains it?

Professor Selina Wray:

It’s perfect, I wonder if I could follow up just with a little bit more detail and anyone can feel free to chip on this. I’m obviously directing it a little bit at Emma at the moment, but what’s doing the tagging and how does the cell know when to tag something?

Dr Emma Mee Hayes:

So if something becomes misfolded or as I said, it becomes damaged in any way, by reactive oxygen species, et cetera, et cetera, et cetera, the cell will use a series of different enzymes to actually tag this protein. So it’s the ubiquitin ligase is like the one, two and three enzymes. So eventually these will, in an enzymatic [inaudible 00:05:57], tag proteins relative for degradation.

Professor Selina Wray:

Super, thank you. So why is this so important in neurodegenerative diseases? Maybe I can bring Georgie into to comment on that. Why is the ubiquitin proteasome a focus of investigation in Alzheimer’s disease and other neurodegenerative diseases?

Georgie Lines:

Well, the main reason that the ubiquitin proteasome system is being more heavily researched at the moment in neurodegeneration fields is because the majority of neurodegenerative diseases, so for example, Alzheimer’s disease or frontotemporal dementia, can be pathologically characterized by the accumulation of misfolded proteins. So with frontotemporal dementia, for example, you get the accumulation of hyperphosphorylated insoluble tau, which actually is not being broken down or cleared by the proteasome system. So this is really implicating a fault in the UPS pathway in disease. Not only do you get the accumulation of these disease-relevant proteins, but some of these accumulations also have ubiquitin in their aggregates as well. So it’s another indicator that the ubiquitin proteasome system is at fault in neurodegenerative diseases.

Professor Selina Wray:

Fantastic. I think also, it’s been really interesting in the past few years, certainly in tauopathy and in progressive supranuclear palsy, that the genome-wide association studies that have been performed in those diseases have also highlighted some of the components of this pathway. So I’m thinking of the [inaudible 00:07:44] locus and that for me, is really important because it starts to indicate that pathway really is central to the disease pathogenesis.

Professor Selina Wray:

That brings me onto my next question, which is a hard one, but I’d love to hear your opinions on it. It seems to me that there’s a bit of a chicken and egg situation going on in neurodegeneration. So we have these unfolded proteins. We know that the ubiquitin proteasome system would normally break down those proteins. Yet, do we know what order things start to go wrong? Is the ubiquitin proteasome system faulty because it’s just overwhelmed by these protein aggregates? Or do these protein aggregates exist because the ubiquitin proteasome breaks down? Does anybody want to chip in and comment on that?

Dani (Katiuska) Pulgar Prieto:

So from the literature, there are publications that say that the ubiquitin proteasome system is overwhelmed by these aggregates and the system is no longer able to degrade them. So that’s definitely there in the literature. However, there are other publications that mentioned mutations in the proteasome subunit. I want to say psmB2, but don’t quote me on that one, and that these mutations actually break down or slow down the ubiquitin proteasome system and that’s what’s causing the accumulation of a lot of proteins, including these, for example, tau aggregates. So I think it’s not exclusive, it’s just individual, depending on the case, in the individual case of the patient. So it can either be the breakdown of the proteases system or the mutation in, for example tau, that causes it to aggregate in a certain way that clogs the proteases system.

Liina E Sirvio:

Yeah, if I can just add on to that?

Professor Selina Wray:

Oh yeah, please do.

Liina E Sirvio:

Another aspect that I’ll at least be talking about later on as well, is that it might also be that post-translational modifications, not just mutations, make these proteins more prone to aggregation. So that’s what my research also focuses on, is does the specific type of modification speed up the aggregation and then form this kind of insoluble aggregate that’s resistant to degradation, and then that inhibits the proteasome system? Or potentially, there’s types of modifications that perhaps aren’t as well-recognized by the proteasome and so this might just proceed from a few different angles in that sense, in terms of whether the modifications lead to the aggregates or whether the aggregate formation actually inhibits the proteasome as well.

Professor Selina Wray:

Brilliant, thank you. So it’s really complicated, I think is the take-home message from that. I think those initial questions, you’ve done a fantastic job at showcasing the complexity of the system, but also its elegance. I think sometimes, I think a lot about these quality control systems and although we are often ask the question, why do things go wrong? Why do we get these protein aggregates? When we think about how complex these systems are, I think it’s a miracle it doesn’t happen more often to be honest, that we manage to have these super complexes in the cells that just functions so well for so long. So it would be great to maybe hear a little bit more about your individual research projects and in this complicated system, what specifically each of you are looking at. So Emma, maybe I can come back to you?

Dr Emma Mee Hayes:

Sure.

Professor Selina Wray:

Can you tell us a little bit about your research? And I know that you’re particularly interested in using a technique called super-resolution microscopy. So again, maybe you could first of all, tell us what that is, particularly for any listeners who might not have heard of it before.

Dr Emma Mee Hayes:

So first of all, I’ll say what I’m actually looking at and then why I’m using super-resolution microscopy, put the chicken before the egg there. So what I’m currently using is actually, I’m also using induced pluripotent stem cells. So for anyone who doesn’t know, these are patient-derived cells that we make into stem cells, which I then make into neurons, which I use then as a model for neurodegenerative diseases. So what we want to do is we want to fluorescently tag the proteasome in these cells and then use live cell microscopy to study the effect of hyperpolarization, or inflammation, or aggregate induction, or any kind of pathological effect on our proteasome and does that affect its localization, or its activity, et cetera, et cetera, et cetera. The reason that we’re using super-resolution microscopy is that honestly, compared to say, something like just your standard florescence, it allows us to look at a single molecule level, what these proteasomes are doing and how they’re then interacting with each other.

Dr Emma Mee Hayes:

I think for the proteasome, that’s critically important because we can then see how it’s interacting with say, something like the cytoskeleton, or specifically how it’s interacting with … We can add in tau fibrils, alpha-synuclein fibrils, or just protein aggregates in general, or block them specifically so we can see on a strictly molecular level what effects that’s having, using super-resolution microscopy.

Professor Selina Wray:

That’s fascinating. I guess it’s something that I actually don’t know the answer to, but I think about neurons as being these … they have this really complex morphology and these long axons. Are the proteasomes everywhere in neuron?

Dr Emma Mee Hayes:

So I think this is a really interesting question. It’s definitely something that I’m personally very interested in because obviously neurons are not just like a bag, they’ve got a very, very extreme morphology. So there is some people showing that they’re very, very concentrated at the synapse which makes sense to clear up neuro-transmitters, there’s a huge amount of activity going on at that point. I think, using standard immunofluorescence or microscopy techniques, we’re just never going to get to that degree of detail to see, are they localized totally at the synapse, or even are they moving from the synapse back to the soma? Are they getting clogged in the middle of the axon? I don’t think that’s really something we can answer without using very, very high powered microscopes, because there’s just no other way to see it.

Professor Selina Wray:

Yeah, super cool. I think one of the things that I really struggled to get my head around is how the proteasome isn’t just a single entity, it can be really diverse and made up of different subunits. Do you have a sense for how diverse proteasomes are within neurons, if there are differences? You mentioned neuroinflammation or looking at the proteasome in response to inflammation. So I just wondered if the proteasomes are similar between the different cell types of the brain, or if there’s more diversity there, for example.

Dr Emma Mee Hayes:

I think that’s a really interesting question. As you said, the proteasome is made up of different regulatory units in core subunits. I think we’re starting to look at that, just seeing is there different expressions? Are they expressed in different areas? We’ve gotten some initial data saying, “Oh, this might be here or this might be there.” It has to be expanded on, but it’s hard to tell what the downstream effects of that would be. I think inflammation now is such a big thing in neurodegenerative diseases, but how that really effects a neuron, which isn’t really an inflammatory cell, or it shouldn’t be at least, so, yeah, we’re starting to look at those inflammation change when proteasomal subunits are expressed in neurons? Because of it does, I think that’s going to be a really, really part of that whole incredibly complex picture of the interplay between the inflammatory systems and neurons and glia and microglia and other kinds of stuff. It makes it much more complicated, but I think it also much more interesting.

Professor Selina Wray:

That’s true, like it wasn’t already complicated enough, right?

Dr Emma Mee Hayes:

Yeah, I know, yeah. I was going to say, I know some point they’re going to be like, “Why don’t you make microglia as well as neurons?”

Professor Selina Wray:

It’s like, let’s try and to figure out this one thing first.

Dr Emma Mee Hayes:

Yeah, one thing first and then we’ll move out and expand.

Professor Selina Wray:

Brilliant, well I’ll come back to you in a second but Dani, I wondered if you could maybe tell us a little bit about your research?

Dani (Katiuska) Pulgar Prieto:

So I focus in microglia, the inflammation pathway in microglia and how this UPS system … So the UPS system is formed by different subunits, as previously said, and when there is some cell stress, such as aggregates or even LPS, you have different subunits substituting this complex. So first of all, I’m looking at how the different subunits play a role in the progression of inflammation and how these subunits control which way inflammation goes and if we’re able to actually understand the pathway, the inflammatory pathway, and how it is controlled by the UPS, then perhaps we can find targets for these particular proteasome subunit to either decrease inflammation, if that is what’s causing the neurodegeneration, or perhaps we can target subunits to enhance degradation.

Professor Selina Wray:

Excellent. I think, so the proteasome, I guess what little I know about it in its function in immune system, it must have really important roles in things like antigen presentation and things like that within the immune system, is that the case?

Dani (Katiuska) Pulgar Prieto:

Yes, it is. So, when there’s a stress, such as interfering gamma or something like that, then you have the immunoproteasome subunits upregulated, almost you have the immune proteasome, that’s really what’s going to get you, that degradation for … the most optimal peptide for antigen presentation.

Professor Selina Wray:

Super, thank you. What sort of models are you using to study with the microglia? Is this using in vivo models or is it also stem cell-derived cell types that you’re using?

Dani (Katiuska) Pulgar Prieto:

Yeah, so I’m first starting in just established cell lines, like BV2, which is a microglia, but actually Emma is supervising me in the IPS side because I also want to study microglia derived from patient brains to see if these mutations affect the degradation or inflammatory pathway. Also, we are looking to collaborate with different lab groups that can provide us with in vivo samples, so brain samples.

Professor Selina Wray:

Oh, amazing, super cool. Liina, can I hand over to you and hear a bit more about your project?

Liina E Sirvio:

Yeah, of course. So my main project, as I go into a bit earlier involves studying how post-translational modifications of tau and alpha-synuclein effect their degradation by the proteasome. So I am focusing only mostly on ubiquitination. So the main question that I want to address with my research is why this kind of post-translational modification that is canonically designating proteins for degradation is so prevalent on these proteins when you mass spec neurofibrillary tangles, or Lewy bodies from diseased patients. So this goes into my earlier answer, perhaps earlier on this kind of modification targets proteins for degradation, but there’s some kind of other stress going on in the cell. Then perhaps, because this makes the proteins more prone to aggregation, this is more of a protective sequestration mechanism in the cell, as the nicest way for the cell to just handle this overload of this amyloid protein if it can’t be degraded.

Professor Selina Wray:

Thank you. I had a general question, which maybe it might be better suited to the end, but I’ll ask it now so I don’t forget, but I think you just said something really nice, which again is one of those big questions in the field about whether these aggregates that we see in the brain could actually be protective and they are sequestering more toxic species to lock them away. With that in mind, the question which I was thinking about is, I guess what’s on all of our minds, is how we can modulate the UPS therapeutically. Do you think that it would be beneficial to restore activity of the proteasome or increased activity of the protein? Or could it be actually detrimental because you might all of a sudden break apart these aggregates and liberate all of these super toxic oligomers?

Liina E Sirvio:

Well yes, of course, that’s the idea in that kind of sequestration pathways that the oligomers are just so much more toxic because they’re much more likely to spread to other cells or recruit other monomers that form other aggregates. So I don’t know how to answer that question about reactivating the UPS, but there’s many papers that have come out recently showing that ubiquitin moieties themselves can stabilize these fibral structures. So perhaps if they are acting this kind of suppressed ration capacity, they are becoming so stable that they cannot be processed by the proteasome, so then that frees them up again to be not so harmful in the cell and then allow the proteasome to perhaps trying to degrade other things, like keep up normal cell function.

Professor Selina Wray:

I should say, if anybody else wants to comment on that, you’re more than welcome to. It’s a tough question to drop on you there, to Liina, but it was a fantastic answer.

Dr Emma Mee Hayes:

Yeah, I think it’s definitely one of the biggest challenges, not just with the UPS system, but with just Alzheimer’s therapies in general, when is the best time to treat? I think you said with the ubiquitin system, I think we’ll find through just basic research, or in animal models, that there is an optimal time and there will be a point where if you start to increase its activity, you could potentially worsen. I think that’s going to be a very difficult thing to translate from an in vivo to even something in an animal model. I think that’s going to be really, really difficult to do.

Professor Selina Wray:

Yeah. I guess within vivo as well, we have full control over when we intervene. Whereas when we think about translation, we can’t identify really exactly where someone is along that spectrum, which further complicates it. I don’t know if this is a stupid question, but I will ask it anyway. I think we’ve heard about different cell types, I’m really interested in the interplay between neurons and microglia. So Dani, this might be a question for you, but again anyone feel free to chip in, do these abnormal proteins that are forming in neurons? Are they always degraded in neurons, or if a neuron is stressed and overloaded, can it actually give some of its protein to an astrocyte or to a microglia and say, “You deal with it?” So the reason I ask about is that I’m thinking about some papers that have been published in the field of stroke, looking at mitochondrial damage and it’s not exactly the same, but what they showed in those papers is if the mitochondria in a neuron are damaged, the neighboring astrocytes can actually replenish the cell with healthy mitochondria, or take away the damaged mitochondria and degrade it. I just wondered if there was any support role for getting rid of these toxic proteins from other cell types?

Dani (Katiuska) Pulgar Prieto:

So, I’m not sure about the astrocyte point of view, but for instance, when neurons have these aggregates and the neurons are stressed, they have more of these inflammatory, let’s call it chemicals, that actually the microglia is able to sense. So there is a level of the stress of the neuron that the microglia can actually phagocytose the neuron. Then these aggregates that were formed inside the neurons would be released into the microglia and then the microglia would become activated, increasing all sorts of inflammatory targets and actually start degrading or eating things they shouldn’t be eating.

Professor Selina Wray:

Wow.

Dani (Katiuska) Pulgar Prieto:

That’s where you get the neurodegeneration.

Professor Selina Wray:

Okay, that’s interesting. So yeah, sometimes this degradation is not a good thing. It’s chomping away synapses and things like that, that shouldn’t be gone. So Georgie, finally, can you tell us a little bit about your project please?

Georgie Lines:

Yep. So my project is focused on investigating tau proteostasis in iPSC models of FTD. So frontotemporal dementia can obviously be caused by mutations in the match gene, which is the gene that encodes the tau protein, and these mutations lead to the deposition of hyperphosphorylated tau tangles, which is one of the main hallmarks of FTD tau. These tangles aren’t cleared through proteostasis, which as I said earlier, is really implicating a failure of protein clearance systems in this disease. So in the lab, I’m differentiating iPSC’s intercortical neurons over the course of around 80 to a hundred days. These iPSCs have mutations in the MAPT gene. So just to give a little bit more detail about their mutations, we have a 10 plus 16 mono-allelic and bi-allelic line, and also a 10 plus 16 bi-allelic line with a P301S mutation. So 10 plus 16 is a splicing mutation that increases the production of 4R tau, leading to the deposition of tau aggregates and P301S is a missense mutation that makes tau more aggregation-prone. So patients with 10 plus 16 or P301S mutations do go on then to develop frontotemporal dementia with these hyperphosphorylated insoluble tau tangles. However, the iPSC-derived neurons with these mutations do not develop any tau tangles.

Georgie Lines:

What we want to know is why. Why are these iPSC neurons resistant to tau pathology? What are they doing or what have they got that neurons in patient brains don’t? If we can elucidate or find out what the differences are there, then that would be really beneficial for potentially looking at treatments for neurodegenerative diseases and tauopathies.

Professor Selina Wray:

Thank you. So I wonder, just following up on that, one of the features of iPS-derived neurons, I guess, is that they’re quite fetal in their identity. So is there anything, known or hypothesized about maybe how the proteasome … does the proteasome also have a role in neurodevelopment and how might the proteasome change from this fetal stage to the point in a person’s life when they might be having these aggregates?

Georgie Lines:

Yes, so fetal neurons in general tend to have, or are thought to have, a higher proteostasis activity or a more efficient proteostasis pathways in them because at that early stage in development, proteostasis is really important, especially for turning over things like morphogens, which are really important for neuronal patterning. So, proteostasis activity is really high during early development and then lots and lots of studies have shown in many different animals, in many different tissues, that proteostasis and proteasome activity declines with age.

Professor Selina Wray:

Excellent, thank you. So maybe if I can bring the discussion back to everyone again, I’m interested to ask a few technical questions, perhaps. So I know a few people on the call are using super-resolution microscopy, and I wondered if you could maybe again, expand on why this technique is better than traditional live microscopy or just fluorescence microscopy?

Liina E Sirvio:

Sure, I can take that one. So, I don’t know too much about other kinds of fluorescence microscopies, other than confocal, but as far as I know, most of those other technologies, they excite the entire sample on your cover slip with a laser to excite the fluorophore so that you can look at this fluorescence. Because of that, you have excitation of an entire sample, like a cell or a whole layer of solution. So you have a lot of fluorophore that you have excited. So even if in confocal microscopy, you’re trying to take an image in one plane, you have a lot of background just because you have excited all of those other fluorophores, whereas in TIRF because of the critical angle that you use with the laser, you create this evanescent field about 200 nanometers above the cover slip, which is the only area in which you actually excite to fluorophore. So then you can exclusively excite the fluorophore in that section of the sample. So then that really improves your signal to background ratio, so you can get a very nice image from this specific plane.

Professor Selina Wray:

That’s a beautiful explanation for someone who knows nothing about super-resolution microscopy, so thank you. Is there anything different, in terms of sample prep and from a technical perspective, that is needed to use this? Or is it just the case of having access to the proper microscope?

Liina E Sirvio:

So I work mostly with in vitro things, I’ll let the others talk about working with cells on this microscope, but for me, there was no changes to the prep really. It’s just, you have to have access to, yes, this very nice, fancy TIRF microscope.

Professor Selina Wray:

And I assume they’re not cheap?

Liina E Sirvio:

No.

Professor Selina Wray:

No, we can’t just go away and buy one if we’re listening to this podcast and we’re excited?

Liina E Sirvio:

Not very easily [crosstalk 00:30:47].

Dr Emma Mee Hayes:

In terms of actually what you do, as Liina said, the standard protocol, you don’t have to change anything because it’s just standard ICC protocol. For certain things, for certain super-resolution you do have to change one of your buffers and that enables you to do certain super-resolution and things. But no, thankfully you don’t have to do any huge involved process for this, which is I’m quite thankful for.

Professor Selina Wray:

Yeah, that sounds quite appealing actually. I always assumed it would involve some sort of complicated sample prep or different culturing conditions. So it’s nice that it’s accessible in that manner. I didn’t ask actually, Dani, if you’re using super-resolution in your own studies?

Dani (Katiuska) Pulgar Prieto:

Yeah, I am starting to use them. I think I started like three weeks ago, but I’m getting there.

Professor Selina Wray:

Nice, easy to learn so far?

Dani (Katiuska) Pulgar Prieto:

Yeah, yeah. As Emma said, it’s pretty much the same particle, it’s just different buffers on once you got to the microscope, we have different parameters, but it’s pretty much the same thing.

Professor Selina Wray:

Super, and Georgie, you’re not doing imaging, but I’m sure you can talk … I have the advantage of knowing your project inside out of course, but I’m sure you can talk or use the opportunity for group therapy, to talk a little bit about some of the technical challenges of working with the proteasome. I wonder if you want to say a little bit about how you’re measuring proteasome activity?

Georgie Lines:

Yeah. So in the lab we’ve been optimizing and in-gel activity assay, which is pretty much the standard bread and butter that most people investigating the proteasomes go to. Yeah, it has been technically challenging, I think. In the process, if I just explain it a little bit, that anyone listening who doesn’t know, what you do is you [inaudible 00:32:43] your sample and you run it on a native gel, but that gel, you have to precast yourself, like the good old days, I guess. Yeah, you precast your gel and it’s quite a low percentage. So initially, we had lots of problems actually just getting these gels to set. Once you can get past that issue and actually run your samples on the gel, there are other problems that come into play in that the gel is very delicate, but once the process is complete, you run your native gel.

Georgie Lines:

You can then take that gel out of the cassette and you can image it on a UV transilluminator and that will illuminate the three distinct bands. So, the three distinct bands, the highest molecular weight of those is the double ECAP proteasome with two regulatory units. The second highest molecular weight is the single ECAP proteasome. Then at the bottom of the gel, you get the illumination of the 20S proteasome. So it is a really cool technique if you’re interested in comparing the activity of the proteasome in different samples, because it really breaks down the activity of each different proteasome species within the sample.

Professor Selina Wray:

Super, thank you. So maybe going back to the individual research projects again, I wonder if maybe everyone could spend a minute or two to tell us, perhaps the thing that they’re most excited about at the moment. That could be something very specific, the experiment that you’re doing that you’re most excited about, or maybe something a bit more general in the field, what do you think the hot topic is at the moment in this area. Does anybody want to go first with that? I realize I’ve put you all on the spot, to think about something, but whoever gets first gets the pick of everything as well, right?

Dr Emma Mee Hayes:

That’s true. I don’t know, I’ll be the brave postdoc and go first [inaudible 00:34:46]. I think from my own research, I’m most interested in seeing is there really a drastic change in localization of the proteasome between the different regions of a neuron? I think that’s going to be really, really fascinating. When you add aggregates on, where do you see that biggest change, how quick does it happen? Can you reverse it? That I think is really, really interesting for me, at least. Then in terms of just the field in general, you guys touched on it earlier, iPSCs are great, I will always think they’re the best model that we can use because I did my PhD on them as well. I’m like, “No, I’ve committed to this now, we’ve got to keep plowing this field,” but there is a disadvantage is that they’re quite fetal in nature, which is just a caveat you have to think about work around when you’re looking at these kind of diseases.

Dr Emma Mee Hayes:

So I’m quite interested in, will the field be able to model age a little bit more in neurons or if other labs are looking at that and how will that relate back to my work and use that to complement mine and understand why I might not, say, see aggregates, but I’m seeing the same effect when I add aggregates and change proteasome levels. I think in terms of the field, that’s definitely what I’m most interested in.

Professor Selina Wray:

I’m obviously quite biased, but I would 100% agree that in spite of their caveats, I believe iPSC are the .. they do have caveats, all models do and-

Dr Emma Mee Hayes:

Yeah, everything has a caveat.

Professor Selina Wray:

They’re our only way to have unlimited human neurons in the lab at the moment. So, in spite of all the caveats, I still think there’s a lot to be said for that. I think the question about age is really an interesting one. There’ve been a few papers recently that have bypassed the iPSC … I can’t speak, iPSC stage and done this direct conversion of fibroblasts to neurons. Particularly with things like nuclear cytoplasmic transport and mitochondrial fitness, they do seem to retain their age very well. So, I think there’s a study begging to be done there about proteasome function-

Dr Emma Mee Hayes:

I know.

Professor Selina Wray:

… and then how it varies, right?

Dr Emma Mee Hayes:

Yeah. I think that’s also a quite critical thing to know, just how this system changes just with age.

Professor Selina Wray:

Yeah, definitely.

Dr Emma Mee Hayes:

Rather than just specifically in neurodegenerative diseases. If we can also answer how it’s just meant to act, I think that will inform our feedback and inform what we think then about where it’s going wrong and neurodegenerative diseases.

Professor Selina Wray:

Excellent, yeah, I agree completely. Dani, what are you most excited about?

Dani (Katiuska) Pulgar Prieto:

I would say this liquid-liquid phase separation project I recently started. I think liquid-liquid phase separation would be a hot topic in every single field right now. So basically what … Sorry.

Professor Selina Wray:

I was just going to say, could you tell us a bit about what liquid-liquid phase separation is?

Dani (Katiuska) Pulgar Prieto:

Yes, absolutely. So I’m just going to refer to it as LPS from now on, but I think the most basic way to explain this is if you can imagine water mixed in with oil, any oil, what you would get is a mixture of this liquid droplets suspended in water. So in biological terms, if you think of the cytoplasm as the water, sometimes proteins or even RNA condenses to form droplets that are almost like the liquid droplets you would see in this water, oil mixture. So that is what liquid-liquid phase separation is. It’s actually two distinct phases in one mixture.

Dani (Katiuska) Pulgar Prieto:

So there has been some research. It’s not very well-established and definitely the verdict isn’t out yet, where these aggregates can undergo liquid-liquid phase separation. So my project is to understand really why, and is the UPS involved in this? So yes, I am very excited about this project, mainly because there’s not that much research into it.

Professor Selina Wray:

I think the LLPS is such an area of intense investigation now, it’s so timely. I agree, no, it’s really important to see how the UPS feeds into that. I suspect that the LLPS could be a subject for a next episode of this podcast too. So maybe you find yourself hosting that, I would certainly like to listen and learn more. Liina, how about you?

Liina E Sirvio:

I am most excited about the TIRF and the super-resolution aspect of my project, which I guess is a good answer for this podcast. We have a really fantastic postdoc in the lab called Michael Morton, who set up our one of our microscopes and we’re currently building another one together. So I’ve got to learn more about this technique in that way, but also my project involving post-translational modifications, it’s definitely not oversubscribed. There’s definitely lots of interesting in this, lots of research has already been done on this. So I’m really excited about studying this because it’s very interesting to me, looking at the different E3 ligases that I have in mind for my project, how this affects aggregation degradation, but also combining that, in a sense standard investigation, if I can say that, with this really new, exciting technique, such as TIRF super-resolution microscopy. I think it’ll make for some really nice papers.

Professor Selina Wray:

I think it will be fascinating. It’s really cool that you’re building your own microscope, that’s pretty awesome. No, I think it will be really exciting. When I was a PhD student, I was actually working on post-translational modifications to tau in post-mortem brain tissue. That work is still ongoing with different groups. I think we’ve got a really good map of different modifications to the tau protein, but we still don’t really have a good understanding of why specific modifications are important and how those specific modifications can change the function or indeed change the clearance of tau. So I think this is really fundamental and critical work that you’re doing. So yeah, good luck, good luck. I’m excited to see the results.

Liina E Sirvio:

Thank you.

Professor Selina Wray:

Georgie, what about you?

Georgie Lines:

One thing, I think, that I’m definitely excited about in terms of my own project is hopefully getting my hands on some post-mortem brain tissue. So we’re interested in looking at proteasome activity, obviously in the iPSC neurons at different ages throughout development, but also in them iPSC neurons with and without the maximum mutations. But I think an extra dimension to that would be to look at the activity of the proteasome in the post-mortem tissue of humans, which then brings things back round to what Emma was saying, in that it’d be interesting to see, is there a difference in proteasome activity in actual brain tissue and these iPSC-derived neurons that it might give us a small indication of how good these models are for modeling proteostasis. Also, obviously we’d like to compare and contrast between the MAPT mutations and controls in the post-mortem tissue and also within our iPSC neurons.

Professor Selina Wray:

Amazing, thank you. I’m very excited to see those results as well. I think that was a gentle reminder for me that I need to look at your draft MTA. I think I’m being called out on a podcast [crosstalk 00:42:45].

Dr Emma Mee Hayes:

Exposed.

Professor Selina Wray:

I will do that when we hang up. Oh well, look, thanks everyone. We’re in the last few minutes before we need to start recording, but before we finish, I wondered bearing in mind this podcast talks about science, but also about careers. I wonder if everyone might have a one line of advice they would give. I know we’re all at different stages, but maybe to someone who was just looking to move in dementia research, looking to find a PhD in this area, what advice would you give them? Maybe Emma, if we start with you again and work around.

Dr Emma Mee Hayes:

Yeah, sure. So I would say for anyone looking to go for a PhD or anything like that, I’d say the most important thing is don’t be afraid to, if you’re applying to a lab, ask to speak to other members of that lab, you’ll probably get … and also to directly question the PI of how they run their lab, what’s day-to-day like in the lab, do you have fun, all that kind of stuff, because if they’re really long days, it’s super, super stressful, but it is all worth it in the end, but I think that’s an important thing, to know the environment you’re going to get yourself into and see if it suits you. I think if you’ve got that, then everything else will fall into place.

Professor Selina Wray:

I think that’s great advice. Dani?

Dani (Katiuska) Pulgar Prieto:

Just as it happened with me, don’t be put off if you come from a different field. So for instance, I have a background in cancer for six years and I wasn’t even sure if to apply for this level, though I really wanted to study the immune pathways in neurodegeneration. I really had no background at all in the new neuroscience field. Even with LPS, before this, nobody was looking at liquid-liquid phase separation in anything to do with dementia. So anybody, I would say, can actually bring their expertise into this field. So yeah, don’t be put off if you’ve never been in this field because it’s certainly fascinating.

Professor Selina Wray:

Absolutely and we really need the additional hands on deck as well because dementia is such a priority problem. So I think having that different skill set and that different perspective, also it leads to innovation. So yeah, we need more people like you moving into the field. Liina?

Liina E Sirvio:

I would say that I would encourage people to apply for, and then accept, positions in labs where they have a few different aspects of the project, because obviously everyone knows that whatever topic you sign on to study might change around you. So, as long as you’re interested in dementia research and you’re at least slightly in the right area, that’s always somewhat safe, but if there’s techniques that that lab is very good at that you want to learn, then I think that’s always a good backup to have so that in case your topic does exchange quite drastically, there are things that you’re still very excited about.

Professor Selina Wray:

Definitely. I think, yeah, exactly what you said, you can never predict whether a certain project might just hit a dead end or a roadblock and knowing that you have enough interest in the general field to be able to switch and still be happy within that group, I think is critical. Georgie, final words of wisdom?

Georgie Lines:

Something that I think is really important, not necessarily if you’re looking to get into the field, but if you’re already in it and you’re an early career researcher, I think that my biggest bit of advice would be not to be scared to reach out to people in other labs and ask for technical advice on how to do things. I think in the first couple of years of my PhD, I was very nervous about doing that and ended up spending a lot of time optimizing things that could have been done very quickly if I’d have just reached out and asked somebody for help. Since doing that more recently, I’ve found that all of the scientists are super friendly, even if you don’t know them on a personal level, people are more than willing to help you out. So yeah, that’s my biggest bit of advice.

Professor Selina Wray:

Definitely good advice as well. I think there’s no sense reinventing the wheel, if somebody already has a protocol that they’re willing to hand over to you, then yeah, use those networks and ask for help, for sure. Well, we’re almost out of time, so it’s time to wrap things up and drive the show to a close. I’d really like to thank all of the guests today. Georgie, Liina, Dani and Emma, it’s been a really fantastic discussion. I’ve enjoyed listening to it and learning from you a lot. If I can summarize, I guess the main points, it would be that the proteasome is very important and also very, very complicated. I think that complexity comes from the proteasome itself, from the multiple subunits that make the complex, the various enzymes that are involved in substrate targeting. There’s more complexity from the substrate and how it’s modified and what role those different modifications might play in leading to the substrate degradation.

Professor Selina Wray:

There’s additional complexity, as if we needed more, in development versus disease and also between different cell types. I think, I certainly still sometimes tend to think of things in a neurocentric way, yet we’ve heard today how important the proteasome is across all different cell types of the brain. But in spite of that complexity, I think we can be optimistic that we’ve got really exciting new tools, including fancy new microscopy techniques that will give us those insights that we really desperately need. So, I’d just finish up by saying that if anybody would like to know more about what we’ve discussed today, there are profiles of all of our guests on the Dementia Researcher website, including their Twitter accounts so you can follow them and find out a little bit more. There’s also loads of other content, blogs, previous podcasts and articles that will be of interest to anyone who’s a researcher in the field of dementia. So, thank you for listening and please remember to subscribe and like the podcast in whichever app you’re listening from today. So, thank you and see you next time.

Voice Over:

Brought to you by dementiaresearcher.nihr.ac.uk in association with Alzheimer’s Research UK and Alzheimer’s Society, supporting early career dementia researchers across the world.

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