Episode 5: Exploring Mitochondrial Dysfunction With Dr. Kat Bowles

Kat: I genuinely think that tau is the most interesting thing anyone could possibly study. It's absolutely wild, like it's so interesting, and so I'm obsessed with it. I don't know why anyone studies anything else.

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Oscar: You've probably heard of tauopathies, brain diseases that change behavior, communication and movement and worsen with time. PSP, CBD and MSA are all tauopathies which have no treatments at the moment. Dr. Kathryn Bowles sees tauopathies as one of the most urgent mysteries in neuroscience. And her team at the University of Edinburgh want to understand how genes change or mutate which causes neurons to die.

With support from CurePSP's Pathway Grant Program, which provides seed funding to support projects studying the fundamental neurobiology of PSP and CBD, Dr Bowles will be studying stem cells made from the blood cells of patients with mutated genes, which will be turned into mini brains, and study to track the early changes in the brain of people who develop a tauopathy by understanding these changes.

Dr. Bowles hopes to develop new treatments to stop them from happening.

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Oscar: We hear the word tauopathy so much in PSP and neurodegenerative diseases, and this might be a simple question, but what makes a primary tauopathy? What does that mean?

Kat: It really just means that tau pathology is the main pathology in the brain, so it's the protein that is accumulating most in different brain cells and seems to be the protein that's being disregulated and causing neurons and astrocytes and our different cells to behave differently.

Oscar: Yeah, and is there evidence to suggest in PSP, CBD and MSA, that irregular tau is the driving force?

Kat: Yes, yeah, definitely. The accumulation of tau is kind of the diagnostic requirement for PSP and CBD. So when people come to postmortem, they have to have that tau that's kind of accumulated in brain cells to have that diagnosis. And if you don't have tau, it's not PSP or CBD or a primary tauropathy.

Oscar: Got it. And so your study is looking at how MAPT gene mutations lead to mitochondrial changes in the brain in the early stages of tau. So what is a MAPT gene mutation?

Kat: Oh, so MAPT, we call it MAPT most of the time, is the gene that encodes for the tau protein. And so there are, there's actually, I think, over 100 different mutations now that have been identified in MAPT or the tau gene that cause frontotemporal dementia, or, you know, a frontotemporal dementia spectrum disorder like PSP or CBD.

And so, I mean, that's one of the reasons why we know tau is so important for primary tauopathies and for PSP and CBD, because these mutations will cause it. So when I say it doesn't just increase your risk, but if you have a mutation, you will get FTD at some point. And so the specific mutations that I'm interested in are known as splicing mutations, so they change the version of tau that's being made by the cells, basically.

So I don't know how in depth you want to go into this, but there are two major groups of forms of tau, so there's something called 3R tau, and there's something called 4R tau, and there's something very special about 4R tau, because that's the one that aggregates and accumulates in PSP, and we don't really know why. And so these mutations that I study force the cells to make more for our tau, instead of 3R tau, and that change is sufficient to cause disease.

And so they're a really nice model to study, because you have that for our tau in cells, which is normally quite difficult to see. And so what we've been seeing from our initial studies is that these mutations are changing the way our mitochondria are functioning, and that different mutations might be doing that in different ways. And so the project is to try and understand, first of all, how these mutations are changing mitochondria function, and also how different mutations are impacting it differently, because they're still getting to the same endpoint of FTD or PSP, but they're getting their different ways which we're really interested in understanding.

Oscar: How long have you been working on these models to study that progression?

Kat: So I actually made these models during my postdoc. So I've been working on tau mutations since 2015 so throughout my whole postdoc, and I made these lines during that time. We've probably been studying these specific mutations for five or six years, and now we're moving them into 3D cell culture models called organoids, and are digging more into mechanism, whereas before, our initial work was just "okay, what's happening?" Like when we put mutation in, what happens? So it's quite exciting now, because we can understand the process a bit more.

Oscar: Yeah, why are you using organoids? Why are they suited for this study?

Kat: Well, they're a really useful model because they — so, I suppose to give some background, this is all IPSC modeling, right? So using stem cells that we've made from patients with these mutations, and we kind of edit them to have the mutation or not have the mutation. And so typically, studies are done in what we call monoculture and just flat in dishes of one cell type. And that's useful. But what you get when you have a 3D organoid model is a much more complex model that's getting a little bit closer to representing the human brain. So it's nothing like an actual human brain.

Just to make that clear, we're not making like a mini brains or anything, but they have more diversity in cell types. They mimic the development of human brain very closely, and so it lets us see effects that are happening in more of a complex system that's more relevant to what's happening in people than just in a flat 2D system with just one or two cell types in it.

Oscar: Yeah, cool. So with that background in mind, I guess, could you give people like me that are not familiar, just a brief overview of the study and what you hope to accomplish, your goals.

Kat: That's a broad question. So yes, okay, my lab is interested in understanding the reasons why these mutations in the MAPT gene, in the tau gene, cause primary tauopathy. So we want to understand what's happening in the cell, what's going wrong that's causing them to die. And so what we found previously by using IPSC models with these mutations is that they're impacting mitochondria function. And so what we really want to now dig into is, how is that tau changing the way the mitochondria are functioning? Is it interacting with them directly? Is it changing the metabolism of the cell? Is it making them much more susceptible to stresses because they can no longer kind of keep up with energy demand?

And so the goal of this project is to really understand this association between tau and mitochondria. And if we can understand that, then we can understand how these mutations are causing disease. We might understand how people without mutations are also progressing towards disease, if we can see this same kind of mitochondrial dysfunction, and then the ultimate goal of that several years on, is to find a way to intervene in the process so that we can stop the cells from having bad mitochondria and to stop them from dying and to stop them from having tauopathy.

Oscar: Yeah, what led you to pursue mitochondrial dysfunction?

Kat: So I mean these mutation lines that we made were new, right? Nobody else had these, these lines with these particular mutations in them. So the very first thing that we did was to just characterize them without any hypothesis, without looking at anything specific. So we did single cell sequencing and bulk RNA sequencing to look at the we call it the transcriptome, right? So all the genes that are changed, or the expression that's changed when you have a mutation present, and doing that, we just saw over and over again this involvement of mitochondria.

So we didn't kind of seek it out, I suppose, just that's where the data pointed us, that the data was telling us that this is something that's really interesting, that's happening, and so that's why we decided to follow it up.

Oscar: How do MAPT gene mutations contribute to primary tauopathies, and why are they so important for your study?

Kat: Yeah so I mean no one really knows exactly how the mutations contribute to primary tauopathies. That's why we studied them. So we're, we're studying the mutations as a model for primary tauopathies to understand how cells are going wrong, what's changing in cells that's causing them to have disease. So that's the big question, right? Why do these mutations cause tauopathy? There's a million reasons mitochondria might be one of them, right? So other things that we're interested in are like synaptic functions, so the way neurons communicate with each other might be another way that these mutations cause disease. It's probably going to be a whole bunch of different processes that are being affected. Why they're so important for this study comes down to the model that we're using.

So my lab uses IPSC, which are induced pluripotent stem cells. So those are cells we so we take blood cells or skin cells from patients with these mutations that have FTD, PSP, and we can reprogram them, change them back into a cell type that has the potential to be anything, right? So that's the pluripotency aspect of it. So once we have IPSC, we can then make neurons or astrocytes or organoids or whatever model we're interested in. And the reason why we use these mutations that are called autosomal dominant, right? So if you have it, you will get the disease. There's kind of no ifs or buts about it is because we then know in our model that it's a disease model, if you see what I mean.

So if we took IPSC from a patient with a sporadic case of tauopathy, so that it wasn't associated with a mutation, it was just bad luck. When we model that in a dish, we're not really necessarily capturing the disease process, because there's no trigger in a dish that might be, you know, environmental tau factors or diet, there might be other genetic components that are contributing to it, but we can't pinpoint what that is exactly, so it's just too noisy of a model for what we do in in cell culture, whereas if we have a specific mutation that definitely causes disease, what we can then do is edit the DNA of that cell line to Remove the mutation. So then what we have is something called an isogenic control. So it's genetically identical to the patient line, but it doesn't have the mutation in it. So then that means, when we compare the mutation with isogenic control, any differences that we see we can be confident are due to that disease process, because the only difference between the lines is the mutation, whereas, if you have sporadic what would your control be? Just someone without disease? But then you don't know if any differences you see are just due to they have a different genetic background, right? There's hundreds, thousands, of genetic differences between individuals that aren't related to disease by having this isogenic pair that are identical, we can control for that. So that's why these mutation models are so important for people who do IPSC work.

Did that make sense? Was that helpful?

Oscar: Yeah. So this might not make sense, but what are like, the characteristics of a gene mutation and what does mitochondrial dysfunction look like? 

Kat: So the characteristics of a gene mutation, I suppose, if I understand your question properly, is the sequence of the DNA changed in such a way that it changes the way the gene functions. So, and we can refer to them as mutations if they are detrimental to human health, we rarely call beneficial things mutations. So in this case, for these MAPT mutations, there's a change in the DNA sequence which makes more for our tau kind of as I was describing earlier, right? So it's changing the way that the gene is behaving that contributes to disease. So, and that's the case for all kinds of different mutations, right? There's Alzheimer's disease mutations that specifically change the way different enzymes can process proteins in the cell.

So every mutation does a different thing, but generally bad things for mitochondrial dysfunction. What that looks like is also another very broad question. So I'm confident you will have heard that they're the powerhouse of the cell, right? So we kind of like the batteries for the cell. And so there's a few different ways that that can go wrong. You can either have the mitochondria not produce as much energy as they should, so they either have to work harder, which means they're under stress, or maybe the cell isn't getting as much energy as it really needs to be functioning properly. It might be that they are able to produce enough energy, but when you do add a stress to the cell, they're not able to cope with it. So instead of being able to increase their production, they just kind of stop and then become more susceptible to stresses, and they die instead of coping with the energy demand. Along that same idea that they're more susceptible to stresses, we find that quite a lot with MAPT mutations that if you stress out the mitochondria deliberately, those with MAPT mutations will just give up quicker. So yeah, then there's lots of different processes within mitochondria that will contribute to that, but it essentially all comes down to how well cells are able to produce energy and cope with changes in energetic demand.

Oscar: Yeah, that makes sense. That's that's helpful background because I'm trying to picture what the process looks like, and just thinking about your study more broadly. Have you had any collaborations in this process? Anyone that you've worked with that's been helpful? Any notable inspirations?

Kat: I've been really fortunate so far with all of my collaborations and mentors and people that have helped me get to this point, really. So, you know, the IPSC models were all introduced by my previous boss, my PI it was Alison Goate at Mount Sinai Sally temple, who's at the Neural Stem Cell Institute. She is driving the organoid efforts in tauopathies. So you know that our work in those 3D models is really directly from the collaboration that we've had with her over the last few years as well. I say the mitochondria is a new departure for us, but there's a few people out there, some new up and coming PIs in the UK, that have some similar interests that we're hoping to reach out to and collaborate with as well to do some really interesting things to complement this project on our end.

Oscar: So the mitochondria is your thing?

Kat: Well, it's starting to be right? This project and the funding from PSP is giving us that stepping stone into it, but it's not something that we've explored before with other collaborators. 

Oscar: What have been the biggest challenges, and just more generally, what do you see as the biggest barriers to research? Because I know this is a tough subject.

Kat: Oh, it's so tricky. There's so many.

Oh gosh, to break it down, the biggest barrier to research is the model. So the model is tricky, right? So to make organoids specifically in IPSC modeling, there's a lot of quality control you need to do to make sure you're working with exactly what you think you're working with. It takes a lot of protocol development, a lot of background work and prep work to do before you actually get to do a fun experiment.

And so for me, I really struggle with that patience, like waiting to do the actual experiment to get the actual result, because there's all of this prep that we have to do first, which is necessary and important, but it's not as fun. Funding always is a thing that's a struggle. I think funding in the UK especially is maybe not as extensive as funding in the US, in my experience, there's fewer pots of money, and the pots of money are smaller, so it's a lot more competitive to get kind of longer programs of work. I'd say those are probably the main issues. I think people are generally very good at sharing these days and collaborating and communication, which makes it not only easier to do the science, but a lot more fun as well. I find the best people for the job some sometimes can be a struggle if you're kind of looking for someone with specific experience in this very niche thing that you're studying. So a lot of the time you have to, you know, get people in with promise and potential and train them up. And so again, that kind of speaks to the time it takes to get to the fun stuff.