Dr Amy Smith is a Research Associate working on microglia heterogeneity in dementia and is part of the Multi-'Omics Atlas Project based at UK DRI at Imperial. In this Q&A, Dr Adesola Bello, a Research Assistant in the microbiome and brain health group, speaks to Amy about the role of microglia and inflammation in dementia, collaborative projects and her expectations for a return to the lab.
The following article is republished from Imperial College London (22 July 2020).
AB: Can you please tell us briefly about your background?
AS: I started off my lab research life in Auckland in New Zealand. I did an undergrad in Biomedical Science, because I was generally interested in science and I thought that that would be a nice way to apply that interest. I really enjoyed it. Towards the end, I started focusing in on Neuroscience, and I did my PhD on microglia in the human brain. I liked the idea of studying microglia because it's a nice link between two different organ systems of the body, the central nervous system and the immune system, and how they link up has always kind of fascinated me. Also, I was fortunate enough to study in a place where, like Imperial, we had a brain bank right next to us and integrated into the labs. It was a great opportunity because not many labs do that around the world. That’s how I got to work with primary human microglia for the first time. Then, after my PhD, I had a look for postdoc opportunities overseas and was offered a postdoc in Oxford. That gave me a really great opportunity to move across to the UK and do my first postdoc at Oxford University. There, I branched out a little bit to study blood cells from people with Parkinson's disease. I was still keeping on the theme of neuroimmunology but it was more of a biomarker-based project. That was a lot of cycling up the hill between the lab and the John Radcliffe hospital with blood samples in my backpack. But I realised that I really missed working with microglia. When I was looking for my next position - the UK DRI was just starting up at the time - I heard that Imperial was going to have a focus in neuroinflammation. I decided that this would be a great opportunity to get back into that area of research and back into microglia. And that's how I ended up here at Imperial working with the UK DRI.
AB: Has research, more specifically academic research, always been the goal for you?
AS: I think for a long time it has. I first had dreams of becoming an Olympic swimmer, but my parents gently steered me away from that. Then, the next phase was marine biology. But for quite a long time now actually, I remember having an idea of medical research. And that probably came from being interested in science and also wanting to find out new knowledge that helped people.
AB: You talked a lot about the microglia. What are microglial cells? And I would like you to explain it to me at three levels of complexity that each of the following people would understand: an 8 year old Primary School pupil, a Year 9 teen and a Neuroscience undergrad in her first lecture.
AS: Wow! Okay, I’ll give it a go:
To the child: A microglia is like a busy cleaner in the brain cleaning up nicely so that our brain can function properly.
To the teen: I’d say it's an immune cell that lives in the brain and one of its main jobs is to get rid of things that aren't meant to be there so that the other cells can do their job properly.
To the undergrad: I’d say microglia are the immune cells of the brain. They're like macrophages in the rest of the body, but they have some brain specific functions too. While they were initially thought to be support cells that clear up debris in the brain, we now know that they also interact with other cell types, including neurons, and are involved in many if not all brain disorders.
AB: That was really good! So, what's their role in dementia in particular and why have you focused on them in your research?
AS: Microglia play many beneficial roles in the brain. One of the main ones is phagocytosis which is to get rid of dying cells, or accumulations of proteins, or organelles, but they have this innate response to foreign objects as well or to things that shouldn't be there in the brain. This response, if prolonged, can have a negative effect and can start contributing to more cell death and perhaps changing the way other cells function. There seems to be a fine balance in the brain but as we age, this balance can change. By the time we have dementia, we know that there are lots of hallmarks of inflammatory processes in the brain. So, we are trying to understand the steps that happen to get to that point; how the microglia lose their beneficial roles and how they gain these detrimental inflammatory roles.
AB: Can you explain how big of a role inflammation plays in dementia, particularly in Alzheimer’s disease? Also, from what you know so far, would you say that microglial dysfunction causes the onset of neurodegeneration or is it a response to something else initiating the pathological process?
AS: There is certainly some evidence for both. And I think in different people, there will be a different group of causative factors. In some people, we know that their genetics predisposes them, and particularly their microglia, to behave differently. But we also know that microglia will respond to something else that occurs, like a dying neuron or amyloid beta. So, it's almost certainly going to be both with different things happening at different stages. And one component might be more important for one patient, and another component might be more important for another patient. About inflammation in Alzheimer's disease, well, we know that it's present at the end stage of disease for sure. In the 1990s, people showed an increase in inflammatory markers in Alzheimer’s brains compared to control brains. So, there's definitely evidence for microglia becoming activated and producing molecules that can have a pro-inflammatory effect. What we're less sure about is how that process comes about, how it starts. But we know that it can have a detrimental effect. We know that microglia can cause neurons to die, and we know that they can also inhibit things like synaptic plasticity. But then we're also learning about ways that you get different types of inflammation. It is likely that the way we use that word will change over time, because ‘inflammation’ in say multiple sclerosis doesn't mean the same thing as ‘inflammation’ in Alzheimer’s disease. And if we learn more, I suppose we might learn that it's not the same as inflammation in Parkinson's disease. But I expect that there will be some common components as well as differences.
AB: Interesting. So, do microglia play same or different roles in the different dementias? How does your research on microglia contribute to the different niches in the field of dementia?
AS: One reason I like studying microglia is because I think knowing more about them could help us with a whole range of diseases, and not just dementia or not just one type of dementia. One hopeful outcome could be that we learn about something beneficial that microglia do, and perhaps that therapeutics targeting microglia could boost that function. And it would be helpful for a whole lot of brain degenerating states or dementias. But there could be other situations, like where there's a genetic predisposition, where there's a particular microglia pathway that is affected. I haven't studied this directly, but I imagine that could also be possible. But there is still a lot of scope for common pathways or functions in microglia that could be targeted to treat many different dementias and disorders.
With a greater understanding of the roles that microglia play in our brains in control states as well as in disease states, we're realising that they do more than just wait around for an infection or wait around for something bad to happen. This new field of neuroimmunology and neuroinflammation is looking at earlier stages than we've ever looked at before to try and find out how microglia could contribute to disease. I also think that, because I'm quite interested in the connection between the immune system and the brain, microglia are an ideal cell type to convey information from the rest of our body to the brain. I see them as part of the link between the peripheral and environmental contributors that several other groups in the UK DRI are focused on, like your metabolomics research, and loss of function in the brain. I think if there are disturbances that we can detect in the blood and in other organs, which I'm sure there are in Alzheimer's disease, then possibly one of the ways that they can affect the brain is through microglia.
AB: Can you describe how you study the microglia? What major technique(s) do you use in your research?
AS: The exciting new technique that we're using in this current project makes use of post mortem human brain tissue and also allows us to look at the level of an individual cell. The microglia are not a large percentage of brain cells, so I’ve been working on a way to isolate microglia to focus our study on them and gain more insight into what they're doing. This new technique is called single nuclei RNA sequencing. It provides information on thousands of genes that each single microglia expresses; giving us a huge amount of data and allowing us to see how different and heterogeneous the microglia in our brains are. Our hope is that this will be useful to develop some more specific therapeutic strategies to target these cells. It's not going to be a matter of just turning the microglia off. They're much more complicated than that and we're going to have to look in a lot of detail to find particular pathways to target.
We start with a block of frozen, archived human brain tissue and have a matched control cohort to study. We select a particular brain region and we break apart the tissue using mechanical force and some detergent. While the individual cells don't stay intact because their membranes break, the nuclei do stay intact, because the nuclear membrane is relatively robust. From this we get a suspension of individual nuclei from a piece of brain tissue. And then what I have spent quite a bit of time developing is a way to sort those nuclei, so that we can look specifically at microglia. Because if we take all of those nuclei obtained directly from the suspension, there's a lot of neurons and oligodendrocytes with a relatively small population of microglia. We use fluorescence activated cell sorting (FACS) - although we've actually turned it into fluorescence activated nuclei sorting, it’s the same thing except a lot smaller- to obtain a population of mostly microglia nuclei.
One reason I like studying microglia is because I think knowing more about them could help us with a whole range of diseases, and not just dementia or not just one type of dementia. Dr Amy Smith, UK DRI at Imperial
AB: How does FACS work? How do you ensure you are looking at a certain cell type (in this case microglia) and not another for instance?
AS: We use a really clever droplet based micro fluidics machine, which takes these single nuclei and encapsulates them in an oil droplet, alongside beads that label the RNA with barcodes. This allows all of the transcripts from a single nucleus to maintain their unique barcode when we isolate the RNA. After the sequencing step, we can then identify which gene comes from an individual nucleus by this barcode, that's the way to get out discrete single cell information. Say it's a microglia nucleus that goes through, some of the genes will be more specific to microglia than to other cell types. First, we identify the type of nucleus that we have. If we're looking at thousands of cells, we visualise this huge amount of data by clustering the nuclei together based on their similarity of gene expression. And then we look at these nuclei in the cluster and we look for specific cell marker genes. Once we've identified the microglia, then we can look at the other genes that are more interesting and see how they're different between say control and AD brains.
AB: That sounds like a lot of data being generated! Who handles all of this data?
AS: Yes it is a lot! So it's a group effort. Firstly, the Imperial genomics sequencing facility process the raw data that comes off the sequencer. And then there are quality control (QC) steps that they partly do, and then we partly do as well. There's a team of us who decide on the QC checks. And then, there's a lot of different analysis techniques that can be done with this type of data. It's quite fun. We're always learning new ways to look at the data and to make sense of it.
AB: That sort of leads on to my next couple of questions about collaborations; specifically about working on an interdisciplinary collaborative project/in a multidisciplinary team and also about initiating collaborations of your own. Firstly, I know you are part of the Multi-Omics Atlas Project (MAP) team. What, if you were involved in the initiation of the project, led to MAP and how does this contribute to your own work and to the advancement of dementia research at large?
AS: I'll talk about the Multi-omics Atlas project first maybe. MAP is a really exciting venture that's based on the preliminary work that I've done with the single nuclei RNA sequencing technique. But it's going to allow us to look in a lot more detail in a lot of different brain regions. It's going to answer some questions that are very difficult to answer by one group alone, and it's a big collaboration, like we were talking about before, involving people who work in brain banks, collecting tissue, pathologists, molecular scientists, and then data analysts. And, we also hope that a lot of other people will do different types of analyses using these brains so that we can look at these brains with as many different angles and techniques as possible and then piece it all together.
AB: I am also aware that you won a grant from the inaugural UK DRI Pilot Studies programme for a joint project with Dr Carola Radulescu, a post-doc in Dr Sam Barnes’ lab. How did you and Carola find each other? How was the collaboration initiated?
AS: For the pilot project, one way that collaborations can start is just by coming across a paper or a concept and talking to someone new about it. I came across a paper on a technique called ‘patch-seq’. And I thought, this other lab is doing patch clamping, I'm starting out learning about RNA sequencing, I wonder if we can link these two. And it was just the start of flicking off an email and saying, Oh, this looks like something that could link our two techniques together. The Pilot Grant has been a really great opportunity to work with a fantastic postdoc, Carola, who's in Sam Barnes' lab. And because we come from quite different approaches to studying dementia, we've had a lot of fun explaining our different techniques to each other. Definitely, we've all learned a lot in the process, and in finding ways to bridge a gap between our two fields. Not only do we have differences in techniques, we also have differences in the cells that we focus on. Where I've been focusing on microglia, they focus on neurons, and through this we both have developed better appreciation for the other cells. It's been a lot of learning about new techniques and about different approaches to studying the same disorder, it's been really interesting.
AB: As an Early Career Researcher, have you found collaboration quite easy or challenging, and in what ways?
AS: I’d say the main challenge is communication. Indeed, it's a learning curve to explain your expertise or your technique to people who don't have the same background as you. But it's great to see things from another point of view as well and be able to communicate the common goal. I think that it's most fun when people have a common goal of finding out new knowledge and asking and answering an important question.
AB: Just out of curiosity, what was the process of the grant writing like? Had you done any grant writing before that one?
AS: That was the first grant writing that I'd done. And we were very lucky to get a lot of guidance from Sam. It was a great way to learn about what to put in a grant and to make it sound appealing, but also well thought through. It took more time than I anticipated. That's probably a general rule with grant-writing.
AB: How long did it take?
AS: We must have worked on it over a couple of months. I have to say that Carola put a lot of work into it and she had some amazing pilot data that really made it look good. Also, Carola is much better at putting figures together than I am - that was really helpful!
AB: Nice, so together, both of you were the Dream Team! How's the project going now? Has it ended or is it still ongoing?
AS: Yes, before we had to start working from home, we had optimised the method which involves doing the FAC sorting on nuclei from animals that Carola has done electrophysiology and imaging studies with. We sequenced one pilot sample, but now we're just waiting to finish off the whole cohort and send it for sequencing to get our large amounts of data but luckily we have a bit of pilot data and it looks like we're on the right track.
AB: That's amazing. When do you have to give feedback to the UK DRI committee in charge of the programme?
AS: They did email us near the beginning of lockdown with information about extending the grant. So, we have until the end of October now, which is a couple of extra months. It won't be a lot of time. And that's probably what I will first have to go back into the lab for is to finish off these samples. I believe if we do some initial analysis, and can show that we have the data, then that will be enough. And then hopefully we can work away at the full analysis with a bit more time.
AB: Are you looking forward to going back to working in a socially distancing lab? What do you imagine that would look like? How do you think that would affect your work and the progress of what you have to do?
AS: What I've learned is having to exercise a bit of extra patience at each step and I feel we'll have to continue to do that. I'm looking forward to getting to work in the new Michael Uren building at the White City Campus. It’s got new labs and an office space. That'll be a fun incentive to go back, and it will definitely be nice to break up the week with being somewhere else. And of course, seeing people again will be the main benefit really. I miss seeing people's faces in person, but working from home does have its upsides. I've enjoyed not having to commute and being able to have the occasional run at lunchtime. Those things have been good. It has also been a time to reflect on what things are important to us. And if there are ways that we can change up our routine for the better, then we can take this opportunity to do it.
AB: You're definitely on Team positive. Going forward, would you want to have like a few days working at home kind of week or like a balance of working from home and going into work?
AS: Yes, I agree, that would be a nice option. I know being forced to work from home certainly hasn't been an easy time for anyone. But when we have more certainty in our daily lives, it'd be nice to have more flexible working arrangements. Hopefully it'll reduce things like rush hour and a packed Central line. It would be nice to get some lasting positive effects out of this.
AB: I really want to thank you for doing this interview. It's reminded me just how much fun science is. This was fun, I hope you enjoyed the chat.
AS: Yes I did too, thank you. It was a nice chat, and I think you phrased the questions really well.
Article published: 27 July 2020
