ResearchPod
ResearchPod
RNA sequencing in Alzheimers investigations
We humans share over 99% of our DNA with each other. This means personalised therapies for diseases such as cancer or neurodegenerative conditions have to be tailored to the most minute differences between us - or even between our own cells.
Professor Mark Ebbert of the Sanders-Brown Center on Aging at the University of Kentucky leads a lab focusing on isoforms of RNA - tiny, short lived molecules - that could open whole new avenues for detection, diagnosis, and treatments of otherwise incurable diseases.
Read the original research: https://doi.org/10.1101/2023.08.06.552162
Find more at The Ebbert Lab
00:00:05 Will Mountford
Hello. I'm Will. Welcome to research pod.
00:00:08 Will Mountford
It's a familiar factoid that human beings as varied as we are share over 99% of our DNA with each other. This means developments in personalized therapies for diseases such as cancer or neurodegenerative conditions have to be tailored to the most minute differences between us, or even between our own cells.
Doctor Mark Ebbert of the Sanders Brown Center on Aging at the University of Kentucky leads a lab focusing on the most minute of variations, in short lived molecules, isoforms of RNA's that could open up whole new avenues for detection, diagnosis, and treatments of otherwise incurable diseases.
00:00:48 Will Mountford
Joining me from the University of Kentucky doctor Mark Ebbert. Mark, hello.
00:00:51 Prof Mark Ebbert
Hello, how are you?
00:00:52 Will Mountford
I'm very well and thanks for joining us today for my sake and for everybody listening at home, could you tell me a bit about you yourself, your lab and the work that it is that you do?
00:01:01 Prof Mark Ebbert
Yeah. So I I joined the University of Kentucky a few years ago in December of 2020, kind of in the in the heat of the the pandemic.
00:01:09 Prof Mark Ebbert
He was really excited to be here at the University of Kentucky and specifically in the Sanders Brown Center on Aging, a really unique opportunity and and truly a privilege to be part of the Sanders ground center.
00:01:21 Prof Mark Ebbert
On.
00:01:21 Prof Mark Ebbert
Aging and you know, for me, you know, performing, you know, disease, relevant research.
00:01:29 Prof Mark Ebbert
Is so valuable because all around us people are struggling with with various diseases, and one of those that is, you know, so impactful to to many of us and people we have known.
00:01:44 Prof Mark Ebbert
Is Alzheimer's disease and your every disease has its challenges. But one of the the most challenging parts, the most painful parts of Alzheimer's disease, is is sort of seeing someone be erased from their own mind and seeing loved ones.
00:02:03 Prof Mark Ebbert
And memories of loved ones being erased from their minds. And that's a really being a part of that is, is is very meaningful, you know, to me and the Members.
00:02:12 Prof Mark Ebbert
Of my lab.
00:02:14 Prof Mark Ebbert
Yeah. So I've been involved in Alzheimer's disease research for, I guess, about 12 years now and.
00:02:22 Prof Mark Ebbert
You know our our lab goals are, you know, I think like most Alzheimer's disease, researchers are to help develop ways to improve disease treatment, even prevent disease if possible through developing therapies. And then the second aspect of our lab that is at least as important.
00:02:43 Prof Mark Ebbert
As developing meaningful therapies is developing ways to detect disease before symptoms onset, so the the importance of a pre symptomatic disease diagnosis.
00:02:56 Prof Mark Ebbert
Because if any meaningful therapy won't be as you as impactful or meaningful, if we can't detect disease before symptoms onset, because by the time symptoms of onset, the disease has already progressed to a point that the brain can no longer compensate for all the cellular death. So those are the two main branches.
00:03:16 Prof Mark Ebbert
Of our labs research and and we do this using both computational approaches and genomics approaches. So and specifically using long read RNA and DNA sequencing.
00:03:30 Prof Mark Ebbert
So that's kind of the the basic background on on what we're trying to accomplish and how we're.
00:03:36 Prof Mark Ebbert
Doing it.
00:03:41 Prof Mark Ebbert
Alzheimer's disease, you know, I think as many people know, is a neurodegenerative disease. So neurons are dying in the brain. And what's really driving that neurodegeneration? As we've learned more more clearly in recent years.
00:03:58 Prof Mark Ebbert
Is the relationship between the amyloid beta plaques and the neurofibrillary tangles. Those are the two hallmark pathologies that are present in Alzheimer's disease, brain at autopsy, and that degeneration in in the disease has actually been going on in the brain for.
00:04:18 Prof Mark Ebbert
You potentially decades by the time the symptoms onset and so you know that just really emphasizes the importance of of, you know, treating disease long before symptoms onset. But you know, ultimately what's what's driving disease.
00:04:34 Prof Mark Ebbert
It's pretty complicated. It can be a range of of genetic factors and environmental factors. And what we're mostly focused on in the Everett lab are the genetic and molecular factors. What's what's happening within the cells. So within the genetic.
00:04:54 Prof Mark Ebbert
Aspects. What is a gene? You know, most people you understand the basic concept of a of a gene which is, you know, from our DNA we get a copy of DNA from Mom and a copy of DNA from Dad and, you know, fundamentally a gene is just a sequence of DNA and that that gene.
00:05:13 Prof Mark Ebbert
Ultimately, typically encodes for a protein, and I like to think of proteins as being all the little workers in our body and in our cells that go about doing things and making life possible, making cells behave how they're supposed to behave. So when.
00:05:33 Prof Mark Ebbert
Genes misbehave. You know that's generally when disease comes about, whether it's cancer or Alzheimer's disease and over the.
00:05:43 Prof Mark Ebbert
Last couple of decades, we've as a community as a research community, have discovered somewhere between 70 and 80 genes that are known to be involved in Alzheimer's disease. The challenge there, though, is that we don't know exactly how these genes are involved. We have.
00:06:03 Prof Mark Ebbert
Basic ideas and understandings, but the exact mechanism is still a mystery, and so we're working to help identify exactly what's going wrong with these individual genes.
00:06:18 Will Mountford
They say these genes go wrong. Does that mean mutations? Is that decay healthy?
00:06:23 Will Mountford
The proteins different from unhealthy ones.
00:06:26 Prof Mark Ebbert
Yeah, that's a great question. So jeans, they can go wrong, you know, through multiple methods, multiple ways it, it could be something that's purely genetic, something that was inherited through.
00:06:39 Prof Mark Ebbert
Your ancestry from your parents, so a variant or a mutation in that gene that makes it so that it cannot behave properly.
00:06:49 Prof Mark Ebbert
Basic concept there would be that it loses its function or on the opposite end of that spectrum it could be a mutation that causes it to do something it shouldn't be doing another mechanism through which genes can can misbehave is that you may be the gene itself is.
00:07:09 Prof Mark Ebbert
Intact and there's nothing inherently wrong with its sequence, but for whatever reason later in life begins to be either overly active or under active.
00:07:22 Prof Mark Ebbert
One of the things that's not.
00:07:24
You know.
00:07:25 Prof Mark Ebbert
Very well talked about.
00:07:26 Prof Mark Ebbert
Or or or very well understood even.
00:07:29 Prof Mark Ebbert
Is that we talk about genes, you know, any given gene as though it has a specific function. But it turns out that.
00:07:37 Prof Mark Ebbert
You know about half.
00:07:38 Prof Mark Ebbert
Of human genes in code for multiple different versions of itself, and that happens through what's known as alternative splicing. So basically a gene.
00:07:49 Prof Mark Ebbert
Is made-up of these individual segments.
00:07:52 Prof Mark Ebbert
That we call exons, and when that gene is being expressed, when it's when it's being activated, different combinations of those exons can be produced, and that results in a different protein. Or, as I like to think of a different worker. But we.
00:08:10 Prof Mark Ebbert
Know very little.
00:08:12 Prof Mark Ebbert
About the individualized for most genes. So for example, there's one Alzheimer's disease.
00:08:19 Prof Mark Ebbert
Gene, that has been long implicated in disease. It's known as Ben 1B IN one that specific gene is actively expressing 8 different what we call isoforms, 8 different versions of that same gene. And we know very little about those different isoforms.
00:08:40 Prof Mark Ebbert
Whether some of them are just noise or junk, I personally doubt.
00:08:46 Prof Mark Ebbert
That or if each individual isoform actually has its own unique function, even if it's a subtle difference in function, there are some genes that you know not necessarily related to Alzheimer's disease, where we know that the different isoforms are performing different functions. Maybe the the classic example.
00:09:06 Prof Mark Ebbert
And again, this this gene is not directly involved in Alzheimer's disease.
00:09:11 Prof Mark Ebbert
But it's known as BCLC X1 isoform of this gene. One version of this, this gene actually promotes cell death, while the other isoform does the opposite. It promotes cellular health. So we know that this definitely occurs, but for most genes, we just don't know. We just don't know.
00:09:31 Prof Mark Ebbert
So that's really a major part of what we're working to do is understand and determine the individual function for genes throughout the body that the ice, the different isoforms for sing.
00:09:44 Prof Mark Ebbert
Gene and in particular, Alzheimer's disease genes.
00:09:48 Will Mountford
And we can hypothesize the nature of those mutations, those isoforms, the alternative splicing, because if it is something that, you know, creates functional or mostly functional versions of a protein, is that offering any particular evolutionary advantage, do you think or kind of like the the long form multi generational view of a species?
00:10:08 Will Mountford
Or is it just kind of the chaotic nature of we are jamming atoms together to make molecules in a cell and this is sometimes just what happens?
00:10:16 Prof Mark Ebbert
The truth is.
00:10:17 Prof Mark Ebbert
It could be a combination of the.
00:10:18 Prof Mark Ebbert
You generally speaking though it has been well established that this mechanism of alternative splicing, this mechanism of a single gene being able to encode multiple versions actually makes complex life possible. So the more complex organisms.
00:10:39 Prof Mark Ebbert
Tend to have greater, you know, isoform diversity among genes. It wasn't until around the the early 2010.
00:10:48 Prof Mark Ebbert
That sequencing became truly accessible to researchers like myself, and it began with what we call short read sequencing and and like you alluded to, it's it's just like it sounds there's, there's short reads versus long reads. And historically, we've we've relied on short read sequencing, which has really been a major boon for reset.
00:11:08 Prof Mark Ebbert
Search but with short read the challenges that were genuinely only sequencing small portions of the DNA or RNA somewhere between 100 to maybe up to 200 nucleotides at a time, and then we're left to kind of piece those back together to try to infer and build the genome structure and and or RNA.
00:11:28 Prof Mark Ebbert
Isoform structure. But the truth is those those reeds are just too short to be able to do this properly.
00:11:34 Prof Mark Ebbert
So with long read sequencing we can sequence up to 10s of thousands of nucleotides in a single go, which means it's a lot. In principle it's a lot easier to construct the full genome structure, which is very important, or at the RNA level we can actually.
00:11:55 Prof Mark Ebbert
Now begin to measure the expression levels of individual isoforms for single gene. Whereas with the short read sequencing a major limitation with RNA sequence.
00:12:07 Prof Mark Ebbert
Is that we've been forced to collapse the expression of individual isoforms for a single gene into a single expression measurement, so it's made it you essentially impossible for us to determine is isoform a involved in disease versus isoform B, whereas now with long read sequencing we can do that.
00:12:27 Prof Mark Ebbert
It's it's truly exciting.
00:12:29 Will Mountford
And the samples that you're working with for these longer sequences, they, as I understand it from actual human samples, postmortem brain tissue, is that right?
00:12:39 Prof Mark Ebbert
Yes, and that's one of the really unique things.
00:12:42 Prof Mark Ebbert
About our work.
00:12:43 Prof Mark Ebbert
And being here at the Sanders Brown Center on Aging at the University of Kentucky is that we have one of the best Alzheimer's disease brain banks really in the world.
00:12:54 Prof Mark Ebbert
It's a very unique cohort in terms of what we've been able to collect. So and what makes that so important in our research and really in understanding Alzheimer's disease is we want to work in the diseased tissue because you know, science and and understanding disease. The truth is it's it's difficult.
00:13:15 Prof Mark Ebbert
It's complicated.
00:13:16 Prof Mark Ebbert
And for various reasons, many researchers have to work very distal from the the diseased tissue. But we have this unique opportunity to actually work in the disease tissue and try to understand what's actually going wrong at the molecular level at the cellular level within the disease, the disease.
00:13:36 Prof Mark Ebbert
Issue.
00:13:36 Will Mountford
I mean, you've mentioned that it's one of the finest cohorts of this nature in the world. Is that just due to the geography or something about the health system you're working in or is that no indicative of more pressing health concerns across Kentucky, that there are that many more people in the area who have a rigid donor, degenerative condition?
00:13:54 Prof Mark Ebbert
Yeah, that's a great question. So you know, one of the interesting things maybe maybe surprising until you think about it, is that the biggest challenge in a lot of disease research is not actually obtaining samples from those that are affected by disease, but those that are not affected. And I think that is even more challenging.
00:14:14 Prof Mark Ebbert
When it comes to obtaining brain samples.
00:14:17 Prof Mark Ebbert
Because we only obtain them postmortem from those that are eager to contribute to the this this form of research to understanding and defeating Alzheimer's disease. So, you know, there are, there are various brain banks around the world that have collected a large number of Alzheimer's diseased brains.
00:14:37 Prof Mark Ebbert
But the Sanders, Brown, Center and aging under the direction of Doctor Bill Marksbury very early in its days back in the 1970s and 80s.
00:14:47 Prof Mark Ebbert
Recognize how important it is to start recruiting individuals who are not affected by disease, and that's one of the main reasons that our cohort is so unique is we have potentially one of the largest cohorts of non diseased brains out there. And of course you know.
00:15:06 Prof Mark Ebbert
Maybe the the obvious question is.
00:15:08 Prof Mark Ebbert
Why does that matter?
00:15:10 Prof Mark Ebbert
Well, we have to know what normal looks like so that we can determine what disease looks like. Right. And and that's really challenging at the molecular level. So so having such a large collection of non diseased brains is so critical and that this is really a, I think, a great opportunity to.
00:15:30 Prof Mark Ebbert
The size and think all those who contribute to this research, the patients, their loved ones, their caregivers, those who contribute so selflessly at every stage of disease, including those and perhaps especially of those that are willing and eager to even.
00:15:51 Prof Mark Ebbert
Allow us to obtain brain samples postmortem.
00:15:59 Prof Mark Ebbert
You we're obtaining these individual brain samples and we go through a very careful process to extract the DNA or RNA as the case may.
00:16:09 Prof Mark Ebbert
Be.
00:16:10 Prof Mark Ebbert
Especially for long range sequencing, because we need the DNA and RNA to be as intact as possible, and it turns out that's quite challenging because.
00:16:19 Prof Mark Ebbert
You are our cells are regularly trying to maintain balance within the cell, maintain health and as part of that, it's really it it quickly after death. It quickly breaks down DNA and RNA. On top of that because these molecules are actually so fragile, getting them out.
00:16:40 Prof Mark Ebbert
Of the tissue intact is.
00:16:42 Prof Mark Ebbert
Typical. So that process just the extraction process can take several hours, and then there's a lot of additional work to clean up the sample to remove all of the excess proteins and cellular debris, and preparing this the the DNA or RNA for sequencing so.
00:17:02 Prof Mark Ebbert
In all, the process can take a couple two to three days before we can actually begin the sequencing, and then the sequencing itself will take approximately 3 days as well.
00:17:13 Will Mountford
I suppose the question now is, what does all of that tell us?
00:17:16 Will Mountford
What are the discoveries? What is the work of the Abbott lab and what kind of breakthroughs are do you think you might be working on or anything that's on the cusp of changing the state of knowledge or care for Alzheimer's?
00:17:29 Prof Mark Ebbert
We just published a paper recently in the Journal of Nature Biotechnology, which is a fantastic journal where we demonstrate this proof of principle for how important these technologies are for how important this specific study is for doing long read.
00:17:48 Prof Mark Ebbert
RNA sequencing in human Alzheimer's disease brains versus controls in that study.
00:17:55 Prof Mark Ebbert
We made several important discoveries, some of which are actually not directly related to Alzheimer's, but but again demonstrate the importance of this where looking across all of the known disease relevant genes, regardless of the disease, we discovered hundreds of isoforms.
00:18:15 Prof Mark Ebbert
Burning isoforms that had never been measured before never been seen before, some of which are very highly expressed. Very, very common in the brain.
00:18:27 Prof Mark Ebbert
And on top of that, we actually discovered more than 200 entirely new genes that had never been described before. And you are those specific isoforms or genes directly involved in disease. You're probably not. Maybe some of them, but again, it's a very important proof of principle for and demonstrates how much.
00:18:47 Prof Mark Ebbert
There is still to learn.
00:18:50 Prof Mark Ebbert
Human DNA and genes, human genomics, human health, and disease at the at the molecular level and then the part that's most directly applicable to Alzheimer's disease is again a proof of principle study where we compared RNA isoform expression.
00:19:10 Prof Mark Ebbert
Between cases and controls, again, this is in human frontal cortex, and we found 99 isoforms that were differentially expressed between cases and controls, where at the gene level it wasn't. And just to clarify what I mean by that, going back to what I was saying earlier.
00:19:29 Prof Mark Ebbert
With standard short read sequencing, we're forced to collapse the expression for individual isoforms into a single expression measurement for that gene, whereas with long reads we can measure expression for those individual isoforms. So at the gene level, basically using the historic approach.
00:19:48 Prof Mark Ebbert
There was no differential expression between cases and controls until we look at the expression at the end of the individualized reforms. So again, a really important proof of principle, and we're actually in the middle of expanding that study into a much larger cohort of hundreds of cases and.
00:20:06 Prof Mark Ebbert
Roles so that we can hopefully identify RNA isoforms that are, if not directly involved in disease, or at least indicative of disease, so that we can develop a pre symptomatic disease diagnostic. And if it turns out that those individual isoforms are in fact involved in disease, then that gives us new.
00:20:27 Prof Mark Ebbert
Targets for therapies.
00:20:30 Prof Mark Ebbert
So there's really a lot of great potential for this study and we're really excited to move forward and very eager to see what comes of this.
00:20:39 Will Mountford
Is there anywhere else in the body that these ice forms might turn up that could be useful for biomarker? I'm just thinking that if I, you know, suspected that I might or a loved 1 might be coming down with something like Alzheimer's, I'm not sure that putting myself forward to having a full brain surgery prospectively to maybe find out.
00:21:00 Will Mountford
Like, that's a lot of commitment, but if there was like a yearing test or a blood draw or something, could it be as simple as that?
00:21:06 Prof Mark Ebbert
Yes, you've touched on a really key point. We're generally not very eager to give up a piece of our brain while we're alive and that's even the biggest limit limitation of what we're doing. But that's actually intentional. So as I alluded to earlier.
00:21:22 Prof Mark Ebbert
Here our goal is to to study the the disease in the diseased tissue so that we can first understand what's going wrong in the disease tissue right from there. The goal is to work backwards and see if we can measure these outside of the brain in a much less invasive way. The most obvious.
00:21:42 Prof Mark Ebbert
Which is also not ideal for patients, is to is to try to measure it in cerebral spinal fluid, donating cerebral spinal fluid is far less invasive than.
00:21:53 Prof Mark Ebbert
Then you know, donating a brain sample, but it's still not pleasant that that requires a Spinal Tap and and it's very uncomfortable both during the procedure and after the procedure. But that would still be a huge win if if that were to work. The Holy Grail generally in medicine is to be able to detect.
00:22:13 Prof Mark Ebbert
And measure things in the blood, because that's generally far less invasive and generally not a big issue for patients. So once we measure these and identify them in the brain, that will be the goal is to work backwards, look in the CSF, the surface spinal fluid, look in the blood and see if we can measure, measure these there.
00:22:36 Will Mountford
What do the isoform findings not tell us?
00:22:40 Prof Mark Ebbert
The central dogma of biology is fairly simple. We have our DNA. That's our we often think of that as the blueprint, so that you know your DNA is the blueprint to build.
00:22:52 Prof Mark Ebbert
You but a blueprint in and of itself is not very useful if you don't do anything with it, right? You need to. Actually you use it. And so DNA is transcribed into RNA, and that RNA is then typically translated into the proteins that I've referred to earlier.
00:23:12 Prof Mark Ebbert
That's being you you typically the workers that, that, that make life possible. So for the most part RNA's and and and and sequencing RNA isoforms.
00:23:24 Prof Mark Ebbert
It's sort of a an intermediary step. So everything that we're measuring at the RNA level is generally just an attempt to extrapolate what's happening at the protein level because it's not, there aren't very any high throughput methods for sequencing protein. That's sort of the Holy Grail and.
00:23:44 Prof Mark Ebbert
In genomics research, but there's really there's so many gaps in our understanding of the central dogma biology, even though we've, we've we've known the basic concepts for decades.
00:23:55 Prof Mark Ebbert
For example, if you measure the expression of a gene at the RNA level and then measure it at the protein level, those measurements often don't agree, and that's something we don't understand very well. But that's a big part of what we're trying to help gap. We're trying to help fill is why is that happening? And?
00:24:15
So.
00:24:15 Prof Mark Ebbert
There's really a lot that we don't know. And so you know, again, you know, science is very, very challenging, very complicated and and generally we're trying to look at these small pieces and try to piece them together or extrapolate what we can't measure based on those.
00:24:32 Will Mountford
Seeing if there are any peers or professionals out there listening to this who want to work with kind of this library of knowledge and all of these DNA sequences that you're missing, the RNA long reads and short reads. Is it something that they can remotely join in with?
00:24:47 Prof Mark Ebbert
Absolutely. In fact, we're already aware of many labs that have downloaded and already been using the data that we generated and that study I I described a moment ago.
00:24:58 Prof Mark Ebbert
Because they're here to see the same.
00:24:59 Prof Mark Ebbert
Things we're here to look at, they want to.
00:25:01 Prof Mark Ebbert
Know.
00:25:02 Prof Mark Ebbert
What's going wrong and part of what's so exciting about the data we're generating you, we have our ideas. We have the things that we want to look at, but you know we don't. We don't have a monopoly in all of the great ideas out there. Obviously, right. There are lots of fantastic scientists with their own ideas.
00:25:19 Prof Mark Ebbert
And ways that they want to explore this data. And so you know, as part of Everett lab policies, we.
00:25:25 Prof Mark Ebbert
Make our data available as early as possible so that other labs can obtain it and they may find things that we miss, and that's that's a very important part of science and and very exciting is that whether we find it or someone else finds it, you know, the goal is to ultimately improve.
00:25:45 Prof Mark Ebbert
Patient health.
00:25:47 Will Mountford
If anyone listening to this is of influence to members of the public, 2 practitioners or two policymakers, and they want to get involved in your research and in outsiders and advocacy more, what would be a good place to start with you?
00:26:00 Prof Mark Ebbert
We'd encourage you to.
00:26:01 Prof Mark Ebbert
To reach out due to me.
00:26:03 Prof Mark Ebbert
Likely to the Sanders Brown Center on Aging, we have obviously, you know, there are lots of other fantastic researchers here doing some really important research that is complementary to what we're.
00:26:15 Prof Mark Ebbert
Doing.
00:26:19 Prof Mark Ebbert
A team effort. Obviously it requires the patients.
00:26:23 Prof Mark Ebbert
Caregivers, the loved ones, the those that are willing to, you know, selflessly donate their time and tissue.
00:26:32 Prof Mark Ebbert
One of the key things that we need that that we're as a constant challenge is funding. You know, these studies are extremely expensive and it takes a lot of resources. We really need the help of everyone. You, especially the patients and their loved ones. And you're really encourage them to reach out to me and to the Sanders.
00:26:53 Prof Mark Ebbert
Citroen aging.
Audio file
Transcript
00:00:05 Will Mountford
Hello. I'm well. Welcome to research pot.
00:00:08 Will Mountford
It's a familiar factoid that human beings as varied as we are share over 99% of our DNA with each other. This means developments in personalized therapies for diseases such as cancer or neurodegenerative conditions have to be tailored to the most minute differences between us, or even between our own cells. Doctor Mark Ebert of the Sanders.
00:00:29 Will Mountford
Town Center on Aging at the University of Kentucky leads a lab focusing on the most minute of variations, in short lived mold.
00:00:36 Will Mountford
Rules isoforms of RNA's that could open up whole new avenues for detection, diagnosis, and treatments of otherwise incurable diseases.
00:00:48 Will Mountford
Joining me from the University of Kentucky doctor Mark Abbott. Mark, hello.
00:00:51 Prof Mark Ebbert
Hello, how are you?
00:00:52 Will Mountford
I'm very well and thanks for joining us today for my sake and for everybody listening at home, could you tell me a bit about you yourself, your lab and the work that it is that you do?
00:01:01 Prof Mark Ebbert
Yeah. So I I joined the University of Kentucky a few years ago in December of 2020, kind of in the in the heat of the the pandemic.
00:01:09 Prof Mark Ebbert
He was really excited to be here at the University of Kentucky and specifically in the Sanders Brown Center on Aging, a really unique opportunity and and truly a privilege to be part of the Sanders ground center.
00:01:21 Prof Mark Ebbert
On.
00:01:21 Prof Mark Ebbert
Aging and you know, for me, you know, performing, you know, disease, relevant research.
00:01:29 Prof Mark Ebbert
Is so valuable because all around us people are struggling with with various diseases, and one of those that is, you know, so impactful to to many of us and people we have known.
00:01:44 Prof Mark Ebbert
Is Alzheimer's disease and your every disease has its challenges. But one of the the most challenging parts, the most painful parts of Alzheimer's disease, is is sort of seeing someone be erased from their own mind and seeing loved ones.
00:02:03 Prof Mark Ebbert
And memories of loved ones being erased from their minds. And that's a really being a part of that is, is is very meaningful, you know, to me and the Members.
00:02:12 Prof Mark Ebbert
Of my lab.
00:02:14 Prof Mark Ebbert
Yeah. So I've been involved in Alzheimer's disease research for, I guess, about 12 years now and.
00:02:22 Prof Mark Ebbert
You know our our lab goals are, you know, I think like most Alzheimer's disease, researchers are to help develop ways to improve disease treatment, even prevent disease if possible through developing therapies. And then the second aspect of our lab that is at least as important.
00:02:43 Prof Mark Ebbert
As developing meaningful therapies is developing ways to detect disease before symptoms onset, so the the importance of a pre symptomatic disease diagnosis.
00:02:56 Prof Mark Ebbert
Because if any meaningful therapy won't be as you as impactful or meaningful, if we can't detect disease before symptoms onset, because by the time symptoms of onset, the disease has already progressed to a point that the brain can no longer compensate for all the cellular death. So those are the two main branches.
00:03:16 Prof Mark Ebbert
Of our labs research and and we do this using both computational approaches and genomics approaches. So and specifically using long read RNA and DNA sequencing.
00:03:30 Prof Mark Ebbert
So that's kind of the the basic background on on what we're trying to accomplish and how we're.
00:03:36 Prof Mark Ebbert
Doing it.
00:03:41 Prof Mark Ebbert
Alzheimer's disease, you know, I think as many people know, is a neurodegenerative disease. So neurons are dying in the brain. And what's really driving that neurodegeneration? As we've learned more more clearly in recent years.
00:03:58 Prof Mark Ebbert
Is the relationship between the amyloid beta plaques and the neurofibrillary tangles. Those are the two hallmark pathologies that are present in Alzheimer's disease, brain at autopsy, and that degeneration in in the disease has actually been going on in the brain for.
00:04:18 Prof Mark Ebbert
You potentially decades by the time the symptoms onset and so you know that just really emphasizes the importance of of, you know, treating disease long before symptoms onset. But you know, ultimately what's what's driving disease.
00:04:34 Prof Mark Ebbert
It's pretty complicated. It can be a range of of genetic factors and environmental factors. And what we're mostly focused on in the Everett lab are the genetic and molecular factors. What's what's happening within the cells. So within the genetic.
00:04:54 Prof Mark Ebbert
Aspects. What is a gene? You know, most people you understand the basic concept of a of a gene which is, you know, from our DNA we get a copy of DNA from Mom and a copy of DNA from Dad and, you know, fundamentally a gene is just a sequence of DNA and that that gene.
00:05:13 Prof Mark Ebbert
Ultimately, typically encodes for a protein, and I like to think of proteins as being all the little workers in our body and in our cells that go about doing things and making life possible, making cells behave how they're supposed to behave. So when.
00:05:33 Prof Mark Ebbert
Genes misbehave. You know that's generally when disease comes about, whether it's cancer or Alzheimer's disease and over the.
00:05:43 Prof Mark Ebbert
Last couple of decades, we've as a community as a research community, have discovered somewhere between 70 and 80 genes that are known to be involved in Alzheimer's disease. The challenge there, though, is that we don't know exactly how these genes are involved. We have.
00:06:03 Prof Mark Ebbert
Basic ideas and understandings, but the exact mechanism is still a mystery, and so we're working to help identify exactly what's going wrong with these individual genes.
00:06:18 Will Mountford
They say these genes go wrong. Does that mean mutations? Is that decay healthy?
00:06:23 Will Mountford
The proteins different from unhealthy ones.
00:06:26 Prof Mark Ebbert
Yeah, that's a great question. So jeans, they can go wrong, you know, through multiple methods, multiple ways it, it could be something that's purely genetic, something that was inherited through.
00:06:39 Prof Mark Ebbert
Your ancestry from your parents, so a variant or a mutation in that gene that makes it so that it cannot behave properly.
00:06:49 Prof Mark Ebbert
Basic concept there would be that it loses its function or on the opposite end of that spectrum it could be a mutation that causes it to do something it shouldn't be doing another mechanism through which genes can can misbehave is that you may be the gene itself is.
00:07:09 Prof Mark Ebbert
Intact and there's nothing inherently wrong with its sequence, but for whatever reason later in life begins to be either overly active or under active.
00:07:22 Prof Mark Ebbert
One of the things that's not.
00:07:24
You know.
00:07:25 Prof Mark Ebbert
Very well talked about.
00:07:26 Prof Mark Ebbert
Or or or very well understood even.
00:07:29 Prof Mark Ebbert
Is that we talk about genes, you know, any given gene as though it has a specific function. But it turns out that.
00:07:37 Prof Mark Ebbert
You know about half.
00:07:38 Prof Mark Ebbert
Of human genes in code for multiple different versions of itself, and that happens through what's known as alternative splicing. So basically a gene.
00:07:49 Prof Mark Ebbert
Is made-up of these individual segments.
00:07:52 Prof Mark Ebbert
That we call exons, and when that gene is being expressed, when it's when it's being activated, different combinations of those exons can be produced, and that results in a different protein. Or, as I like to think of a different worker. But we.
00:08:10 Prof Mark Ebbert
Know very little.
00:08:12 Prof Mark Ebbert
About the individualized for most genes. So for example, there's one Alzheimer's disease.
00:08:19 Prof Mark Ebbert
Gene, that has been long implicated in disease. It's known as Ben 1B IN one that specific gene is actively expressing 8 different what we call isoforms, 8 different versions of that same gene. And we know very little about those different isoforms.
00:08:40 Prof Mark Ebbert
Whether some of them are just noise or junk, I personally doubt.
00:08:46 Prof Mark Ebbert
That or if each individual isoform actually has its own unique function, even if it's a subtle difference in function, there are some genes that you know not necessarily related to Alzheimer's disease, where we know that the different isoforms are performing different functions. Maybe the the classic example.
00:09:06 Prof Mark Ebbert
And again, this this gene is not directly involved in Alzheimer's disease.
00:09:11 Prof Mark Ebbert
But it's known as BCLC X1 isoform of this gene. One version of this, this gene actually promotes cell death, while the other isoform does the opposite. It promotes cellular health. So we know that this definitely occurs, but for most genes, we just don't know. We just don't know.
00:09:31 Prof Mark Ebbert
So that's really a major part of what we're working to do is understand and determine the individual function for genes throughout the body that the ice, the different isoforms for sing.
00:09:44 Prof Mark Ebbert
Gene and in particular, Alzheimer's disease genes.
00:09:48 Will Mountford
And we can hypothesize the nature of those mutations, those isoforms, the alternative splicing, because if it is something that, you know, creates functional or mostly functional versions of a protein, is that offering any particular evolutionary advantage, do you think or kind of like the the long form multi generational view of a species?
00:10:08 Will Mountford
Or is it just kind of the chaotic nature of we are jamming atoms together to make molecules in a cell and this is sometimes just what happens?
00:10:16 Prof Mark Ebbert
The truth is.
00:10:17 Prof Mark Ebbert
It could be a combination of the.
00:10:18 Prof Mark Ebbert
You generally speaking though it has been well established that this mechanism of alternative splicing, this mechanism of a single gene being able to encode multiple versions actually makes complex life possible. So the more complex organisms.
00:10:39 Prof Mark Ebbert
Tend to have greater, you know, isoform diversity among genes. It wasn't until around the the early 2010.
00:10:48 Prof Mark Ebbert
That sequencing became truly accessible to researchers like myself, and it began with what we call short read sequencing and and like you alluded to, it's it's just like it sounds there's, there's short reads versus long reads. And historically, we've we've relied on short read sequencing, which has really been a major boon for reset.
00:11:08 Prof Mark Ebbert
Search but with short read the challenges that were genuinely only sequencing small portions of the DNA or RNA somewhere between 100 to maybe up to 200 nucleotides at a time, and then we're left to kind of piece those back together to try to infer and build the genome structure and and or RNA.
00:11:28 Prof Mark Ebbert
Isoform structure. But the truth is those those reeds are just too short to be able to do this properly.
00:11:34 Prof Mark Ebbert
So with long read sequencing we can sequence up to 10s of thousands of nucleotides in a single go, which means it's a lot. In principle it's a lot easier to construct the full genome structure, which is very important, or at the RNA level we can actually.
00:11:55 Prof Mark Ebbert
Now begin to measure the expression levels of individual isoforms for single gene. Whereas with the short read sequencing a major limitation with RNA sequence.
00:12:07 Prof Mark Ebbert
Is that we've been forced to collapse the expression of individual isoforms for a single gene into a single expression measurement, so it's made it you essentially impossible for us to determine is isoform a involved in disease versus isoform B, whereas now with long read sequencing we can do that.
00:12:27 Prof Mark Ebbert
It's it's truly exciting.
00:12:29 Will Mountford
And the samples that you're working with for these longer sequences, they, as I understand it from actual human samples, postmortem brain tissue, is that right?
00:12:39 Prof Mark Ebbert
Yes, and that's one of the really unique things.
00:12:42 Prof Mark Ebbert
About our work.
00:12:43 Prof Mark Ebbert
And being here at the Sanders Brown Center on Aging at the University of Kentucky is that we have one of the best Alzheimer's disease brain banks really in the world.
00:12:54 Prof Mark Ebbert
It's a very unique cohort in terms of what we've been able to collect. So and what makes that so important in our research and really in understanding Alzheimer's disease is we want to work in the diseased tissue because you know, science and and understanding disease. The truth is it's it's difficult.
00:13:15 Prof Mark Ebbert
It's complicated.
00:13:16 Prof Mark Ebbert
And for various reasons, many researchers have to work very distal from the the diseased tissue. But we have this unique opportunity to actually work in the disease tissue and try to understand what's actually going wrong at the molecular level at the cellular level within the disease, the disease.
00:13:36 Prof Mark Ebbert
Issue.
00:13:36 Will Mountford
I mean, you've mentioned that it's one of the finest cohorts of this nature in the world. Is that just due to the geography or something about the health system you're working in or is that no indicative of more pressing health concerns across Kentucky, that there are that many more people in the area who have a rigid donor, degenerative condition?
00:13:54 Prof Mark Ebbert
Yeah, that's a great question. So you know, one of the interesting things maybe maybe surprising until you think about it, is that the biggest challenge in a lot of disease research is not actually obtaining samples from those that are affected by disease, but those that are not affected. And I think that is even more challenging.
00:14:14 Prof Mark Ebbert
When it comes to obtaining brain samples.
00:14:17 Prof Mark Ebbert
Because we only obtain them postmortem from those that are eager to contribute to the this this form of research to understanding and defeating Alzheimer's disease. So, you know, there are, there are various brain banks around the world that have collected a large number of Alzheimer's diseased brains.
00:14:37 Prof Mark Ebbert
But the Sanders, Brown, Center and aging under the direction of Doctor Bill Marksbury very early in its days back in the 1970s and 80s.
00:14:47 Prof Mark Ebbert
Recognize how important it is to start recruiting individuals who are not affected by disease, and that's one of the main reasons that our cohort is so unique is we have potentially one of the largest cohorts of non diseased brains out there. And of course you know.
00:15:06 Prof Mark Ebbert
Maybe the the obvious question is.
00:15:08 Prof Mark Ebbert
Why does that matter?
00:15:10 Prof Mark Ebbert
Well, we have to know what normal looks like so that we can determine what disease looks like. Right. And and that's really challenging at the molecular level. So so having such a large collection of non diseased brains is so critical and that this is really a, I think, a great opportunity to.
00:15:30 Prof Mark Ebbert
The size and think all those who contribute to this research, the patients, their loved ones, their caregivers, those who contribute so selflessly at every stage of disease, including those and perhaps especially of those that are willing and eager to even.
00:15:51 Prof Mark Ebbert
Allow us to obtain brain samples postmortem.
00:15:59 Prof Mark Ebbert
You we're obtaining these individual brain samples and we go through a very careful process to extract the DNA or RNA as the case may.
00:16:09 Prof Mark Ebbert
Be.
00:16:10 Prof Mark Ebbert
Especially for long range sequencing, because we need the DNA and RNA to be as intact as possible, and it turns out that's quite challenging because.
00:16:19 Prof Mark Ebbert
You are our cells are regularly trying to maintain balance within the cell, maintain health and as part of that, it's really it it quickly after death. It quickly breaks down DNA and RNA. On top of that because these molecules are actually so fragile, getting them out.
00:16:40 Prof Mark Ebbert
Of the tissue intact is.
00:16:42 Prof Mark Ebbert
Typical. So that process just the extraction process can take several hours, and then there's a lot of additional work to clean up the sample to remove all of the excess proteins and cellular debris, and preparing this the the DNA or RNA for sequencing so.
00:17:02 Prof Mark Ebbert
In all, the process can take a couple two to three days before we can actually begin the sequencing, and then the sequencing itself will take approximately 3 days as well.
00:17:13 Will Mountford
I suppose the question now is, what does all of that tell us?
00:17:16 Will Mountford
What are the discoveries? What is the work of the Abbott lab and what kind of breakthroughs are do you think you might be working on or anything that's on the cusp of changing the state of knowledge or care for Alzheimer's?
00:17:29 Prof Mark Ebbert
We just published a paper recently in the Journal of Nature Biotechnology, which is a fantastic journal where we demonstrate this proof of principle for how important these technologies are for how important this specific study is for doing long read.
00:17:48 Prof Mark Ebbert
RNA sequencing in human Alzheimer's disease brains versus controls in that study.
00:17:55 Prof Mark Ebbert
We made several important discoveries, some of which are actually not directly related to Alzheimer's, but but again demonstrate the importance of this where looking across all of the known disease relevant genes, regardless of the disease, we discovered hundreds of isoforms.
00:18:15 Prof Mark Ebbert
Burning isoforms that had never been measured before never been seen before, some of which are very highly expressed. Very, very common in the brain.
00:18:27 Prof Mark Ebbert
And on top of that, we actually discovered more than 200 entirely new genes that had never been described before. And you are those specific isoforms or genes directly involved in disease. You're probably not. Maybe some of them, but again, it's a very important proof of principle for and demonstrates how much.
00:18:47 Prof Mark Ebbert
There is still to learn.
00:18:50 Prof Mark Ebbert
Human DNA and genes, human genomics, human health, and disease at the at the molecular level and then the part that's most directly applicable to Alzheimer's disease is again a proof of principle study where we compared RNA isoform expression.
00:19:10 Prof Mark Ebbert
Between cases and controls, again, this is in human frontal cortex, and we found 99 isoforms that were differentially expressed between cases and controls, where at the gene level it wasn't. And just to clarify what I mean by that, going back to what I was saying earlier.
00:19:29 Prof Mark Ebbert
With standard short read sequencing, we're forced to collapse the expression for individual isoforms into a single expression measurement for that gene, whereas with long reads we can measure expression for those individual isoforms. So at the gene level, basically using the historic approach.
00:19:48 Prof Mark Ebbert
There was no differential expression between cases and controls until we look at the expression at the end of the individualized reforms. So again, a really important proof of principle, and we're actually in the middle of expanding that study into a much larger cohort of hundreds of cases and.
00:20:06 Prof Mark Ebbert
Roles so that we can hopefully identify RNA isoforms that are, if not directly involved in disease, or at least indicative of disease, so that we can develop a pre symptomatic disease diagnostic. And if it turns out that those individual isoforms are in fact involved in disease, then that gives us new.
00:20:27 Prof Mark Ebbert
Targets for therapies.
00:20:30 Prof Mark Ebbert
So there's really a lot of great potential for this study and we're really excited to move forward and very eager to see what comes of this.
00:20:39 Will Mountford
Is there anywhere else in the body that these ice forms might turn up that could be useful for biomarker? I'm just thinking that if I, you know, suspected that I might or a loved 1 might be coming down with something like Alzheimer's, I'm not sure that putting myself forward to having a full brain surgery prospectively to maybe find out.
00:21:00 Will Mountford
Like, that's a lot of commitment, but if there was like a yearing test or a blood draw or something, could it be as simple as that?
00:21:06 Prof Mark Ebbert
Yes, you've touched on a really key point. We're generally not very eager to give up a piece of our brain while we're alive and that's even the biggest limit limitation of what we're doing. But that's actually intentional. So as I alluded to earlier.
00:21:22 Prof Mark Ebbert
Here our goal is to to study the the disease in the diseased tissue so that we can first understand what's going wrong in the disease tissue right from there. The goal is to work backwards and see if we can measure these outside of the brain in a much less invasive way. The most obvious.
00:21:42 Prof Mark Ebbert
Which is also not ideal for patients, is to is to try to measure it in cerebral spinal fluid, donating cerebral spinal fluid is far less invasive than.
00:21:53 Prof Mark Ebbert
Then you know, donating a brain sample, but it's still not pleasant that that requires a Spinal Tap and and it's very uncomfortable both during the procedure and after the procedure. But that would still be a huge win if if that were to work. The Holy Grail generally in medicine is to be able to detect.
00:22:13 Prof Mark Ebbert
And measure things in the blood, because that's generally far less invasive and generally not a big issue for patients. So once we measure these and identify them in the brain, that will be the goal is to work backwards, look in the CSF, the surface spinal fluid, look in the blood and see if we can measure, measure these there.
00:22:36 Will Mountford
What do the isoform findings not tell us?
00:22:40 Prof Mark Ebbert
The central dogma of biology is fairly simple. We have our DNA. That's our we often think of that as the blueprint, so that you know your DNA is the blueprint to build.
00:22:52 Prof Mark Ebbert
You but a blueprint in and of itself is not very useful if you don't do anything with it, right? You need to. Actually you use it. And so DNA is transcribed into RNA, and that RNA is then typically translated into the proteins that I've referred to earlier.
00:23:12 Prof Mark Ebbert
That's being you you typically the workers that, that, that make life possible. So for the most part RNA's and and and and sequencing RNA isoforms.
00:23:24 Prof Mark Ebbert
It's sort of a an intermediary step. So everything that we're measuring at the RNA level is generally just an attempt to extrapolate what's happening at the protein level because it's not, there aren't very any high throughput methods for sequencing protein. That's sort of the Holy Grail and.
00:23:44 Prof Mark Ebbert
In genomics research, but there's really there's so many gaps in our understanding of the central dogma biology, even though we've, we've we've known the basic concepts for decades.
00:23:55 Prof Mark Ebbert
For example, if you measure the expression of a gene at the RNA level and then measure it at the protein level, those measurements often don't agree, and that's something we don't understand very well. But that's a big part of what we're trying to help gap. We're trying to help fill is why is that happening? And?
00:24:15
So.
00:24:15 Prof Mark Ebbert
There's really a lot that we don't know. And so you know, again, you know, science is very, very challenging, very complicated and and generally we're trying to look at these small pieces and try to piece them together or extrapolate what we can't measure based on those.
00:24:32 Will Mountford
Seeing if there are any peers or professionals out there listening to this who want to work with kind of this library of knowledge and all of these DNA sequences that you're missing, the RNA long reads and short reads. Is it something that they can remotely join in with?
00:24:47 Prof Mark Ebbert
Absolutely. In fact, we're already aware of many labs that have downloaded and already been using the data that we generated and that study I I described a moment ago.
00:24:58 Prof Mark Ebbert
Because they're here to see the same.
00:24:59 Prof Mark Ebbert
Things we're here to look at, they want to.
00:25:01 Prof Mark Ebbert
Know.
00:25:02 Prof Mark Ebbert
What's going wrong and part of what's so exciting about the data we're generating you, we have our ideas. We have the things that we want to look at, but you know we don't. We don't have a monopoly in all of the great ideas out there. Obviously, right. There are lots of fantastic scientists with their own ideas.
00:25:19 Prof Mark Ebbert
And ways that they want to explore this data. And so you know, as part of Everett lab policies, we.
00:25:25 Prof Mark Ebbert
Make our data available as early as possible so that other labs can obtain it and they may find things that we miss, and that's that's a very important part of science and and very exciting is that whether we find it or someone else finds it, you know, the goal is to ultimately improve.
00:25:45 Prof Mark Ebbert
Patient health.
00:25:47 Will Mountford
If anyone listening to this is of influence to members of the public, 2 practitioners or two policymakers, and they want to get involved in your research and in outsiders and advocacy more, what would be a good place to start with you?
00:26:00 Prof Mark Ebbert
We'd encourage you to.
00:26:01 Prof Mark Ebbert
To reach out due to me.
00:26:03 Prof Mark Ebbert
Likely to the Sanders Brown Center on Aging, we have obviously, you know, there are lots of other fantastic researchers here doing some really important research that is complementary to what we're.
00:26:15 Prof Mark Ebbert
Doing.
00:26:19 Prof Mark Ebbert
A team effort. Obviously it requires the patients.
00:26:23 Prof Mark Ebbert
Caregivers, the loved ones, the those that are willing to, you know, selflessly donate their time and tissue.
00:26:32 Prof Mark Ebbert
One of the key things that we need that that we're as a constant challenge is funding. You know, these studies are extremely expensive and it takes a lot of resources. We really need the help of everyone. You, especially the patients and their loved ones. And you're really encourage them to reach out to me and to the Sanders centre on aging.