Alexander Disease Research Update
Discussion of recent publications from the research literature on Alexander Disease, hosted by Albee Messing from the University of Wisconsin-Madison.
Alexander Disease Research Update
Alexander Disease Research Update - Episode #17: 1) new iPS cell line, 2) acetylation and GFAP, and 3) STAT3 as a key regulator driving GFAP accumulation
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Albee Messing, Rachel Battaglia, and Tracy Hagemann discuss the following recent publications:
[01:45] A defined roadmap of radial glia and astrocyte differentiation from human pluripotent stem cells
Stem Cell Reports 18, 1701-1720 (2023)
[includes new iPS cell line from an individual with Alexander disease] [full text]
[05:39] Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications
Journal of Visualized Experiments 18, 55655 (2017)
[includes data on acetylation of lysines in GFAP] [full text]
[10:26] STAT3 Drives GFAP Accumulation and Astrocyte Pathology in a Mouse Model of Alexander Disease
Cells 12, 978 (2023)
[identification of one of the key mechanisms responsible for the increase in GFAP levels in Alexander disease] [full text]
[22:35] Questions from email: does Alexander disease affect menstrual cycles?
Send your questions and feedback to: axdrupodcast@waisman.wisc.edu
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Hello, and welcome to Alexander Disease Research Update, episode number 17, recorded on September 5th, 2023. I'm Albie Messing from the Wastman Center at the University of Wisconsin, and with me today are Rachel Bataglia from Brigham Women's Hospital and Harvard Medical School. Rachel's been with us before, and a new guest, Tracy Hagman from the University of Wisconsin, who is the lead author on one of the papers that we're going to discuss. Tracy doesn't like to talk about herself, so I'll do it for her, at least this part. Tracy received her PhD in molecular genetics and immunology from Rush University Medical Center in Chicago in 1995 and joined my lab in Wisconsin in 2001 as a postdoctoral fellow, just as we were beginning to focus all of our efforts on Alexander Disease. And I think it's fair to say that Tracy's had her hand in almost every significant advance that we've made over the past 20 plus years. She's now a research associate professor and head of the Alexander Disease Research Laboratory at the Wasteman Center, having inherited that role from me. Before we get started, please send feedback to AXDRU podcast at Waistman. That's W-A-I-S-M-A-N.wisc.edu. Today we're going to talk about three publications, two of which came out earlier this year and one from 2017.
A defined roadmap of radial glia and astrocyte differentiation from human pluripotent stem cells
SPEAKER_00The first is by Jovanovic et al. and it's entitled A Defined Roadmap of Radioglia and Astrocyte Differentiation from Human Pluripotent Stem Cells, published in Stem Cell Reports in 2023. And honestly, I didn't even know about this paper until I think Tracy told me about it, because I thought it was really just basically a methods paper for how to use IPS cells in studying GLIA. But it turns out, to try to illustrate one example of how useful this these techniques could be, they decided to use Alexander disease as a model. And they turned these IPS cells into astrocytes expressing mutant GFAPs. So first let me ask Rachel, what did you think of this paper? Did they do a good job? Did they tell us anything new?
SPEAKER_01Well, I mean, the first part looking at the method to differentiate astrocytes, I thought was probably the most interesting part of the paper. Back when I was working on this with Natasha, it was certainly a struggle to make astrocytes. It takes a very long time. It's expensive, you know, it's complicated. So it could be interesting for people in the field to try this method. Um it would be nice to nicer to have a shortcut, but I don't know how these cells compare it to the ones that Su Chen works with, of course.
SPEAKER_00In your hands, how long did it take to get cells into astrocytes?
SPEAKER_01So it was about 90 days, about three months. Um, so it's similar, but I think the cells in these paper uh were more astrocyte-like than the ones that I was working with. Yeah, the ones I had could be more fibroblast-like at some time. So the cells were spread out sometimes and they didn't have those star-like shape that astrocytes should have. So it would be interesting to try this method.
SPEAKER_00Tracy, you have any impressions from this paper?
SPEAKER_02I I believe the senior author is now working at um James Thompson's company here in Madison. And, you know, so I think there is probably some interest in accelerating astrocyte differentiation, uh, just because we all know it takes so long to generate these things, and that's half the problem of working with them.
SPEAKER_00Well, you know, they touched on a lot of properties of the astrocytes that you would want to see in a model of Alexander disease, such as claiming that they had Rosenthal fibers. I wasn't so sure they were really Rosenthal fibers. They had increased GFAP, enlarged endoplasmic reticulum, uh, you know, a lot of things that really had been published before for IPSL models of Alexander disease. So that wasn't anything really new. They did have some data on TDP43, and that's a protein that we're interested in, certainly, because it's coming up again and again in a number of other neurodegenerative disorders. So it'll be interesting to see if any of these authors continue to work on Alexander disease, which would be great, and what they do with the cell line. Rachel, do you know if this is your cell line or did they generate their own? It's the same patient.
SPEAKER_01Right. No, they weren't using our IPS line. They generated their own cells. Um, and they also didn't use the control that we made from those cells.
SPEAKER_00Do you want to explain what that control is?
SPEAKER_01Yes, yes, that control is an isogenic control, meaning that we corrected the Alexander disease causing mutation in GFAP. Um, so it's basically the same exact cells from that patient, just one small change in the GFAP locus.
SPEAKER_00And in general, isogenic controls are considered uh much better controls than simply using a different cell line from a quote-unquote healthy donor.
SPEAKER_01Yeah, they're the gold standard of the field.
Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications
SPEAKER_00All right, let's move on to the second paper. Uh, and this is one that Rachel is actually the leader on from 2017, entitled Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging Associated Post-Translational Modifications in the Journal of Visualized Experiments, which is a really interesting journal that I like not only reading but watching the videos for because it's a really helpful way to teach people about methods. So this is really a methods paper, how to isolate and study biochemically intermediate filaments. But the reason I wanted to talk to you about it today is that it's got tacked on at the end of it a small item about post-translational modifications, and one that doesn't get much attention uh with GFAP, uh acetylation. So, Rachel, could you first talk a little bit about what post-translational modifications are? And then, secondly, acetylation in particular, and what we should be thinking about that.
SPEAKER_01Of course, yeah. So post-translational modifications are a change to the protein that happens after it has been translated from the RNA into protein. So this change can be lots of things. You can add small fats, small sugars, or even other proteins on top of the protein that just exists at baseline. And in the case of acetylation, we're adding a very small modification that's just a small carbon chain with an oxygen atom attached. Um, but this acetyl group is added to a very specific residue on the protein, which is a lysine. Um, and lysine is a special amino acid because it has this positive charge. So, one thing that happens when the acetyl group gets added is that the positive charge is taken away. Um, so this can affect the actual uh interaction of the protein with itself or with other proteins, which can affect its function. And the second thing that's special about lysines is that it's also the site of another post-translational modification called ubiquitanation, where a protein called ubiquitin is added to the lysine. And this is an important signal, it can be an important signal for protein degradation. So there's kind of this theory in the field that if there is too much acetylation, then you're blocking residues for ubiquitanation and you could disrupt protein turnover.
SPEAKER_00Well, that's interesting because there is data about impairment in protein degradation for uh GFAP and Alexander disease. And so maybe acetylation of lysines would be part of that mechanism. I don't believe it's ever been looked at uh for Alexander disease yet. Tracy or Rachel, do either of you know about that?
SPEAKER_01No, we we looked at it during aging. Um, so what we saw is that in in normal mice, when you age them up to two years, in the paper we reported that we see increased acetylation. But in the context of Alexander disease, um we haven't published anything yet, but I know Natasha and I have done some mass big studies. Um, so acetylation is definitely something that came up that was modified in Alexander disease.
SPEAKER_00Okay, well, hopefully we'll see that soon.
SPEAKER_02Are there specific glycine residues that are acetylated or is it just a general phenomenon? I don't recall.
SPEAKER_00Well, I will mention one thing. I went back and looked at a paper from 2013 on proteomic analysis and amyotrophic lateral sclerosis, where the authors were looking at acetylation of GFAP. It's a it's a nice paper, but unfortunately they got all the numbers wrong. So even in the abstract, they cite uh one, two, three, four, five, six lysines in GFAP that are acetylated, and all the numbers are wrong. And the mistake they made, which I actually commented on uh in the journal website at PLOS 1, is that they use the number of the beginning amino acid of the peptide that was being analyzed. They forgot to focus just on the number of the lysine. So eventually that should be changed. But you know, if you look at those numbers, you're gonna wonder what those aren't lysines.
SPEAKER_02Well, it's interesting that there is a recent paper on keratins showing that deacetylation helped mitigate protein aggregation and with mutant keratins. Um, that was by Bishop O'Merie and JCI Insight that just came
STAT3 Drives GFAP Accumulation and Astrocyte Pathology in a Mouse Model of Alexander Disease
SPEAKER_02out.
SPEAKER_00Let's move on to the highlight of today's podcast, a paper that Tracy and I started working on, I don't know when, 10, 11, 12 years ago. Uh Tracy's the lead author. It's entitled Stat 3 Drives GFAP Accumulation and Astrocyte Pathology and a Mouse Model of Alexander Disease. And it was published earlier this year in the journal CELS. So, Tracy, could you start out by talking about what the goal of this research was? Um, and then we'll move on to say a little bit about how you did it, the results, and what it really means. So, first the goal.
SPEAKER_02Well, at some point during our research, we came up with the idea that the expression levels of stat, oh, sorry, of GFAP were very important, and that elevated GFAP was part of the problem in Alexander disease. And I think that goes back to the very early models where you just before I was in the lab, where you over-expressed wild-type human GFAP, and those animals developed Rosenthal fibers and GFAP pathology. So uh, and that led to the discovery that GFAP was involved in Alexander disease. So I think that was always in the back of our minds, and uh maybe collectively or individually, I can't say, but um uh so we were always trying to think of why GFAP became elevated in Alexander disease and could we stop that? And I think it was around 2010 or maybe earlier that Mike Safraniev came out with a paper that talked about STAT-3 regulating astrogliosis. And astrogliosis is what occurs when astrocytes become stressed. And part of that process involves upregulation of GFAP. It's a marker of reactive astrocytes that's been used throughout the years, and that kind of tied into our narrative that we already had about GFAP expression levels, and so I think that led to an initial question of well, is this what's regulating GFAP in the context of Alexander disease?
SPEAKER_00Well, what is stat three?
SPEAKER_02Stat three is a signal transducer and activator of transcription three, and it is a protein that regulates the transcription of other genes, and mostly those genes are involved in growth and development. Um, it's also thought of a it's often thought of as an oncogene or a gene that's involved in cancer, uh, because when it becomes dysregulated, then you see an increase in growth and cell proliferation. So uh it's it's got a lot of different functions. Uh, it's not just a transcription factor, it also regulates uh mitochondrial function and autophagy. So autophagy is part of the clearance of proteins that uh and small organelles and debris in the cell. Uh, so that may be related to issues with uh Alexander disease as well. Uh but I think most people think of it as a transcription factor and one that has a major role in the immune system. So it controls cytokine expression and also in the central nervous system, it can controls uh neuroinflammatory responses. So it's it's a pretty diverse uh protein. Uh you can't get rid of it uh without it, it's a necessary protein. If you get rid of it uh genetically, then it's it's a lethal um outcome.
SPEAKER_00If you get rid of it in all cell types.
SPEAKER_02Right, right, right.
SPEAKER_00What took us so long on this project was uh trying to figure out a way to get rid of stat three without killing the mouse. Um so it is such a widely expressed gene, it controls so many different other genes that if you just get rid of it globally, it's an embryonic lethal. So you had to develop ways to just limit its effect to astrocytes. So why don't you talk a little bit about how you did that?
SPEAKER_02Well, that's one thing that's important here, too, is that stat three also regulates the differentiation of astrocytes early on in development. So if you don't have stat three, you won't have astrocytes. Um and it in that process, it upregulates or turns on GFAP when astrocytes differentiate. So that's part of the regulatory response. And of course, I mentioned that it also regulates the astrocyte response to stress. Um, and what we were able to do is use mouse genetic tools and to target specifically STAT-3 uh activation in astrocytes. And we actually did a number of different experiments. So we used tools to manipulate um the expression of the gene where we could turn it off as soon as the cells became astrocytes. So as soon as GFAP was turned on, stat three would be deleted. So once stat three had done its action and turned the cell into an astrocyte, then stat three would be turned off. And in that case, when we did that in animals that modeled Alexander disease, those animals never upregulated GFAP. They never became the stress, the astrocytes never became stressed, and we pretty much prevented any of the pathology uh that mutant GFAP causes, I believe, because GFAP stayed at a low enough level that the astrocytes could continue to get rid of it and turn it over, and it never accumulated and became a problem. So that was one set of experiments. Um, but I suppose you know, if you think about it, the real question is can we turn off stat three later on once pathology has already developed in our models, and then see reversal of GFAP accumulation? And I think that's more important because then you're not only looking at the regulation of stat three developmentally, but you're looking at the reactive astrocyte response, which is I believe what causes most of the problems uh in astrocyte pathology and Alexander's disease. So again, we could use our mouse genetics to answer that question as well by using what's referred to as inducible transgenes, uh, where you can treat the animal with a specific drug, and that will genetically delete stat three at the time in which you inject the animal with the drug. So in this case, we treated the animals at eight weeks of age, which in the mice they already have established pathology. And then we checked them another month later and we found that all that pathology was gone. There was no GFAP accumulation, uh, we did not see Rosenthal fibers, GFAP levels returned to close to normal, there was no stress response. So essentially, that told us that indeed STAT-3 does seem to be driving uh this GFAP accumulation. And if you get rid of it, you can get rid of GFAP pathology.
SPEAKER_00Let me just clarify one thing, though. The drug that you're talking about here is tamoxifen. And that is a drug that is designed specifically to interact with some of the genetic tools that have been engineered into these mice. So tamoxifen itself is not something that we would pursue as a treatment for Alexander disease. But the general finding that reducing stat 3 is beneficial opens up many other ideas about how you might be able to achieve that in a patient. Uh so, as I understand it, there's a lot of interest in the pharmaceutical industry in finding stat 3 inhibitors, partly to address uh many of these other diseases you mentioned where stat three is involved, such as cancer. So there are drugs out there that are potential stat three inhibitors. Are any of them ready for prime time in Alexander disease? What's your approach for thinking about that?
SPEAKER_02Well, there's a couple of different things that you have to think about. Safety, I think, is one. Stat three is very important in development, and as I said, it all it regulates astrocyte differentiation. Um, I think the cancer community is sometimes happy when stat three inhibitors don't cross the blood-brain barrier. And of course, we need one that does. And so we do have a few candidates that we'd like to try out. Those are the two big concerns. They have to cross the blood-brain barrier and they have to be safe. And preferably, they'd be safe in a pediatric population. So those developmental roles that stat three plays have to be taken into consideration.
SPEAKER_00Yeah, you know, I would point out two other things about STAT-3 in comparison with ASOs that are currently in clinical trial. So the ASO suppression of GFAP that's in clinical trial is not specific for astrocytes because the ASOs are picked up by many other cells as well, but it is specific for GFAP. And so that's really uh the main appeal of the ASO approach. In contrast, stat 3, if you introduce a stat-3 inhibitor by sort of typical pharmacological means, it won't be specific for astrocytes and it won't be specific for GFAP. So that's where you run into this question about what are the side effects going to be, uh, and you'll have to balance those side effects against whatever the benefits are. I suppose there are other genetic techniques coming down the line, such as viral vector approaches, where you could perhaps selectively suppress stat three in astrocytes, but nobody's done any of that yet. And it's a long way in the future before any of that would be ready for uh for human use. Rachel, did you have any comments you want to make about this paper?
SPEAKER_01Yeah, I was just wondering if you envision using it down the line once you get stat three inhibitors that are working, using that in conjunction with the ASO. Since they're perhaps worried about some developmental effects, maybe the ASO would be something you use early on and then follow up with the stat three inhibitor later in life.
SPEAKER_02That's something I thought about, but it's not it's a good idea. We always think about one pathway, but I think that might not be the best approach and thinking of multiple pathways to try to get to reducing GFAP would be a better one.
SPEAKER_00Yeah, and I think um there's always going to be interest in combination therapies because the the alternative is that you look at one pathway only and you try to have a 100% effect. And that may be limiting in a lot of situations because of side effects and toxicity. But with combination therapies, one of the really appealing prospects of that is for each of those pathways, you wouldn't necessarily need 100% efficacy. Uh, since you're coming at it from multiple directions, you know, perhaps a 50% effect, but in two or three different pathways, would end up being very beneficial in a way that uh avoids a lot of the toxicity and side effects you get with just a monotherapy approach. So combination therapies are are really interesting. They're also exponentially more complicated to design and test.
SPEAKER_01I guess another thought I had was that maybe this could be a better treatment specifically for Alexander disease type 2 patients that are older.
SPEAKER_02Yeah, there's some drugs that are very safe, at least in clinical trials, but they've only been tested in people who are 18 or older.
Questions
SPEAKER_00Now for some email. This time there's uh just one question that we're gonna look at. Is there any connection between alexander disease and menstrual cycles in girls? Uh the short answer to that is nobody has reported any such connection. So we really don't know. I suspect not. It's an interesting issue, though, because certainly there are astrocytes throughout the hypothalamus and even in the pituitary, where they get a specialized name called pituicytes. And you would expect that these astrocytes in Alexander disease will be dysfunctional just like they are elsewhere in the central nervous system. But so far nobody has reported any specific effects on uh pituitary or reproductive functions. That's all for today's episode of Alexander Disease Research Update. Thanks for listening, and thank you to Rachel and Tracy for joining me today. Our theme song was written by Charlie Allenson, special technical assistance from my daughters Zoe and Rebecca, and from Clark Kellogg at the UW's Wastement Center. And thanks to our donors for these podcasts, the Barrent Riddle family. I'm Alby Messing. See you next time.
