 I'm Megan Donaldson, CEO of the Hereditary Disease Foundation, and I'm delighted to welcome you to our first research spotlight webinar. We fund innovative research to identify treatments and cures for Huntington's disease and other brain disorders. Before I begin my introduction, I'd like to acknowledge the news from Roche Genentech that the dosing on their clinical trial has been halted. We are all incredibly disappointed about this, but it points to the need for us to continue and intensify our support for research to develop treatments and cures for Huntington's disease. Our webinars will feature brilliant scientists who are unlocking the secrets of the brain and making game-changing discoveries. We are thrilled to launch the series with a presentation by Dr. Ayama Motto, Associate Professor of Neurology at Columbia University, and recipient of the 2020 HDF Leslie Gary Brenner Prize for Innovation in Science. From a creative graduate student with HDF funding to a prestigious NIH grant recipient, to a lab of her own mentoring a new batch of young researchers, and as a member of our Scientific Advisory Board, Dr. Ayama Motto has become an acclaimed expert in her field. Her path as a scientist illustrates how HDF provides not only funding, but an international family of collaborators who help nurture and lift the trajectory of each scientist's work. It is my pleasure to introduce Dr. Ayama Motto discussing What's It All About, Elfie and Aggregates. There will be a question and answer session at the end of I's talk. Please submit any questions in the Q&A box on the bottom right hand of your screen. Thank you for joining us. My name is Ayama Motto, and I'm here today to share with you what we've been doing in the lab. And of course, one of the things that we've been focusing on is whether or not we can undo the course of Huntington's disease. So just to begin, what I'd like to do is kind of set the stage. So for us, Huntington's disease is all about a protein known as Huntington, or HTT, and what happens to it in the course of the mutation that leads to this devastating disorder. So here on the slide, what you can see is a beautiful yellow cell. This is actually a picture of a brain cell taken from the Lispata lab. And in blue, what you can see is another brain cell kind of reaching out and touching this other cell. And this is what these brain cells do. They kind of reach out and touch and talk to each other. And so let's pretend that this cell is inside your brain. And we're going to zoom inside there and pretend that we can see what's going on inside these individual cells. And here inside the cell, you can see this big circle in the middle, and that is known as the nucleus. And the nucleus is the place where all the blueprints for the various machines that do the things inside the cells that make the cell be able to do what it does resides. And in there resides the blueprint for this very important protein, Huntington, or HTT, here in green. And Huntington is an essential protein. That means that it's necessary for life, and it helps to make the brain and make the brain work properly. Now in the brain of people with Huntington's disease, there's a little bit of an error in this blueprint that makes HTT and makes a different form here in pink that I've called toxic HTT. Now that might be a little bit of a cruel thing to do. I mean, this form of Huntington is actually doing a lot of the things that normal HTT can do, but it's just gained a new personality as it were. It does things that it wouldn't normally do. And one of the things that it does is it becomes very, very sticky. It's sort of like if you walk through a briar patch or something and you try to avoid all of the thorns and everything, but you can't. It just keeps snagging your clothes. Well, that's what this toxic HTT does. It just starts to stick to things. And one of the things it does is stick to itself. And by doing so, it starts to collect inside the cell, accumulate, and form these clumps that we call aggregates. Now, what it is about this new form of Huntington, its stickiness, this tendency to collect and accumulate and form these clumps, what of it is toxic. What of it causes Huntington's disease is not fully clear. But what we know is that something poisonous happens, ultimately leading to the demise of these cells. Now, whatever it is that this toxic form of Huntington does is unclear. But it is so robust that we can even capture it in a humble little mouse. And this is really from pioneering studies by Dr. Gillian Bates, who found that if you introduce this blueprint, the blueprint with the mistaken form of Huntington, and put it inside a mouse. The mouse will make toxic Huntington and then start showing features that mimic Huntington's disease, such as compromises in the way the animal can move, its cognitive ability, and such. And so what I show in this graph is kind of just a picture way of showing what could happen. So toxic Huntington would be expressed over the lifetime of a mouse. And as the mouse ages, they'll start showing various symptoms, such as the ones I just described. Now, even at the level of the brain, you can start seeing how this toxic form of Huntington starts getting sticky. So I'd like to show you a picture of that. So this is an image from a patient brain. And you can see here labeled something called an INI pointing to this brown clump. This is one of these protinaceous clumps that I was talking about, about Huntington sticking to itself. There's also things known as CI's that form cytoplasmic inclusions. And these are similar clumps that are forming outside of the nucleus inside the cytosol. Now we can actually see very similar structures inside of the mouse brain. So here's the picture of that, with the black showing these INIs, intranuclear inclusions, these clumps that are forming inside of the nucleus where all the blueprints reside, and then read the cytoplasmic inclusions, these clumps that are forming throughout the other parts of the cell. So as I mentioned, we can capture all of these features in the little mouse. And because of these studies by Dr. Bates, we were able to really propel how to study the relationship between this toxic form of Huntington and these symptoms that are associated to Huntington's disease. And so way back when over 20 years ago, when I was a graduate student in the laboratory of Renee Henn, together with a postdoc named Jose Lucas, we create our own mouse model to try to understand the relationship between toxic Huntington and these symptoms. And to do this, what we did was simply put a little switch, an on-off switch, right in front of the Huntington gene. And what we had found, of course, is that if the gene is on, this toxic Huntington is formed and the mouse gets sick, much like Dr. Bates' study. But of course, we were able to turn the switch off. And when we turned the switch off in mice that were already showing symptoms, the mouse got better. So this finding really set up the question of, how did it happen? And can we figure out what happened so it can bring this about to patients? Well, one of the things that happened was the clumps. So here we have, as I mentioned before, this form of Huntington that gets sticky, forming this protonaceous clumps throughout the cell. And then this mouse model as well, we could see these clumps. As you can see these dark kind of brown dots kind of littered throughout the image from the mouse's brain. Well, when we turned off the expression of this toxic Huntington and the mouse didn't make it anymore, we were very much surprised to see that these clumps went away. And so we were wondering, well, where in the world did they go? And does this mean that if the clumps go away and the symptoms go away, are those two things related? Does it mean that if we can get rid of these clumps, can we somehow also get rid of the disease? So to go about this, we had to first try to understand where those clumps could have gone. And that kind of took a while for us to figure out. But ultimately, it brought us to the sky. So for those of you that might remember back in the 80s, there was this creature that kind of was all over the place known as Pac-Man. So Pac-Man is this creature that lives inside of a dark box and goes around and gobbles things up and eats anything in its path. I wonder if it would surprise you to tell you that we actually have a Pac-Man like thing in every single cell of our body. Of course, we don't call it Pac-Man, we call it autophagy. So what autophagy does is actually clear out the debris throughout the cell. And it can clear out all sorts of things, including these progenacious clumps. Now, the problem about autophagy, to try to harness the power of kind of clearing out these clumps, is that autophagy can degrade all sorts of things. And so it's faced with the kind of conundrum that you might be faced if you are faced with a dish full of macaroons just like this one. So for me, I don't know which macaroons to begin with. I would want to eat the strawberry ones, the lime ones, the chocolate ones, the coffee ones, all at once. But of course, we're trying to stay focused here. We want to focus Pac-Man autophagy onto the clearance of these progenacious clumps, like the chocolate macaroons. So how can we do that? Well, studies suggested that actually Pac-Man doesn't just randomly eat things. In fact, it eats things in a very, very deliberate way. And it does so because the cell has a way of labeling those things that need to get eliminated. And so we wanted to understand how is it that autophagy knows how to eliminate these clumps, these progenacious aggregates. And that introduced us to something known as alfie. So alfie stands for autophagy link five domain containing protein, which is not as fun to say as alfie. Alfie is actually like a little flag. What it does is that it will find the aggregate, the clump, label it. And as it labels it, it sort of acts as a beacon for, in this case, Pac-Man, our autophagy, to eat the inclusion and clear it away. So we identified the flag, how to attract Pac-Man to eat the clumps. And so now we wanted to test whether or not we can actually take advantage of that. So can we take the autophagy that's in all of ourselves and now label the progenacious clumps and say, OK, Pac-Man, go eat it. And clear out those clumps. And let's see whether or not it can undo the course of Huntington's disease. So how do we go about doing that? Well, Catherine Croce and my laboratory is doing just that. What she did is she's taking a mouse model of Huntington's disease. And now, instead of adding a genetic switch like I did so many years ago, she's actually introducing increased levels of alfie. So now we hope that all of those aggregates will get labeled, right? And then we can see whether or not autophagy can go and clear them out. So let's see what happened. So here is a picture from a Huntington mouse brain. Again, you can see those dark brown dots and kind of like pepper flakes, kind of like sprinkled around the sample. These are, of course, those aggregates. Now we next like to look at a brain that has a lot of alfie. What happens to those progenacious clumps? And look at that. Where did the aggregates go? It was very much like what we saw when we turned the switch off. The aggregates went away. So it looked like alfie is doing its job. It's finding those clumps and helping to attract that Pac-Man to eat it all up. All right, so mission one was accomplished. We can clear out the inclusions. But of course, the reason we wanted to do this to begin with was to see whether or not we can have any impact whatsoever on those symptoms that are associated with Huntington's disease. So in order to do that, Catherine next started and is continuing to do some very painstaking studies of trying to understand how this is impacting the course of disease in the mouse. So the place to start is looking at movement. And so we do a variety of tests. So one of the tests that we use is known as the Accelerated Rotorod. And that's just a picture shown here. What you can see is a dark bar kind of going through the center there where the mice are sitting on top of it. This bar is like a log roll. And it just starts to roll, turn, turn, turn. Accelerating, accelerating through the course of 300 seconds or five minutes. And what the mouse has to do is scurry along and try to stay on top of this rod. Otherwise, kerplunk, it will fall down just like some of the ones that you see here. And so what we can do is actually quantify how long the mice are able to stay on this rod and be able to successfully do this task. So here are some examples I show here. So what you have is we're measuring how long the mice are able to stay on the rod. And this is a two month old animal. And what you can see in blue is the control mice, a normal mouse. And normal mice are able to do this task with no problem. They just stay on for 300 seconds. Well, then if you look at the Huntington's disease mouse here in red, what you can see is that the mouse has a more difficult time staying on the rod. And they have a tendency kerplunk to fall at about 220 seconds. Well, now what happens if the mouse has Huntington's disease but also expresses a lot of alfie? Remember, these mice have fewer clumps in their brains. Does this have any impact whatsoever on the movement? And so far what Catherine is showing, very excitedly, of course, is that indeed, these mice look just like the normal mice. They're able to stay on the rod for the full duration of the 300 seconds. Now, we don't only look at this at one age. Catherine's been looking at this continuously across many ages. And I hope you can appreciate that if you look at the green line, that they are able to look just like the blue line. That is to say that the Huntington's disease mice with a lot of alfie look just like normal mice and are not showing the motor symptoms that we see in the Huntington's disease mouse. Now, this is already very exciting. But as scientists, we tend to be very, very cautious with our conclusions. So we had to, of course, do another test. And luckily, it's a relatively simple test. It's known as the open field. The open field is literally a box. And inside the box, you put a mouse, just like I show here. And what we do is we just measure how much it runs around the box, explores the box, to try to get a sense of how well it can just simply walk around in its environment. Here is an image just depicting some of the data we have so far on that. Here, again, in blue are the normal mice. And they tend to walk about 8,000 centimeters over the course of two hours. That's a lot for a little old mouse. Now, in the Huntington's disease mice, they still can move around. But they move around a lot less, closer to about 6,000 centimeters. And so what happens if we have alfie, the mice that can do the accelerated rotor rod, the mice that have fewer clumps in their brains? Well, in this task, they don't do as well as the normal mice. But they sure do a lot better than the Huntington's disease mice, suggesting that alfie seems to have an overall impact on the way the animal is able to move, that somehow, in the absence of these clumps, these mice can move very much like control. So if I take it back now to this graph that I've been showing you, what we think is happening is that in the presence of alfie, even though toxic Huntington is continued to be expressed, the clumps go away thanks to alfie, thanks to that pack man that can gobble those things up. And then when it comes to the actual symptoms that we've measured so far, it's significantly delayed. And when we do start seeing it, we anticipate that we'll see a very, very blunted symptomatology. Of course, it's going to take a while before we can really complete our studies to see the lifecycle of this mouse. But so far, we're very excited about how promising it looks. Now, this in and of itself is very exciting for our Huntington's disease research. But I wanted to just simply raise the point that there's more to this than just Huntington's disease. The appearance of these abnormal proteinaceous clumps, the accumulation of protein, is something that we see across a lot of different diseases, from Parkinson's disease, ALS, and Alzheimer's disease. This is just a mosaic I like to show of various images I've taken from different sources showing how these proteinaceous clumps can be found in different diseases of the brain. And so our next goal also as we continue our studies with Huntington's disease is to really try to understand to what extent alphae and autophagy can impact proteinaceous inclusions, these clumps, inside brains of different diseases and see whether or not it can have similar reparative effects as we saw in HD. So that's all I have for you today. I just like to mention that this work was not literally done by me. I don't do it anymore. I have all these people that trust me with their futures and they really work hard for me and I want to mention them. I only have here highlighted the people that have contributed directly to the work with Alfie and Catherine's work is really what I highlighted today. Leora, Kyrgyz, Joanna, Aveline, they've also all contributed significantly to the progress that we have made over the years with Alfie. Also, there are people outside my laboratory that we work with very closely and Siemensen is the person that named Alfie so we can credit her for giving us something fun to say. William Yang, we're doing this to phenotype the mice that is to characterize the symptoms more deeply. David Hausman, Nancy Wexler, Jean-Paul von Sattel, where we're actually looking at how Alfie might be impacting age of onset in HD patients. And Bev Davidson and Frank Bennett represent a new line of work where we're trying to actually augment Alfie therapeutically in people. Thank you very much. Well, thank you very much, I, for that outstanding presentation. We have quite a number of questions here and I'd like to start with the first question that someone wrote that's not directly related to your presentation but I did want to just address it in case anyone else is thinking about this. The question is, I'm not sure if this is the right forum for this but I'm coming close to early onset age with Huntington's and my family. What is your recommendation for testing and are there any benefits to finding out early? What I would say to this is if anyone is having similar questions about this topic, please email me, Megan Donaldson, M-E-G-H-A-N, Donaldson, D-O-N-A-L-D-S-O-N, at hdfoundation.org and I'd be happy to speak to you about this. Now with that, I'll leave it to I to answer the questions about Alfie. Thank you. Great. Thank you, everybody. First, thank you all very much for coming to the webinar. I hope you enjoyed it and I'd like to thank, of course, the Hearty Territory Disease Foundation, their supporters, which is many of you out there, for your continued support because without you we could never have gotten this work off the ground. I'd also like to thank the Alexander Boyd and Jane Starboy Charitable Foundation for their support, especially of this ongoing work we've really entered. I hope you all agree, an exciting phase of this research. So there are several questions that have been imposed. A lot of anonymous attendees, I was going to list everyone's name, but of course, I'm happy to start answering them. Some of the questions are similar, so I'll put them together. So I think one of the continued themes in the questions has to do with, if this looks so promising and nice, how are we going to bring this to humans? Can we label these protein clumps in human cells? Can we ever test this in humans? Can we bring this to a therapy? How would we go about doing that? And that is, of course, a very big, typically, actually, to be perfectly honest. First of all, we would usually say we just put the ideas out there and hope someone brilliant will come up with a plan. But thanks to Nancy Wexler, we already have a plan. So I'll get into that in just a moment. So first of all, this protein is present in all of our cells as well. It's not something unique to mice. And so this was one of the reasons why we really started to focus on it is because this is something that we actually saw was active also in human cells. So it was something that we could translate potentially to patients. So this was something that was very interesting for us. Now, how do we bring about doing this? And so as we're doing our mouse-based studies, we're also looking at human-based model systems. These are always in cells. We are not yet at the stage of bringing this to patients, of course, to make sure it's working in the right environment. And it seems to be working, which is very exciting for us. Now, in terms of how are we going to bring about this to patients? So this is where, for those of you who know Nancy Wexler, you know that when she wants you to get something done, it's going to get done. And so very much thanks to her, she got me to be able to kind of ask people. And thanks to the Scientific Advisory Board and members of the Davidson, Frank Bennett, we're actually looking at ways by which we might be able to increase Alfie levels in patient brains. And whether or not we use an ASO-based approach or kind of a more typical medicine-based approach is something that is left to be seen. So we can add Alfie to human cells. And we can also put this into human cells. We're studying Alfie in the context of other diseases. So one question is, Alfie being studied in other diseases yet, another related question, hi there. I'm curious, Dr. Yamamoto, is there an Alfie for Alzheimer's disease? Another question that was very similar. Do I saw somewhere along the way? I'll answer these two questions. Is that we are currently actually looking at Alfie in the context of mouse models of Parkinson's disease, mouse models of Alzheimer's disease, and mouse models of frontotemporal dementia and ALS. The reason we can look at all of these different diseases is because they all share a feature, which is the formation of those clumps. And so if we can direct Alfie to those clumps as well, maybe we can bring about the same kind of promising outcomes that we're so far seeing for Huntington's disease. Now, there was a question about how do you get Alfie to go to the aggregate? And that is a really good question. This is something that we are trying to understand. Basically, what we believe happens is that the clumps get labeled by the cell, kind of like a postage stamp equivalent. And Alfie can find that postage stamp. That actually is a whole other set of machines that we have to kind of bring into play. And what we were hoping by just focusing on Alfie was that Alfie was the part that was missing. That is to say that these clumps are getting stamped, stuck all over them, saying, please get rid of me, please get rid of me. But we thought maybe Alfie was the one that was kind of slow to respond. And that's why we thought boosting Alfie levels will actually help. So what is the current research emphasis from Gloria? Well, the current research emphasis is really about trying to understand increasing these Alfie levels can indeed drive not only the change in behavior, the positive change in behavior we see in Huntington's disease, but we want to understand that more deeply. There's always a question, how much of the mouse really mimics what's happening in patients? And so we really want to understand that translation better. So that's really one of the big emphasis right now in our work. Let me see, the questions are starting to come in. So for those of you who can't, it's like the scroll, it's so hard, it's hard to read. I have a good one here. Comes from an anonymous attendee saying, how is the research with Alfie applicable to those with HD now? Do you think this research will be useful to them within the next 10 years? Wow, within the next 10 years. That's a very good question. Thanks Megan, that was really hard. Okay, let's see. So it's always hard for me to know how long these things take, right? I mean, so first of all, I guess one of the things that I can definitely say is that we all have Alfie in ourselves and we know that the Alfie that we have currently is working as hard as it can to do the best it can. That is to say that we did experiments where we actually removed Alfie from otherwise seemingly healthy animals and it made them sicker. So we know that Alfie, the Alfie is there is helping to sustain whatever movement we can. Right now, there is nothing that we know of that can boost Alfie levels with any kind of known medicine or known vitamin or something like that. So I would say that right now it's still very much in the experimental phases. We are working very hard to see how quickly we can apply what we're finding in these animals in patients. As I mentioned before, it is a current focus of ours. And so I would hope that in the next 10 years we will be somewhere where we're actually talking about what is happening potentially in patients. And given the breadth of Alfie's function, hopefully we'll be able to get there soon. But I have to say that this is really outside actually bringing this kind of experimental work to the lapse of patients is very a bit removed from my expertise. And so it's difficult for me to give a real timeline on this, unfortunately. I following on from that is an interesting question from another anonymous can be, are these experiments being done in a rescue model where the Alfie are gone after the mice have developed symptoms as would be the case in a human treatment? Yes, so those experiments are currently ongoing. So Catherine Croce and the lab who's leading these experiments, who's wonderful by the way, she is currently doing experiments where she's taking mice and waiting for them to become, to have clumps in their brains especially. And then turning on Alfie, right? And using our genetic tricks. This is not with a medicine, but with a genetic trick to see whether or not it can affect change. Of course, these are the kinds of experiments we would like to do because of course, this is how we would envision a therapeutic being used. And so this is how we're trying to address these questions in a way that we know best. Okay, and another question from Christopher Pearson who is on our scientific advisory board and is going to be presenting our next research spotlight webinar in June. Christopher writes, does Alfie alter levels of pelvis in the brain? If so, is there a specific cell type that is affected? Okay, so, and for those of you listening, I'm gonna go into a little bit of jargon here because I'm just gonna take advantage of the fact that Chris should understand. So basically when we did a loss of function experiment, it's almost like talking in a different language. I feel like I'm talking in Japanese all of a sudden. When we did the loss of function experiment, so that is to say when we decreased the levels of Alfie, we actually did not seem to affect cell death. That is while the patients, the mice got sicker, we did not accelerate cell loss. Now, and this has a lot to do with the fact that the mouse models we are using do not experience cell loss. So it's very difficult for us to study. For those experiments that I was showing today where there were fewer inclusions and then the onset of the symptoms in the mice were slowed. Here we, again, don't know if we're affecting cell loss because at least the ages that we've looked at thus far do not show cell loss. So this kind of question is something we would like to answer in the cell-based and the human-based models. These are the direct conversion, so where we take fibroblast skin cells from patient with patients and turn them into neurons. That model actually shows cell death and we are trying to figure out a way to administer Alfie to those cells to see whether or not we can affect cell death. So I can't answer a question about cell type specificity but hopefully we'll be able to with the direct conversion-based model. Okay, I have another somewhat scientific question for you to answer from another anonymous attendee. Is there any evidence from the human GWA studies that variation in Alfie affects the age of motor onset? Yes, so this is the very exciting work by David Hausman, Nancy Wexler, Jean-Paul Vonsetel in collaboration with the New York Genome Center, turning to one of our most precious, precious resources, which is of course the Venezuelan cohort. And there, I mean, it's like unbelievably enough, but really what really galvanized our work and gave us kind of the courage to really go after this full throttle, had to do with the fact that it was found that in patients, Alfie, like all proteins in everyone's body can be slightly different. They're called variants, they're slightly different from one person to the next. And there was a group of patients in the Venezuelan cohort that actually showed a variant of Alfie, and Alfie that was slightly different. I mean, super, super slightly different that actually had a delay of onset. So instead of say at 40 years of age had the disease onset at 50 years of age, average age of onset delay of 10 years, the maximum delay age of onset was 23 years. So this is what really got us excited because we were like, oh my goodness, sometimes when we do experiment at a bench and play with mice all the time, we start losing track of what's real and because we hope so much of what we're seeing as real. This was what we call independent confirmation, a study outside of our realm telling us that we're onto something. It looks like, and Catherine's also exploring this, how does that little change boost the way Alfie can work is something that we're exploring currently because this is really going to give us insight into how we can potentially increase Alfie in patients. So yes, the GWAS studies were very valuable to us. Okay, thank you, Matt. I hear a couple more related questions from two anonymous attendees. If we already have Alfie to clear the aggregates, why do they form to begin with? And related to that is, are clumps the only thing Pac-Man eats in ourselves? Yes, so for the first question, yes. So basically what we know is that if we didn't have Alfie, the clumps would form a lot sooner and we would experience patients, experience symptoms we would argue sooner. That is to say that the Alfie we currently have working in our brain works for as long and as hard as it possibly can, but somehow Huntington's disease is stronger than Alfie. That's why we need to kind of give Alfie a boost. I don't know, a Superman cape, something, something to help it work better. But what's promising is that there's something in there that's already working and we can potentially take advantage of it. Now, of course, Alfie shuttles the aggregates to Pac-Man. So does Pac-Man eat me? Well, we have to tell Pac-Man to eat the aggregates, the chocolate macaroons, because actually Pac-Man likes strawberry macaroons, lemon macaroons, all sorts of macaroons. That is to say Pac-Man goes around because it is important in keeping ourselves nice and clean and healthy. It has to clear out all sorts of other things. And so in essence, what we've been trying, what Pac-Man can do is eat other things. And that's why we have to boost Alfie and say actually you have to like the chocolate macaroons a little bit more. We of course don't want to distract Pac-Man. Pac-Man needs to do its other things as well because that's also very important. But we know that for some reason those aggregates are much of a distraction for our brain cells. And so we're trying by maybe just making them like chocolate a little bit more than strawberry. Maybe we're hoping that that's really what's affecting change in these brains. Okay. Thanks for that. I, another quick question. I'm calling on an anonymous attendee. What was the HT mouse use in these studies? Oh, wow. We used a couple of mouse models. So for the slowing, so for the elimination of the aggregates and the slowing on the behavior that we showed in the video, we use the mouse model known as the Borschelt model, the N171 model, which is a fragment model which uses only a portion of Alfie. And then we also use the CAG 140 knock-in model. That is to say it is a mouse uses mouse Huntington where the Huntington's disease mutation is put inside. There are experimental subtleties as to why we use both models. But what's very exciting is that Alfie seems to be able to work in both situations, which was very important because of course, in patients, one could argue we could see one, if not both of those forms of the protein. So we wanted to make sure that Alfie can act on both. Okay, thank you. And then another question, a bit on the more scientific side from a participant named Boone Mabam Malou. I'm sorry, I probably got that totally wrong, but his question or her question is, could the targeting system used be applied to epitopes presented on other inclusion body disorders? And I think you may have touched a little bit on this already. Yes, so we think so. So what we do know is that, like what we've tested explicitly is we've looked at Huntington's disease like polyglutamine inclusions, and we've looked at alpha-synuclein-based inclusions. And Alfie seems to be able to work in both in cell-based systems. For synuclein, we are now looking in the adult brain, so in the mouse brain, so we're doing this in mice. In theory, we think that it should work generally. The way protein clumps are viewed by Pac-Man, it doesn't care what is in the clump. So in theory, we believe it should work more broadly. We're currently looking now at TDP 43 inclusions and we're also starting to look, which we haven't fully started yet, but we're starting to look at intranu- intracibata that's still inside of the cell. So we think it should, but we have to actually test to make sure that it does work inside the brain as well as it does inside the cells in a dish, which has been a lot of, where a lot of our experiments had been done thus far. Okay, we have another question about the mice test. Did you use any other bolder cognitive tests to evaluate the mouse symptoms? And if so, what were the results? So the tests we showed are thus far, I believe the only tests we have run are the two motor tests. We have not done cognitive testing on the mice yet. That would be a good idea. That would be a good idea. And here's another interesting one from an anonymous attendee. Are there other proteins like alfie that are also in our cells that aid in clearance of aggregates? I mean, there is a whole lot of proteins inside the cells that kind of all play into what alfie does. However, in terms of actually working like alfie, people have looked at, there are a couple of proteins that might work like alfie. And at least for a couple of, at least one of them in cell-based systems, it doesn't seem to work like alfie in the sense that if you eliminate the proteins, they all cause more aggregates to form and accumulate inside the cells, just like alfie. But what set alfie apart in our very early studies is that if you increase levels of other proteins, it doesn't seem to affect the turnover. That is to say, it doesn't seem to drive Pac-Man to eat the aggregates faster or more. It doesn't increase its preference for chocolate macaroons, as I like to say. So, alfie was unique in that increasing its levels is what actually drove the elimination of the clumps. And that's why we focused on it. But I've always, you know, never want to kind of rule out the possibility that there's like, you know, more than one way to shoot a duck or whatever is the metaphor. Maybe not a duck. I don't mean to kill any ducks, but whatever is the expression. So, but as far as we know, we've only, in the screens we use, only identify alfie. It's what, it was a factor that made it stand out. Okay, thanks for that. We have another question from Casey Fox asking, is alfie a large molecule and therefore difficult to increase its concentration in the brain? Is that one of the reasons that it isn't closer to being used as a drug therapy at this time? Oh, hello, Casey. Thank you for a very, very good question. In fact, alfie is very big. This is one of the reasons why, you know, we're trying to think of alternative ways to increase the levels of alfie in the brain. We can't just put it in because it is very big. And, you know, this is why, you know, with Nancy's health, and of course, I'm sure they would have helped anyway. It's just that I guess I was a little shy. But, you know, with the help of doctors, Bev Davidson, Frank Bennett, I get to use their brain power to help, you know, figure out how it is that we might be able to tell the cells themselves to actually make more alfie. And this might be one of the more effective ways to actually boost the level of this very large protein. I do want to address a question. I think it just happened to be floating in front of me by Sandra Fienko, which I think is an interesting question, as many of the others have been, is alfie labeling other slash physical proteins for autophagy? And could this contribute to possible adverse effects, right? We've only talked about, you know, how increasing alfie can be good, you know, could increasing alfie be bad? And, you know, this is something that, of course, we are very concerned about, right? If Pac-Man goes around eating a lot of other things, and this is where I could think it's especially, it could potentially be bad, that first alfie might distract Pac-Man from doing what it needs to do. And that could be adverse. It could cause, you know, just garbage to start piling up in the cells and then affecting it, affecting cell function. The other thing, though, is, of course, alfie might call other things chocolate macaroons, right? Call things that the cell actually needs and make Pac-Man eat those things by accident. So these experiments that we're doing in mice, where we're increasing alfie levels in mice, I think is the most telling. So so far in Catherine's experiments, where she's increased the level of alfie in the entire body from the beginning of time, she's also doing it in later in life. So far, these animals of just alfie, higher alfie levels are not showing any issues. They are not showing any signs of stress, discomfort. They are aging apparently normally. They're breeding normally. They're behaving normally. So it looks like while the possibility is always there, at least in the approaches we have used thus far, we are not seeing any detriment in the presence of increasing alfie levels. But in all fairness, you know, understanding how alfie works, what alfie does inside the cell is still actually quite early science. It's not very well studied. And so there's still a lot to be learned in that regard. So thank you for the question. Okay. I had another couple of questions about CRISPR. Here we go. One from an anonymous handy who writes, I know research is slow on this disease as my mother passed away from it 21 years ago. How does CRISPR factor into experiments? And then related to that. Is one again from Booney, my bomb balloon as a CHE expansion disorder with anticipation becoming significant around 40 years. As you said, would it be possible to utilize embryogenic CRISPR to delay presentation of this glutamate channel disorder? Okay. So for the first question, can CRISPR somehow play a role in this? So I guess, you know, the way I would answer this question is that it will go to those experiments looking at what I was referring to earlier when someone asked about the GWAS GWAS studies about alfie. And I was talking about how in the Venezuelan cohort, there were a group of patients that had a change in alfie, a small change, but that a single nucleotide change, as small as you can be. And that was sufficient to potentially boost alfie function. So one place where CRISPR could play a role is to make that little change in alfie, right? And if, in fact, that change is sufficient to drive alfie, increase the way alfie works, that's somewhere where CRISPR could play a role. Now as to the second point in CRISPR and kind of like eliminating the CAG and embryogenesis, because this is where I guess, I think that's what the question was asking. I mean, I guess in theory, I think embryogenic CRISPR, there are a lot of other concerns and ethical concerns that are related. And this is really, I have to say outside of my wheelhouse, because this is a technology that we actually are not using regularly. We kind of use more traditional approaches. So as we are not developing CRISPR-based approaches. So as I said, you know, we just put the information out there. And so if there's someone that can do it, please, please go do it. Because we can't do everything ourselves. Okay. I have Thomas both, the HDI, who asked the question. Hi, Tom. In addition to your histopath and behavioral studies, have you performed any molecular comparisons by RNA-seq, SCN RNA-seq, and proteomics and controls versus alfie over expression mouse studies? So thank you for this question. So we are collaborating actually. So the reason we're using the CAG 140 mouse model has to do with the fact that we're collaborating with William Yang, who has of course done a huge study characterizing the impact of the trinucleotide repeat expansion in Huntington mouse models and using the full panel of omics-based approaches to characterize the impact of potential variants and modifiers on the transcriptomics and proteomics of HD. And we are going, we are basically using his design, much in consultation with active consultation with William, where we're doing both a loss of function, partial loss of function of alfie, as well as gain of function of alfie and looking, doing RNA-seq. Now, one of the things that we are, we have some hints that the impact that we are having on the HD phenotype in these mice is beyond just the histopathology and the behavior, but those studies are really early going and we would like to do kind of the full blown RNA-seq. And I believe Catherine has finished collecting the sample so that we can do so. Or we're almost there. I don't want to, I don't want, if Catherine's listening, I don't want her to like dive a heart attack because I guess she did more than I claim. Sorry, Catherine. I think we're in process. That's for sure. And so we should be getting the RNA-seq data soon. Okay. I think we're going to need to wrap it up. We've got about five more minutes. Do you want to just take a quick look and see if there were any questions that you would like to address before we wrap it up? Sure. There was just one quick question. Because I do like to say, you know, there are possible issues. There is a question by an anonymous attendee, were there any mouse models where Alfie did not work? Of course, we always focus on the positive data. But so far, no. But that's because we've only done it on the models that I showed. So there's still plenty for failure, I guess. Those scientists, we kind of prepare ourselves for failure. It's basically, it's something where we train. And then there was another kind of technical question. What kind of cells, for example, astrocytes and microglia are important for the Alfie mice, clear, misfolded proteins. And I would say that it's within neurons. It can also be through microglial engulfment or potentially autophagy and microglia, as well as potential autophagy and astrocytes. We still don't know. But Alfie is expressed in all of the cells. And our experiments are also targeting all neural cell types and not just neurons. There are a lot more questions, but I think it's just like lots of them. I'm happy to answer them offline if people would like. So if there's a mechanism by which that is possible, I'm happy to do so and address any of those questions as well. Yes. If anybody would like to send additional questions, you can send them to me, Megan, M E G H A N, Donaldson at hdfoundation.org. And I can pass them along to Dr. Yamamoto. So just one last question that I'd like to address, especially given the news from Rose, Roche and Genentech. I just want to put it out there. It's from Robert Burgess. Robert, thank you very much for your question. If a trial has risk, but the risk is greater to live with Huntington's without any treatment. Shouldn't a patient be able to choose to get treatment now? I think that's an excellent question. And thank you so much for asking it. It's a very difficult question for us to address. And in light of everything, the news last night, I think we just have to wait and see what happens with all of the data from that trial. And there are lots of other trials that are continuing to go forward. And let's just hope that those have good results and that we'll find some good results from the, the Roche, um, Genentech trial. I think that's something that, um, I would recommend you discuss with your family and your, your doctor and your genetic counselor. I did you want to add anything? No, no, I mean, that sounds great. Yeah. I'm also anxious to understand what happened. I think we all are and hopefully we'll get some more, um, information soon. But to wrap up, I, um, the predatory disease foundation would like to thank, um, triplet therapeutics for underwriting our research spotlight. Webinar series and also Novartis for sponsoring today's webinar. We'd especially like to thank I am a moto for her incredible research and the time that she's graciously spent with us today. Thank you also to eyes colleagues, especially Catherine crochet, who has performed much of this work that I discussed today. With the valuable insights that we gain across the medical and biological professions, we're better able to serve victims and other families of Huntington's disease and so many other brain disorders. Thank you also to all of today's attendees for meeting, learning and sharing with us to continue our research spotlight webinar. We will be hosting our next one on June 15th, noon Eastern featuring Dr. Christopher Pearson. Of the hospital for sick children at the University of Toronto. Registration is open. www.hdfoundation.org slash webinars. Thank you again. I for this incredible presentation and thank all of you for joining us and for asking your excellent questions. Great. Yeah. Thanks for having me. This was a lot of fun, everybody. Thank you.