 Okay everyone we're going to get started and I want to welcome everyone to our virtual grand rounds today with with Sheila Baker who's on the screen here right now. And I'd also like to welcome I think we have our first cohort of applicants for our residency here and many of them will be online this morning so this is our typical and somewhat atypical as well grand rounds we needed eight o'clock on Wednesdays, usually in person but. And, but today we're having a guest lecturer from University of Iowa, and it's Sheila Baker, and Sheila just to give a little bit of background because I don't think Sheila and I have ever met in person. As far as I know so she's she's from the Midwest, or at least trained did her undergraduate degree at university University of Wisconsin, Stevens point and then got her PhD at the Medical College of Wisconsin. She did a postdoctoral fellowship from 2003 to 2005 at mass I near, and then was a research associate at Duke University. And since then she's been in Iowa at where she became an assistant professor into in 2010, and was promoted to associate professor, and she is in biochemistry so biochemistry of eye disease, and she'll be talking about KCNV to retinopathy a story of mice and men. And for those of the residents here we're doing our retinobole tomorrow so this could have been an it could have been a question on there because this. What's important about KCNV to, and she will. We'll talk about that and what's interesting in terms of electrophysiology today. And since she's a biochemist she focuses, obviously on mice and other animals. But when I saw the title of our talk and said, as a clinician, I do know something about that and, even though it's a rare disease I think it's on the order of a one in a million disease. I'm going to base here of our patients and I do see patients with this and all I had to do was write a quick email to Emily Spoth our genetic counselor, and she came back within 10 minutes and said, These are the three patients that we have with this disease. So, this is translational this can be a translational talk and to start things out. I'm going to have Lydia Sauer, give a just a very brief presentation of our and some pictures of our three patients before we go to Sheila. Thank you very much. I'm just going to share the screen here. I can everybody see this. So I'm going to talk about just the clinical cases of Casey and me to write an opposite that we have seen at the more and I center. And as Dr. Winston mentioned, there are there are three cases that we have seen all three of those have been genetically. Analyzed. The first one is a 23 year old male with a compound hydrozygous Casey and the two mutation with two pathologic alleles. The second one is a 37 year old female that is heterozygous in the US. And the third one is 16 year old female with a compound heterozygous that is likely pathologic. And with the US has a very similar phenotype and we think that she actually has the disease. And here are the three fundus photographs, as well as auto fluorescence fundus auto fluorescence intensity images. And I just want to highlight the clinical features and the general things about this mutation. So it's an autosomal recessive rod cone disease that is not up. One set is in the first to second decade of life, and it has a prevalence of one in 865,000 people with an incidence of five new cases per year. So it's pretty rare. And Casey and me to encodes for a voltage gated potassium channel that sets the photo receptor resting potential and is responsible for the voltage response. Symptoms that the patients will experience are central scatomas for visual acuity variable photophobia and red queen does comatopsia with a relative loose bearing. And then the findings that we see on imaging would be a ring like a wall side change in the macula central atrophy increased for real auto fluorescence and variable OCT findings with like variable auto retinal integrity loss of the ellipsoid zone and all hypo reflective spots. And all three patients showed this in both of the eyes. And the very important passive economic finding is the electro retinogram signature where in the dark adapted state, the a wave is normal to normal with a late negative component. The wave has a relatively high amplitude and it's called or can be super normal. And these are just two of our patients who had EIG imaging. And that's a very classic finding that's very specific to Casey and the two mutations. And I think that Sheila is going to talk more about that. And with that, I'm actually going to have Nana come up because he has some fun facts about her. And then we will give the word to Sheila. Thank you. Awesome. Good morning everyone so we couldn't let her start without a couple of fun facts. So she has two cats we might be able to get a glimpse of during her presentation, named Dungey and monkey girl. And then she has. She's an avid reader, a big fan of the murder bot diaries. So with pleasure, we'll pass it over to Dr Baker. And so you can begin your presentation here. All right. Thank you very much. That was lovely. Let me just share my get my screen set up here. Okay. Hopefully now. We should be good you should be seeing my title slide and able to hear me. Is that all good. All right, thank you. So, first of all, I want to thank you for accommodating my request to have this meeting virtually there are a number of personal reasons why travel is difficult for me. And one upside for me out of the whole chaos that is the pandemic is that we learn how to do virtual meetings better so I really appreciate you, allowing me to do this. I'm going to tell you about work we've been doing in my lab recently investigating the biochemistry molecular biology underlying casey and V2 retinopathy. And I didn't know we'd have such a lovely presentation so I have my real quick intro to the disease. So we commonly call it in the field casey and V2 retinopathy. And it's in that name is very sensible it's a disease of the retina caused by mutations in the gene casey and V2. And I'll be telling you all about how that affects the protein whose name is Katie 8.2. And more formally at least according to OMIM the disease that should be called retinal cone dystrophy 3B, but the only other name I have ever come across is cone dystrophy with supernormal rod responses. This name is very cumbersome but I like it because it helps me understand what's going on. And I also like it because it gives me a short acronym I can put on all my title slides. I will be going back and forth using CDSRR or casey and V2 retinopathy interchangeably. As we just saw the key features of this disease is that it starts early in the first and second decade of life. It's characterized by a macular degeneration that typically can start as early as the second decade of life and it's progressive. Sometimes it starts later. And when you have a macular degeneration you tend to get a lot of phenotypes consistent with cone dystrophy such as loss of visual acuity loss of color vision and photophobia. So this part helps with the name of the disease the first part is cone dystrophy. This is part of genetic explanations for why a person might have a cone dystrophy. What sets this particular disease apart is that it's also has variable amounts of night blindness in patients and most strikingly it's characterized by abnormal ERG. And that's where the part of the disease named supernormal rod responses come from. This is truly unique. And this, because the ERG is a recording of the electrical activity the retina this really gives me some insight into what's going on. And then I have one pitch fundus image from a patient seen at Iowa so as we just learned this disease is incredibly rare, but there doesn't seem to be any particular genetic or ethnic group where the disease shows up it just happens all around the world you'll find cases everywhere. And we have them at Iowa, it was actually, I mean, obviously feel bad for the patients but I'm quite glad to hear that you've seen them and you can help me understand this disease from your perspective. I will also make a point that I think that there's a high likelihood that this disease is under diagnosed. And to help explain that I want to tell you my own experience, sort of with a patient. So I'm a basic fundamental scientist. I'm a lab rat myself, and I don't interact in the clinics but when I first got this grant to study kcmd2 retinopathy I have someone contact me was really, really quite nice they just started by thanking me for actually studying their incredibly rare disease. But through a series of communications this particular patient volunteered their medical history in an effort to help me better understand what was going on and this had a huge impact on me and well as my lab very motivating I want to encourage you that when you do see maybe rare patients that that perhaps the PhDs in your eye center are studying, go ahead and invite them into the clinic and let them see exactly how the disease is affecting the life of their patients it can be really rewarding. So this particular patient, their history starts as young as five years old. They went into clinic they were treated for amblyopia, also reported at that time and he was already struggling with tracking small objects had poor color vision, especially under low contrast conditions. In his early teens he was really struggling with reading in dim light, and he went in for testing he was given a visual field test but he failed it. But there was no diagnosis given at that time that he was just told, yeah you just did a bad test, and they let him go on with his life so a few years later. And he went back for more testing because he was starting to notice progressively failing central vision, which he described as there's a dark blob over people's faces, which is exactly how my grandmother with amd describes her vision. Adaptation for this patient is delayed and he described that as anytime he moves from a bright room to a dark room, or conversely a dark room into a bright room it takes has to pause and it takes quite a bit of time for him to adjust. And he was a little cutie is not too bad at that point. And then, just a few years later, he was diagnosed with something similar to star guards disease. And the main complaints that he had is he continued to have difficulty tracking objects. When he was a young child that was tracking small objects that were hard for him. And now it's becoming harder and harder, even the large objects, including cars. So I was very glad to hear that he had stopped driving, although his excuse for stopping to driving is that he's having a hard time seeing stop lights road signs and potholes. I think you should be more concerned about the other cars more than the potholes, but there you go. He was under dim light, meaning rod media vision was becoming worse in his visual cutie was dropping off a little bit more. Finally, when he was 25 years old, he underwent genetic testing and finally had an ERG done, and that ERG and the genetic testing is what allowed him to move from you have something wrong with your retina that kind of looks like this other disease to an accurate diagnosis cone dystrophy with super normal rod responses, CDSRR or ACMV2 retinopathy. At this point, we really can't do very much for the patient. He's working with various low vision devices he uses a lot of contrast inversion works under very bright light, and on the and can still work on the computer had into use zoom and large fonts. I'm in a position where I'm a photoreceptorologist and I think that I know enough about photoreceptors that I was really intrigued by this disease and I thought I might have some ideas and be able to explain what was going on. And if I could do that, I thought that I could work with people such as yourselves and we might be able to come up with better options for patients like this person as well as your own patients and any others in the future. For those of you who are particularly interested. This patient did when he had his genetic testing done it's found that he was heterozygous for two variants in case you need to. One is pathogenic and I have a good explanation I believe for why that is. But if you're really interested in the molecular details, you'll have to stick around for my research seminar later on today. And then there's a mutation where we really don't know what's going on, which is unfortunate for the patient but good for me because it means there's more work I have to do. All right, so now what exactly is Casey and you to. It is a relatively small gene I'm sorry I think I'm sharing this window there we go with that a little bit easier. So it's a relatively small gene, simple two exons coding sequence is a little bit less than two kilobases. That's good anyone who's thinking about gene therapy like aha that can fit into a a b viruses if this seems like a good idea. And again it's very rare, but in the last 15 years or so I've come through everything in pub bed and I found at least 61 variants that I think it's reasonable say that they are definitely linked to causing the disease. When you look at the types of various show up in the gene, about 50% of them are nonsense mutations or frame shifting mutations that are going to result in the absence of protein expression. And I think that's interesting this is a case where such patients would be the best candidates if anyone were to develop a gene therapy for this disease. The other 50% of the mutations are missense mutations, and I've drawn on to this cartoon showing the topology of the protein Katie 8.2. So this is a membrane protein it has six membrane spanning domains. So what is the organization is that of a typical voltage gated ion channel. So it has a domain that functions, or can function in sensing voltage, and then it has a domain that will form a poor has a third domain that's a little bit unique to this particular ion channel as it has it's called the T one domain, and that's used to interact with other ion channels that are related to it. And I'll be telling you a lot more about that in a second. The missense mutations have been reported for the gene will affect the entire length of the protein they're scattered throughout the different domains, but the bulk of them that we know about today, impact the T one domain. So what is that T one domain. How does it affect the protein and then how does the protein affect our vision. Right. This T one domain is going to be important for assembly. So I have to tell you about now, you have to learn about more than just one item channel today. You have to learn a little bit about the larger family of voltage gated potassium channels. A family that subdivided into different groups you have KB one, two, three, and four. These all form ion channels that are expressed throughout the nervous system in the heart, the pancreas, and in several other places at lower levels. I'm always familiar with these channels because they are responsible for generating a propagating action potentials in the nervous system. Consequently patients with mutations in the genes and coding these channels, typically present with epilepsy. One of the things that I find interesting is that you can sort of fine tune neuronal signaling by adjusting the exact type of ion channels that a neuron will express in diversity in signaling within this family is achieved by mixing and matching different sub units. So it affects the biophysical properties of the channel. You get slightly different. Transmission effects. This family is divided. So this is a family where no kissing cousins are allowed. So for instance, this, the genes within the KB one subfamily can mix and match and assemble assemble with each other, but they don't interact with KB for KB three is alone. So KB two is the interesting exception. KB two has a is closely related to a large subfamily that is called the KBS for silent. And I will explain that in just a moment. I will say that in the retina, even though we have all of these different KB genes and the genome in retina. We only have two to worry about one from this KBS family are 8.2 culprit for the topic today, as well as KB 2.1. I think that they form an ion channel that would look something like this with the transmembrane domains for me to voltage sensor here, pour in the center, and then that T1 domain hanging down like a gondola and cytoplasm. So these ion channels are expressed in photoreceptor inner segments, and just a word of warning here. We all know that culturally we like to put things that are most important to us at the top. When we present slides, and as a photoreceptorologist, all my retina pictures have photoreceptors at the top. So you need to take a just a brief second and flip that image in your in your head to have it make sense. But this is the outer nuclear layer photoreceptor layer outer plexiform layer, inner nuclear layer game themselves are cut off. So what you see here is labeling for KB 8.2 and 2.1. They're found in the inner segments of photoreceptors. Now, what's the deal with having both of these. They're having interdependence and first let me tell you a bit more about KB 8.2 and why it's called the silent channel. The easiest way to explain that is to show you this nice experiment from a paper in 2012. These are heterologous cells have been transfected with protein. You can see if you if you express KB 8.2 all by itself forms this beautiful reticular pattern, but there's none of it out at the plasma membrane. So if an electrophysiologist puts a patch pipette on that cell they will record no current. Hence, it's an electrically silent channel. So there's just no channel actually at the surface. However, when you co express 8.2 with 2.1. Now you do get it released from internal stores and it traffics out to the surface and you record a current. But having 8.2 assembled with 2.1 does affect 2.1 as well. So it's trafficking, but it's activity. So what happens in this is of getting a figure from the literature and so the paper in 2007. And this is a little bit complicated but I don't just take you through the key observation here, at least in my mind. So the upper plot is the normalized current plotted as a function of membrane voltage. What you need to pay attention to is this range down here. So photoreceptors in the dark adaptive condition have a resting numbering potential about minus 35. And then when they hyperpolarize a response so like they go more negative minus 70. So, there's a little bit too much information in this graph but if you'll pay attention to the lines with the square symbols this is the activation of data for this channel. The big squares low are just KV 2.1 in this channel forms a beautiful well functioning homo tetramer channel throughout the nervous system, but its activation kinetics are such that in a full receptor, it would be closed at all times. However, when you add in 8.2. Now the activation curve shifts the left. So that at photoreceptor operating potentials you do have open channels. Now there's not a whole lot, but that's exactly what you want because if you only have a small population of channels that have a high open probability. That means small changes in the number of channels that are open or closed are going to have a big effect on the output of the cell. So adding 8.2 changes the membrane potential at which the channels will open. The other thing that happens is it affects when they will close. So in the lower plot you're looking again at normalized current as a function of time at this time with 8.2 channels are open, they will inactivate with a set kinetics, but when you add in 8.2 that is slowed by nearly 50%. So I have taken you through this in this details, not because I'm an electrophysiologist and therefore you can't be fascinated by this even though it is really cool. But these two features, a channel that opens at more negative potential and has a very slow inactivation is characteristic of at the time this was published a channel for which we did not know the molecular carrier. It's called receptors carrying a current known as IKX. So just a potassium current with the designation of X because the molecular identity of the channel is not known. So the work that I'm describing here led to the proposal that the molecular carrier of this particular current was this heteromeric channel. What I hope I convince you of today is that when we look at mouse models, where we disrupt this channel and this has worked on my lab as well as two other labs in the last three years, hopefully you'll be convinced that this is indeed true. We know this. So the significance of that is more impactful if we go back take a step back and think about how photoreceptors signal. So, I'm glad that there are some younger students in the audience so hopefully this won't be too much review for for everyone. So we've got a cartoon here of a rod photoreceptor and we're zooming in now in the inner outer segment. As we know we have outer segment is responsible for light detection right this is our adoption this is a photo transduction cascade that biochemists love to ask you detailed questions about just to see if you were paying attention. The top shot is if you think about photoreceptor signaling in terms of the currents experienced by the cell is easiest to describe the dark adapted photoreceptor as having a circulating dark current. There's positive ions flowing into the outer segment in the dark. And this is balanced by positive ions flowing out of the cell from channels in the inner segment. So, the bulk of that positive efflux comes from itx or KB 2.18.2. Also need to point out that because the sodium potassium ATPase exchanges an unequal number of sodium for potassium ions. This is actually a net efflux of positive charge from this transporter. So it also contributes in a smaller way to this net circulating current. This is what keeps the photoreceptor depolarized in the dark went too fast that's okay when the lights flash on what's going to happen is activation of the photo transduction cascade. It's closed the channels in the outer segment, breaking that circulating current. There's no immediate response to what's happening in the inner segment. So the membrane potential moves more negative. Now the channel does deactivate it just does so a little bit slower, and while it's open and there's an opportunity for this channel to sort of help shape and filter the ultimate response of the cells. As the light continues or gets brighter, the cell will driven into more and more negative potentials until there's other channels that will help reset the cell until you reset the photo transduction cascade. And I mostly mention this current right here is on this slide, because in the last few years, 10 years we've actually done a lot of work on hdn one. So if you have questions about that, and you want to talk about it and you're one on one, we can do that. But coming back to Casey and the two disease, understanding that this channel is helping to control the resting membrane potential in the dark. And the fact that doesn't immediately switch off when the lights are turned on allows it to filter responses. Make let's us make some predictions about what would happen if you have a non functional channel, which is what we think is the case in our disease of interest. It should reduce or delay the photo response. So, in order to test this. I am not someone who's going to poke around with human beings and said I'm going to do this in mice. But I want to address big elephant room first of all. This disease is characterized by a macular degeneration and mice don't have a macula. So some of you might be thinking, why are you wasting my time with this. Well, I don't think I'm wasting your time I think we can learn a lot from mice, even though they don't have a macula. The only other argument is this in the mice, we know that they have a very beautiful array of rods that's actually quite similar to the human distribution and structure and function distribution. So whatever's going wrong in case in me to retinopathy that the rods would hopefully be phenocopy. In terms of the cones, we have roughly the same number of cones to rod ratio mice is in humans, we're just missing the macula and that high density for central vision. But that means that if what's going wrong in the diseases intrinsic to cone physiology, or is either driven by coupling to dysfunction rods, we should still be able to recapitulate the basic mechanism in mice. On the other hand, if all of your assumptions that it's silly to study a macular disease in mice is correct that actually by testing that in mice, I can confirm that. And we can say if we don't see a few copy of the disease, it tells us that there is indeed something unique by the structure or function of the macula that's contributing to the disease. And that will help us focus in on future efforts to understand the disease and treat it. So I hope I made my case. With that, we made a knockout mouse. This is a time when our mouse genome editing facility was fairly new so it's very fun to play with designing the, the CRISPR guides and making our mouse but we set this up to cause a deletion that would result in truncation of the protein very, very early on. We should get no protein. We do Western blots indeed KV 8.2 is gone. We looked at a few related channels and there were some minor changes we don't really know what those mean yet. So next I'll draw your attention over here this is our immunohistochemistry again photoreceptor site up in the wild type we have labeling in the inner segment. And in the knockout the labeling is gone. You'll also notice that in the knockout the outer nuclear layer is thinner so we have retinal degeneration. I don't know about that, but first I want to address the ERG phenotype because that's the diagnostic characteristic of this disease. And if this mouse model is going to be useful at all it has to copy that aspect. So here we go you saw some ERGs really quickly. Thanks to Lydia really appreciate that. These are some on the left hand side you have sample ERGs again taken from the literature. Here you have a stimulus condition that will evoke a rod dominated response. You have hyperpolarization of the photoreceptors reflected in the downward a wave. And then you have the upward reflection of the B wave that comes from the response of the deep polarizing or on bipolar cells in the inner random. So what you see here is that the a wave response is fairly similar all of that varies across patient. But what's unique and definitive is that the B wave is larger. This is the super normal rod response part of the name of the disease. If you use a stimulus condition that evokes more cone dominated responses that get normal patients give you a robust B wave here's the a wave beautiful B wave. But the B wave is now reduced when it's elicited by cone signaling. And if you use a flickering light stimulus to isolate cones, you see that again the cone responses reduce. What we have on the left hand side are ERGs from the mouse. And the simple description is they're the same as humans, where you see differences, they're mostly just mouse human. So for instance, on the rising B wave of the mouse ERG you see a lot more of these oscillatory potentials. That's a mouse human difference. But what we have is we have the super normal B wave response for the rods, and we have reduced signaling coming from cones. Now what you can do with the mice that difficult to do with the patient is you can put them through more extensive ERG testing, and try to learn more so we did quite a lot of that. I'm going to show you just two sets of experiments. The first is asking more detail about the broad response. So here's what we're doing is we're using dark adapted mice we give them a flash of light that elicits a response. And then we repeat that with increasing intensity of light so you get that what's called this flash family. And the really amazing thing here is when you compare the wild type traces which are black to the knockout traces which are in red, you see that the effect is different depending on the lighting. So here early on in response to very low light, you'll see that the red traces are delayed for when you see the B wave and then that B wave is small at the very most dim light. Just a slight increase in light that will start moving us into that super normal B wave response. And what you can also see here is I'm going to focus in on the a wave because the a wave is reflecting hyper polarization is that closure of channels and outer segments, where KV 2.18.2 is really contributing to regulation. And so the way it's easiest to see what's happening here in these lighting conditions and they're very dim light. So this is very slow it's delayed to respond, you do get a full amplitude response just takes a long time to get there. However, when you go to brighter light, it speeds up, and you now start seeing the onset of this wave matching for wild type and knockout. But if you're going to go fast, you can't get as big of a response so the a wave plateaus out. Here's the summary, under dim, very dim response, lighting conditions, the a wave is delayed, and a brighter flash and keep in mind this is bright for a rod, the amplitude is reduced. Okay, how does that make sense. Let me give that a try. I'm going to come back to my cartoon of signaling that's happening. I'm skipping the dark adaptive condition we're just looking at the light in the light the channel in the outer segment is close. And the IKX channel should be there and slowly inactivating, but in this disease where you don't have 8.2, you're left with just 2.1. And remember that means it's voltage of activation is shifted so it should not be open. Effectively you have zero channel here for IKX. And the only thing supporting the light response here in this knockout would be the activity of sodium potassium ATKs. And forgive me for being maybe a little bit too simple, but one way I like to describe this is, you're really messing up the amount of potassium you have in the extracellular space around the photoreceptor really don't have enough go juice to get the response you need. I don't know why under some conditions that photoreceptor sacrifices speed for amplitude and vice versa. But the effect is the same if you don't have enough positive charge in the extracellular space. One of those two things is going to happen and we happen to see both of them. Okay, I'm going to throw in here just a quick note now about the B wave mostly because that's just a characteristic of the name right it's the supernormal rod responses, which is actually a little bit deceptive because under the most dim lighting conditions for rods. The B wave is actually underperforming. And it's only after this you make this switch between sacrificing time versus amplitude that you start to see that supernormal response. So, this is a little bit hard to think about, but the way that it makes sense to me is I want to come back to one of these traces over here, because the onset of the B wave is always delayed, even though this a wave is not reaching its, its full amplitude you don't have any wave response. And it's, and it sort of square off it keeps going, you don't, and that means you can't really drive the cell all the way to its full hyper polarized condition you don't have the other mechanisms to help return the cell to its resting normal timing. So in some ways, even though you don't have a large amplitude you have more continuous drive for the signal and I think that's why you get the larger B wave, not because there's an effect specifically in the bipolar styles, but just because the information coming from rods is not stopping. So you get an accumulated large or supernormal B wave response. So the first set of more set of ERGs I want to show you here we're using a different stimulus assign you soil to flicker stimulus. So the first set of experiments were designed to be more similar to what you might see in the clinic. This one is a little bit more helpful for me. So it'll flicker we're just taking a step closer to a more normal lighting experience. And we can, we can analyze the waveforms you get from this and one of the things that we've learned is that across various frequencies. The response is always reduced in the knockout mice. And because this is a flickering stimulus, you know this is a large part driven by cones so this is absolutely be producible see that across the board. When you're when we use this particular ERG protocol under very low frequencies we can actually isolate information from the rods. And here this is what the raw waveforms look like below this is that light stimulus. So the white would, when the lights go on we elicit the positive B wave. Again black is our wild type and red is our knockout. And then because the signal continuously flickers we actually isolate. So we are able to see another component of the ERG that we didn't see in our first set of experiments and that is a second positive going wave that is called the C wave. Now this is important in this disease because the C wave is generated by the Mueller cells, and they do this in response to the light induced drop in exercise or potassium. So without KB 2.1 KB 8.2 that extra cellular potassium amount should be lower. Therefore the change between light and dark should be smaller, and indeed the C wave is smaller. So we can see that, and this then conforms to our hypothesis. Right, we did more ERG but I'm going to spare you. We're going to now move on to the degeneration. Now, here in mice. We used OCT again retinas upside down to the way most of you guys look at it. But here in the yellow bars this is the, the outer nuclear layer for receptor layer so we just measured that over time and wild type and knockout. And you can focus in here on panel C. What we see is that at two months of age we have something in the retina, not to submit, not too big video, about 10% thinner at this point, but that progresses fairly slowly so that by about 10 months of age we've lost about 30%. Now, this is a little bit different because the majority of patients that I've read about for KCMD 2 retinopathy don't have any obvious thinning of the peripheral retina. So this might be a mouse specific thing here. The other thing is an information I have on this slide is labeling for GFAP this is a marker for reactive gliosis. And indeed you can see that that is upregulated in the retina and you can see that the mule cells are starting to express the GFAP and it's starting to fill some of the processes in the retina. This is very commonly seen anytime you have a photoreceptor degeneration, but this is happening very early on in this particular disease that at two to three months when we're just starting to see photoreceptor drop out. We're seeing the mule cells respond, which might be sensible because they're electrically part of the problem as we saw with the C wave and the ERGs. And here's a really important question. What about the cones in cones are only 3% of the photoreceptors so for losing 30% of the retina know a lot of this is coming from the rods. What's happening to the cones. The answer that we did retinal flat mouth and we labeled for code arrested. Now this is obviously very low magnification picture you can't see individual cells. But what we did is we collected regions of interest at different eccentricities. And that's because even though the mouse right now does not have a macula there's a very slight increase in density of cones as you go more towards the center. So we did get these different eccentricities, comparing the cone density. And what we saw is absolutely no difference between wild type and neck out. We first did this experiment with six months old mice. And then my colleague slash competitor from Australia who had a different line of KBA point to mice published her own analysis. You can see a slight loss of cone density. So we repeated this experiment our mice, but this time aging them out to one year of age. And this is the data from the one year old mice, no difference, no loss of cones. At this point, though, I wanted to push a little bit harder on what would be happening with cones. And we have in the lab, a strain of mice, which you may or may not have heard of before. So if you've heard of the NRL mouse, take a moment today dream I'm just going to explain this to those of you haven't. NRL is a transcription factor expressed in the retinas required for specification of cones. So if you have a knockout mouse, all of the photoreceptors are cone like photoreceptors instead of being rods. The NRL mouse is used throughout our field to study cone dominant biology in the retina, but it's a difficult retina to work with because as you can see from this picture, it's morphologically abnormal, the cone and nuclei, instead of being a nice smooth layer form of polypholdings and rosettes. So instead I got a variation of the NRL knockout from Christian Grimm, where he crossed it with another line of mice and obtained a normal beautiful morphology. Boyer the outer segments, or where the outer nuclear layer happens me just all cones. So to make sure I'm very clear that this is not the pure NRL knockout line. We call this the cone full mouse. Here's our own histology. The retina looks pretty good. The outer nuclear layer is not as well organized. It's not as compact, but that's because it's cones not rods, and the outer segments are obviously much shorter. This is not a degenerated retina. It's just missing long rod outer segments. So we took this mouse, we crossed it to our KV 8.2 knockouts. And what did we get? Well, when we did ERGs now targeting the cones, you can see again black as wild type, red as knockout, the responses reduced exactly by 50%. So one of my questions, which you may remember early on was, is it possible that the defect in cone driven signaling, that reduction in the B wave is actually driven by the dysfunctional rods? And the answer from this experiment is no. It's intrinsic to cone signaling. B and C are just quantitations of the A wave. The A wave in this protocol is very, very small. There's a lot of variability. We don't see anything that statistically significant, but we do see this reliable reduction in the B wave. So this experiment was done at one month of age on the mice, because we know that in the anteroom knockout in the cone full mice, there is a degeneration that occurs. So this experiment was done at one month of age to check and make sure everything was okay. We did histology at two months, and then we followed it up with it four months. In two months, the cone full mice with a normal copy of KB 8.2 looked as fine, as well as the animals that are missing 8.2. But when we looked at four months here, the outer nuclear layer, it's a little bit more disorganized in this particular image, but it's actually within statistical normal range. However, by four months, the animals missing 8.2 have a striking degeneration. So in this particular environment, cones do degenerate in the absence of KB 8.2. This brings me to my summary of the mouse work, and this is work that we've all published. So I'm going to do a quick summary and then move on to unpublished data so you can learn what I'm thinking about for the future. We have a mouse model where the ERG phenotype is common to mice and humans. Those features I've described in the name of the disease, and it supports the hypothesis that the problem is insufficient extracellular potassium. Can't support the dark current required for resting membrane potential and they can't filter responses in response that come from light. In the mice, we see clear degeneration of rods. I don't really understand if this is consistent with the human disease as it's described in literature right now. Most patients do not have a rod degeneration. That said, the macula, even though we tend to think of that as being an area of really high cone density is also an area of really high rod density. And if we ever have access to histological samples from donor patients, maybe we will see that there are indeed rods dying more frequently. But it could also just be a mouse specific thing. This is something we just have to be mindful of as we continue. When we looked at this unusual mouse model with the all cones, we were able to learn is that the cones will be generate, but only in the absence of rods. And I want you to be patient with me, but this is one idea that this sparks in me and it requires further testing but perhaps the way to think about translating this to the humans, perhaps the fragility of the foveal cones right so in the center of the foveal is pure cones. Maybe the absence of rods there is what makes that particular population cells more sensitive and they degenerate which then propagates out into the progressive macular dystrophy we see. And again, could be mouse only so we need to test that, because as you saw from those retina pictures, even though this cone foe mouse has an all cone retina, it is still not the morphology of a foveal. So we still have to figure out exactly what is making cone so sensitive to degeneration in this disease. With that caveat clearly stated and clearly in mind, I think that as I go forward if I use both of these models normal mice and our cone foe mice, and I pay attention to these caveats think we can still use them and make progress and understanding the basic biology what's happening. Okay. See I'm starting to run short on time so I'm going to speed up but feel free to interrupt me. Oh, we need to stop and ask questions. One thing I'm doing to go forward in the future is, I was wondering what else is changing in the retina. One experiment we did is we looked at, we're measuring the various metabolites in the retina. The vast majority of things did not change but we saw changes in two metabolites. One is an AA or an acetyl aspartic acid, and the other is in Tori. What these two. These are free amino acids, meaning they look like amino acids but they don't actually get incorporated into proteins. And the common is that they both function as osmolites. So, changing levels of these molecules can reflect water stress or stress to your protein folding. What's strange to me is that in one case, the NA levels are going up, but in the other case Tori and the levels are going down. And this is an effect due to genotype and not age. Our young mice are one and a half months old. Our old mice are a little over a year old, in this case, and you see the effect at both ages. So I was going to tell you just a bit more about what we think we know about NA and Tori. I'm going to go really really quick through that. We really don't know very much about NA in the retina per se, but we do know that increased levels are hallmark of the disease called Canavan disease. Canavan disease is caused by mutation in an enzyme that breaks down NA, releasing acetate that is used to build lipids in all the dendrocytes. So you have a defect in myelin synthesis in terms of the retina that's obviously going to affect the optic nerve, but we don't know if it has any effect on the retinal neurons themselves. Tori is more fun. Sorry. We think we know a lot about Tori. If you look in the literature does a whole bunch of things regulate cell volume, forms bile salts in the digestive system. It's been purported to act as an antioxidant at the inflammatory blah blah blah everything. So criminals will sell it to you until you eat it. This is a very dangerous thing. So beware. However, when you look at the scientific literature. There's some interesting things. Every few years as a paper on Tori and retinal health. Tori depletion causes rapid retinal degeneration effects cone for receptors ganglion cells rods. If you mutate the transporter required for bringing Tori into photoreceptors you get a rapid retinal degeneration. So as I go forward, I want to study more about the biochemistry of the channel and I'll be telling people more about that in my research talk. And from this talk, I want to say we've learned more about the basic biology of this channel. In some patients gene therapy might be a viable option. The big hurdle there is going to be the very rarity of this disease gene therapy is incredibly expensive to develop and deploy. Maybe what we learn in mice about various metabolites will give us an alternate or complimentary tool though to help patients sooner. So stay tuned for that. And if I have time I have just one more slide. I wanted to show to you guys because I want feedback on this. When you think about the future we always have to ask what don't we know. So Casey and be to so causes a retinal disease. But Casey and be to is not only expressing photoreceptors. There's some evidence that at least at lower levels it's expressed for instance in the hippocampus. This is RNA expression data from the Allen brain Atlas. Don't know the significance of this but in a paper from about 10 years ago, it was reported that within a group of pediatric epileptic patients. There were Casey and be to polymorphisms. So we know that from this standpoint of retinal disease, you have to do ERG to accurately diagnose the disease. But if a patient is subject to his prone to seizures, you don't want to put them under extensive ERG testing. So we don't know if if epileptic patients with mutations and Casey and be to might have a vision defects, for sure. Conversely, we don't know do our Casey and be to read out the few patients are they prone to perhaps having seizures or other cognitive impairments. I don't know. It's pretty rare but I'm really curious and feedback on that to determine if it is worth taking the time and effort to look more deeply using our mouse models at what's going on in other parts of the brain. With that, I will stop and take any questions I have time to answer and just stay on my. Thank you slide this is my, my group and my collaborators might flood the course. Very nice. And a translational talk of how we can go back and forth between human and mouse disease. I know we have other things that other people have to go to the next minute or two are there any quick questions for anyone here. And if not, I would encourage you at noon she will be speaking on online. Yeah, so there's even more detail and research people will be having it. Anything more that you want to say, Jim. Okay, all right, well thank you very much. Okay, thank you for your attention.