 Welcome everyone to our Grand Rounds. A few of the Zoom ground rules in this particular Grand Rounds. If you'd like to be unmuted, please raise your hand or you can ask questions in the chat. Dr. Kramer will be monitoring that chat. If there's anything kind of timely for the topic that you're speaking on at that moment, would you prefer to be interrupted or save questions until the end? It is fine to interrupt me. Hopefully I'll notice. I'll be looking at the slides and I'm not sure I'll... Very good. And I did not, perfect. Thank you. All right. Well, I will do a brief introduction. So my name is Jeff Petty. I'm the vice chair of education. I'm in charge of our Grand Rounds series. We're honored to be joined today by Dr. Richard Kramer. He's a professor of neurobiology at UC Berkeley and where he's a professor in the Department of Molecular Cell Biology. And he's a CH and Anne Lee, Chair of Molecular Biology and Diseases and Director of the Vision Science Corps. Among his research interests, he really focuses on exploiting neuroplasticity to restore vision and retinal degenerative disease. There's certainly others in our department who could go on and wax eloquently on you and your accomplishments. But with that brief introduction to our clinical group, welcome and thank you for joining us. Well, thank you, Jeff. I originally was supposed to be in person there today and giving a research talk as well in the afternoon, but a couple of things intervene. First of all, I have a grant proposal due in about four days. Secondly, last week I came down with COVID. So it's just as well that I didn't come right now, but I'm recovered now. And all of you, none of you need to wear a mask because this is all electronic anyway. So, yes, I'm gonna talk to you about sort of the most recent work that we've been doing in my lab. And this is gonna be much more sort of science than that is it. But let's see if I can share my screen. Is that, let's see, a second here. Trying to use just two screens and it's always complicated. Share screen, am I sharing? We do not see the screen. What do I need to do to share screen? And I'll just verify that you have the permission. Yeah, maybe that's the problem here. Is it showing the share screen in green? So there we go. I'm sorry, I see it now. How's it going? So. All right, we do see the screen. Audio is great. It's not present review. So we do see the sidebar. Now that's the wrong screen. I'll be right back to this in a second. In fact, I'm gonna close this screen. We do see suppressing retinoic acid just. Oh, you see that? Okay, great. Okay. I will now go to slide show late from start. So now you should see the whole screen and all its glory, correct? Yeah. It's great, perfect. So yes, I'm gonna talk to you about what might be a new idea for how to address the problem of vision loss and degenerative blindness. And, you know, I'm sort of, this is, I guess the British expression is coals to Newcastle. You are one of the world's centers of understanding what happens after photoreceptors die in disorders like retinitis pigmentosa. Robert Mark and Brian Jones and their colleagues. So, you know, what happens is that in these disorders, rods and cones degenerate. People go through a stage where the outer segments of photoreceptors are largely missing. The vast majority of people in these diseases are stuck in this sort of middle situation where they've lost a lot of photoreceptors, but there are still some left and not completely gone. And then a small minority of cases, all their photoreceptors disappear and people lose all light perception. And it's these people that have been the target so far of new technologies that are intended to restore vision. And these technologies include things you all know about retinal implants, either positioned in front of the retina epiretinal implants or behind the retina subretinal implants. Of course, the epiretinal implant that's in clinical use is the Argus II made by Second Sight. And the subretinal implants are designed by a company called Pixium and they're still under clinical trials in Europe. So that's one approach. Another approach is to use optogenetic tools. Microbial opcins like channel rodopsin express them artificially in the downstream neurons of the retina to try to provoke light sensitivity in these cells. Of course, there's a huge effort by the National Eye Institute to develop stem cells to actually to regrow new photoreceptors and get them integrated into the circuit. And then finally, there's something that we've worked for 20 years on now, which are chemical, photoisomerizable chemical photo switches that get into neurons and impart light sensitivity on action potential firing. So all these approaches are really because they're all risky and also because they tend to supplant the activity of whatever rods and cones are left, they're really at this point restricted to people in this terminal sort of end stage degeneration population where people have no light perception. And that leaves behind this huge population of people that might be legally blind, have severe vision impairment either from RP or perhaps more locally for AMD that where the function cannot be restored. And those are the patients that we're primarily interested in. So I should say that to get these technologies to work, there's an assumption or there's at least the hope that the downstream retinal circuitry remains intact. Most importantly, the retinal ganglion cells have to remain connected to the brain and that the way it processes information needs to be more or less unchanged. And then if you put back light sensitivity you have some hope of restoring visual perception. But largely due to the work of people at Utah, we know that this isn't exactly the case. And so classic studies I mentioned by Brian Jones, more recently by Rebecca Pfeiffer as a student in his lab and Robert Mark have elucidated a process called remodeling. This takes several forms. First of all, there's the morphological aspects of this that you can see in animal models of retinitis pigmentosa. It develops slowly. So in mice, we're talking about at least six months after the photoreceptors have died. And in humans, we're probably talking about years or decades after the photoreceptors have died. You start to see sort of stereotypical changes in the structure of the retin. And there's a variety of changes that you see. One of the first things that you see is that some of the neurons, particularly bipolar cells, they lose their dendrites that formerly were contacting the rodent confotoreceptors. They start to sprout new branches of those dendrites. Some of the cells slowly start dying. Again, these are late events. The cells start to migrate out of their normal positions and beautiful lamina in the retina. And the entire retina can become disorganized and glial cells, particularly muller glial cells swell and they form a seal over the inner and outer margins of the retina. So these things, like I said, are happening late after photoreceptor death. But what has emerged more recently is the understanding that there are much more rapid physiological changes that start immediately as the photoreceptors are dying. And here's one big example of a physiological change. If you take a retina out of a mouse model of RP and you put it on a multi-electro-recording chambers that you can record from lots of retinal ganglion cells at once, here is the recording, extracellular recording of spikes from these ganglion cells. And this is spontaneous activity just with the retina in the dark. You could see that a wild-type retina, there's some spontaneous activity, but the retina that comes from this model of RP shows something like six to 10 times higher frequency of spontaneous firing. And so that's something that's been noted in mice and rats and dogs. And it probably is true in man that there's just hyperactivity of downstream neurons. And we've done a lot of work trying to understand what the basis, what the molecular mechanism of this hyperactivity is, and this involves single cell electrophysiology with patch clamping methods. And I'm not gonna go into the details here, but we see different kinds of ion channels that seem to be upregulated. We also have molecular evidence for this. So there are changes intrinsic to the retinal ganglion cells, there's also changes in other neurons in the retina that lead to this hyperactivity. And in addition, there's this rhythmicity that emerges from the degenerated retina. Again, when you record at sort of higher time scale, these are milliseconds instead of seconds, you can see that the spiking of these ganglion cells tends to occur in rhythmic bursts in the degenerated retina. And there tends to be synchrony across different cells. So somehow they're either being driven by a common input or they're linked up through gap junctions or both. And so all of these things you can imagine are, they're all happening as the retinal's degenerating. What if there's still some photoreceptors left? They're trying to get their message across. They've got a much more noisy background to get through, which includes this sort of rhythmic activity. And the idea is that this is gonna degrade the ability of the retina to encode information about light and dark, at least as long as there is still some photoreceptors. And this is an illustration of that, maybe more explicitly, here's a healthy retina, here's a bar of light that you shine on the retina, and here's a recording from one retinal ganglion cell. There's some spontaneous activity, but you can clearly see the light response above that activity, this retinal ganglion cell. But in a degenerated retina, where you have fewer photoreceptors, you also have this higher level of background activity. And now that background activity can obscure the light response. And so if we understood what it was that was causing this hyperactivity, maybe we could develop some kind of treatments that would suppress the hyperactivity and allow the light response to sort of reemerge. This isn't rescuing when I'm talking about a treatment that is preventing the degeneration itself. We're talking about something that is making the retina work better in the face of this signal corruption process. And that's really the theme of the entire talk. Trying to understand what causes this physiological remodeling and trying to do something about it. So the idea is that when the photoreceptors die, the question is how do the downstream neurons even know that the photoreceptors are dead? And I would say the prevailing idea until recently was that the synaptic signal that the photoreceptors are producing is sort of propagating through the neural circuit. And in the absence of that synaptic signal, there may be compensatory mechanisms that kick in in these downstream cells that change the way they operate. So for example, in the absence of an excitatory neural transmitter glutamate, maybe the downstream cells start to develop their own hyper excitatory activity to compensate for it. Maybe they also start to form aberrant excitatory synapses for the same reason. So that idea is called homeostatic plasticity. And we know that it operates in all neural circuits, but we do not think that that's what's responsible for the hyperactivity of the retina. And I'll, I'm raising the suspense level here. What is the degeneration signal that's causing hyperactivity? And I'm just gonna cut to the chase. And I'm gonna tell you that the hint for what this signal was, again, came from Robert Mark's lab. And there's a theme here. And the work of Brian Jones and his colleagues. And this was a, you know, we sort of had this epiphany when Robert came to give a seminar at Berkeley about 10 years ago. And he was talking about work that already was quite old at that point. And I guess the paper came out 10 years ago, but he was here maybe five or six years ago. And what they had found is that in one particular model of light induced retinal degeneration, this is a mouse that was particularly sensitive to light and exposure to bright light would cause the photoreceptors to die. They implicated retinoic acid as the signal that was causing these bipolar cells to sprout new dendrites. And this is just a diagram of cultured, you know, cell cultured bipolar cells that have been dissociated from the retina and applying exogenously retinoic acid, you can get them to grow new axons and new dendrites. They also had results showing you can block the sprouting of dendrites in this mouse model that I mentioned. So that got us thinking maybe the hyperactivity we see is also a consequence of retinal acid. And so before I start showing you some of the evidence that I should tell you how retinal acid is made and something about the retinal acid pathway, retinal acid is a downstream metabolite of vitamin A, also known as retinol. Of course vitamin A is critical for vision. It's sort of extracted from the bloodstream. It's processed in the retinal pigment epithelium, which have enzymes that convert vitamin A into retinaldehyde, including 11-cis retinal, the chromophore for opcins. And this retinaldehyde is normally absorbed to a great extent by the rod and cone and her segments. Each rod contains a hundred million copies of rhodopsin. So there's a very large sink for absorbing all of this retinaldehyde. And ordinarily there's sort of cycling between the retinaldehyde absorbed by the photoreceptors and vitamin A metabolites. But when the photoreceptor outer segments, particularly the rods, start to degenerate, now this gigantic sink for absorbing retinaldehyde starts to disappear. And even though the RPE down regulates how much it produces, there's still some retinaldehyde that escapes. There's also sort of a breaching of barriers in the retina. And the retinaldehyde can reach cells that express this enzyme called retinaldehyde dehydrogenase, which is a member of a big family of other aldehyde dehydrogenases, which I'll talk about in a few minutes. So, and what RAL-DH does is convert retinaldehyde into retinoc acid. So retinoc acid is a really important regulator of growth in embryonic development. It exists in gradients in the developing embryos. It sets up patterning of limb embryogenesis. So it's a really critical morphogen early on. But then the system is largely in most parts of the body, including the CNS, retinaldehyde acid and its effectors are down-regulated in adulthood. And the way this retinaldehyde acid works is by binding to a retinaldehyde receptor. Here it's RAR-alpha, which dimerizes with another protein called RXR. And this dimer binds to specific sequences in DNA and turns on gene transcription. So this is a transcriptional regulator that turns on programs of transcription in cells that it acts on. And so this is the system that we think is engaged as photoreceptors degenerate. And as there is an sort of an excessive amount of retinal gas. And so I'll show you some of the evidence for this. First of all, we can detect the higher retinalg acid induced gene expression in degenerated retinas. So we do this with a reporter construct. This is a gene that we introduce into retinal neurons with an AAV virus and the gene has two parts. First of all, I'll get to the business part of it first. First of all, there's GFP that's under the control of this retinalg acid response element. So if there's elevated retinalg acid that activates the receptor, the retinalg acid receptor and it turns on expression of GFP. And then there's another module in this gene which expresses red fluorescent protein and that's just constitutionally made. So that's just a let's us that's sort of a control that tells us which cells have been transduced by the virus and controls for the possibility that maybe normal and degenerating retina are of different susceptibility to be infected by viruses. So if we compare, for example, in mice, the wild type mouse retina versus a degenerated mouse retina, the virus infects cells equally in these two retinas but it's only in the degenerated mouse retina where we see GFP being expressed to a large extent. And this is even more dramatic in a rat model of retinitis pigmentosa. This is a truncation in rhodopsin itself which leads to the rods dying. And you can see once again, GFP is greatly upregulated. We're looking at the level of retinal ganglion cells. So this is expression of RA induced genes in the retinal ganglion cells. So there really is more retinal acid in these retinas and it's capable of inducing gene expression. And if we inhibit that retinal acid receptor, this is with a small molecule originally invented by Bristol Myers Squibb, BMS 493. If we inject that into the eye of one of these mice and the contralateral eye gets just saline, what we can see is that the, and it takes a few days for this to happen, but we can see the level of this hyperactivity dramatically decreases. We see this in different animal models. Again, we see it with different kinds of inhibitors, not just this one. So, you know, there's evidence that retinal acid goes up, inhibiting its activity reduces this, inhibiting its receptor reduces the hyperactivity. I'm not gonna show you the evidence that we can mimic hyperactivity in a wild type by injecting retinal acid itself. So that's one way to inhibit the retinal acid receptor is with drugs like BMS 493. I'm calling it a drug, but it really was just a molecule that they made. The reason they were interested in retinal acid receptor inhibitors was as possible cancer chemotherapies. That program was sort of dropped, but there's a lot of, there's a rich pharmacopeia of candidate blockers of RAR. And we'll get back to that a little bit later because we have strong evidence now that we can improve vision dramatically in these mice with this strategy. An alternative strategy instead of blocking the receptor with a small molecule, you can use gene therapy. So it turns out over the years, people studying retinal acid the way it works have come up with a dominant negative version of this retinal acid receptor. So this is a mutant of RAR alpha, which not only doesn't bind to DNA, but it sort of takes down the whole complex so that if you express any of this in a cell, you interrupt the possibility that retinal acid is gonna turn on gene expression. And this can be delivered to neurons by encapsulating the gene in AAV, introducing it into the eye, having it infect retinal ganglion cells. So there are two alternative approaches, either small molecules or gene therapy for blocking the retinal acid pathway. We'll get back to that again at the end. So I've been talking about retinitis pigmentosa as the main sort of application for all of this. What about AMD? So in AMD, there's a much more localized loss of photoreceptors from the macula. And of course, mice and rats do not have a macula. They have a rod dominated retina. And so they don't have any sort of specialized region with all cones. And so you can't really study AMD. In fact, you can't study it in any animal model yet. So the best you can do, I suppose, is just ask what happens if you very locally ablate photoreceptors? In RP, we're talking about mutation that causes the generation of photoreceptors. Here, we're talking about a lesion, if you will, that is caused by implanting some foreign materials subretinally. In this case, it's actually a retinal implant, a subretinal implant. And this doesn't say so here, but this was the result of a collaboration with Daniel Polonker, who's at Stanford. If you implant one of these implants, I'm not showing you this here, but one of the sort of unfortunate consequences of these implants is that they actually destroy the photoreceptors that are remaining in that little region of retina. So we basically have a local ablation of an island of photoreceptors. And what do we see? We see that the retinal ganglion cells overlying that area show enhanced RA-induced gene expression, just like we saw on the RP animals. And they show hyperactive firing. So this is recording from this area where the lesion has occurred and it's not marked here, but from control areas outside with the lesion has occurred and the change, the retinoic acid-induced change is restricted to the area where you've caused this lesion. So it just may be that the same phenomenon applies really to any disorder that causes loss of photoreceptors. The downstream consequence of that may be that retinoic acid is elevated and that there's changes in the operation of the downstream circuitry. So, okay, so let's get back to the question of what all this has to do with vision and how it impacts vision. So to address this question, we use another RP model mouse, but this is one that degenerates slowly. So you can actually catch the degeneration and the remodeling in the act. And you can see physiologically what that looks like in these three recordings. These again are multi-electrode array recordings. This is from a two week old mouse. And what you see here is the response to these periods of light and dark. And you can see that this retin is responding quite normally to illumination. When light turns on, you see this high frequency firing of retinal ganglion cells. And when light turns off, there's a burst of firing from some of the retinal ganglion cells as well. And these presumably reflect activities of on and off retinal ganglion cells. And there's some adaptation that occurs in the continued presence of light. So this, I'm not showing it to you, but if I compare this to a wild type light response, this would look quite normal. Those same mice at one month of age, you can see the story starts changing quite dramatically. The light responses are diminishing greatly. And by the time we get to two months old, the light response is completely gone. So as I said, this is a mouse that has a progressive form of RP. It loses its photoreceptors between two and three months. But if you look carefully, you'll see that the level of background firing is increasing during the same time period. So coincident with the loss of the light response is this sort of game of hyperactivity. And that's shown here. Here's the light response in blue diminishing over time. Oops, sorry. And here is the spontaneous firing of ganglion cells rising over time. And so there's a time period where the retina still has photoreceptors can still generate a light response, but it's already becoming quite hyperactive. And so what if we intervene in retinal gas and signaling right then, you might think that the hyperactivity, if it's obscuring any light responses, maybe the retina would be better at detecting responses to light, maybe the animals sort of behaviorally would have better vision as well. And so we addressed this, we answered this question. We took these RD-10 mice. Here's a mouse, I think this is about 45 days of age or so. When degeneration is progressing, it's lost a lot of its vision. There's a very brief light flash here. You can see in the recording, I can't really make out any particular response to light. That's the eye that was injected with vehicle. And here's the eye that was injected with BMS 493. And you can see that it's a very brief light flash, but there is some spikes that occur right during or after the light flash. And this is the summation of all the spiking in that particular recording. This is if you take nine flashes of light and you average them together from this same recording, you can clearly see that the light response has emerged from the background activity, which by the way is reduced by in the eye that's been injected with BMS 493. And this is now comparison of five different mice, the eye that was injected with drug versus the eye that was injected with vehicle. In every single case, we see quite a significant response to light expressed as firing rate light over firing rate in dark. So we've really sort of pulled this signal out of the noise, if you will. And what this looks like, if you use a variety of light intensities is that particularly at lower light intensities, you dramatically boost the response to light. And these kind of equalize out as you get to more saturating intensities of light. So blocking the retinoc acid, receptor increases, the light response increases sensitivity of light. By the way, it doesn't do anything in a healthy retina. We've done those controls. So that's the electrophysiology of vision. What about vision itself? Do the mice actually see better? And I've already mentioned, how we intervene in this drug pathway at the level of the retinoc acid receptor with BMS 493. So just to run through this again, their vitamin A is converted into retinaldehyde. When photoreceptors die, there's an excess of retinaldehyde which leads to excessive production of retinoc acid. And that turns on gene transcription by binding to the retinoc acid receptor. And that's what causes hyperactivity and hyperpermeability. But in addition to being able to block the retinoc acid receptor, there's another target in this pathway for pharmacological manipulation. And that's the retinaldehyde dehydrogenase itself. And it turns out a drug there as opposed to RAR, there actually is a real drug that is FDA approved, that's been used very commonly for at least 30 years. And it comes from a completely different world than ophthalmology. It's called dysulfurum. It's more common name is ant abuse. And it's used to treat alcoholics who have sort of hopeless addiction to alcohol. And the way this works is when you drink ethanol, ethanol is broken down by, or is converted by alcohol dehydrogenase into acid aldehyde. And then that happens in your liver. And then there's an enzyme or a family of enzymes I already mentioned, aldehydehydrogenases that metabolize the acid aldehyde into acetic acid and water. And disulfurum blocks aldehydehydrogenases. And so if you're on disulfurum, which is taken orally on a daily basis and you drink alcohol, it's converted into acid aldehyde, but then it's stuck at that stage. And that acid aldehyde builds up in your bloodstream and it basically gives you an instant severe hangover. It's called the disulfurum reaction. It's very, it's a, I haven't experienced this personally, but it is very unpleasant. It can even be dangerous. And the idea is that disulfurum then becomes an alcohol deterrent. It was at one point, the first line treatment for severe alcoholism, it's kind of fallen out of favor because maybe punishing people for drinking is not the best approach. So there's other kinds of therapies that have emerged, but it's quite safe when taken for this purpose. And like I said, it's been FDA approved for many, many years. And the prediction is, it turns out that retinaldehydrogenase is one of these aldehydehydrogenases. So if you give people disulfurum, perhaps they'll prevent retinog acid induced remodeling of their retinas and they will see better if they have a degenerative disorder. And so we've tested this in mice using an operant conditioning task where we train mice to recognize a visual stimulus, which is a drifting grating shown on a computer screen. The mice are trained to run on this running wheel and when the image pops up, they learn to associate that image with the availability of some reward that they can get by poking their nose into this little noseboat device. And that delivers a drop of sugar water. We start out with mice that are water deprived. So they're very motivated to get a drop of water and they learn by trial and error to recognize that the image represents the availability of reward. And so we train them over a period of several days by pairing the stimulus, the image, with the reward availability. And with each training session, we decrease the time that the reward is available for. So the mice more and more stringently learn, they've got to go and do that nose poke to get their reward. And after a few days, they've got it. We do all this training when mice are really young. RD10 mice are really young and they still have their normal complement of photoreceptors. And then we put them aside for a month or two while their photoreceptors start to degenerate. And then when that period is done, we can come back and test not only whether they can do this task, but how sensitive they are to changes in the image itself. So here we're changing the contrast of the image and we can measure contrast sensitivity in this manner. So here's an example. Here is the behavior of a single mouse over the course of one testing session. Let's start over here where the stimulus is a full 100% contrast grading. And you can see the mice very reliably jump off that wheel and go and poke their nose with it about two seconds. There's very little variability in the latency of that response. They get it right just about 100% of the time. And as we lower the contrast, you can see that they get worse and worse at getting this correct. And more and more variable. And so this is sort of how we determine contrast sensitivity. We could do this right after training and we could do it a couple of months later when they're starting to lose their vision. And what we see if we do this right after training is that here is the contrast sensitivity curve of the mouse at about five weeks of age. And here it is at about 10 to 12 weeks of age. The gray line here represents the untreated mouse where it's lost a considerable amount of its vision. In fact, it's barely succeeding at all in responding to even 100% contrast image. But if we've been feeding the mice disulfurum during that time, their vision is dramatically retained almost as good as it was if there had been no degeneration at all. And the same is a little less true of BMS 493, but in this case, instead of feeding the mice, the disulfurum were injected in their eye and it's only on board for five days or so before the test. So both of these manipulations really dramatically improve vision. So we've done this at the level of behavior. We've also have collaborators at UC Santa Barbara, Michael Gord, who's a wonderful neuroscientist and friend. And he's looked at the activity of neurons in the visual cortex V1 and using a calcium indicator molecule called G-Camp, which he expresses in these neurons. The idea is that you would express it and the neurons wait a few weeks for the fluorescent indicator to be expressed. And then you make this cranial window that lets you peer into the cortex and then you use different visual scenes that you can shell onto the mouse, either simple things like a drifting grading or more complicated things like a movie. We'll get back to that in a second. And here's what you see in some of these neurons in the cortex. As you change the orientation of the visual image, different neurons tend to light up. These neurons are orientation-tuned. This is what Hubel and Wiesel initially found decades ago in the cat visual cortex. You can see the response of this G-Camp dye in different neurons to different orientations of light. They all have different orientation tuning. And when you lose your photoreceptors, of course, among other things, the cells become worse at discriminating between different orientations. But the whole point is if you've treated these mice with disulfurum or BMS493, the orientation tuning is dramatically improved. This is orientation selectivity index. That's improved both by treatment with disulfurum or BMS493. This is just one of the many things that these guys have evaluated in these cortical neurons. Here's something else that they've evaluated. This is a much more, or the stimulus in this case is much more complex. In fact, instead of being a static image, it's a movie. And the movie happens to be one of the classic film noir movies from the 1950s directed by Orson Welles. It's a movie called Touch of Evil. And this has been used traditionally in neuroscience because it's black and white. And this particular scene I'm about to show you is one continuous scene in the movie which has no cuts and it has a lot of contrast and it's complicated. And I'll just let you watch the movie. It's only a few, it's only about a minute long. Okay, so at the time this was actually pretty technically advanced this particular shot because like I said, it's one piece of camera work and the camera's on a boom that has to go way up in the sky and catch everything at different angles. And I've never, I actually must admit I haven't seen the rest of the movie. And so I don't know whether that bomb in that car blows up or not. And I probably should go and watch it at some point. I'm very curious to know what happens. Anyway, so you wouldn't think that Orson Welles would be so interested in film noir but if you shine that, if you, sorry, play that movie to either one eye or the other of these mice here's, this is the duration of the movie it's about 30 seconds long. And it's hard to see but these are different scenes in the movie. And what you're looking at here is a raster plot of many, many different neurons. I'm sorry, this is three individual neurons but the movie has been shown 30 successive times. And you can see in this neuron there's one scene in the movie where the cell tended to respond. And this neuron it's a different scene in the movie with a different neuron tended to respond. And finally, this third neuron responds at different scenes. And so what it turns out in a healthy mouse with normal vision, a lot of the neurons are tuned to some features of this film of certain scenes. It's hard to know exactly what the features are but the reliability of the response to a particular scene is pretty high. You can see that consistently the cell responds when the movie gets to this particular point in time. This is true of a normal healthy mouse but an already 10 mouse that's losing its photoreceptors it's a whole lot less true. So here's the, I'm not comparing normal to healthy but it goes way down the reliability is greatly reduced. You can see it's only 0.1 or less than 0.1 but disulfurum or BMS dramatically improves the reliability of responding to a particular scene. So this is all evidence that these drugs are actually improving, I realized this was a long roundabout way of saying this but they're really improving vision per se in these mice. And so that's kind of the end of the data of this presentation. But the idea here is that if your retina is losing photoreceptors that's only part of the problem. There is much more going on that involves the rest of the circuitry of the retina. And I didn't go into our more mechanistic studies but we have evidence now that it's not only the ganglion cells becoming hyperactive there's also dramatic changes in bipolar cells in particular their ability to synaptically transmit information to the ganglion cells. And that's also degrading tremendously and that's also rescued by blocking retinoc acid receptors. And so whereas these kind of manipulations are may end up being effective for restoring vision in cases of complete blindness they're not gonna address this other problem which is that the retina itself and maybe even further downstream the way that the system is processing information is getting corrupted. But now we have strategies for correcting that problem. And at least in mice you can correct that problem after the fact. You know, these changes have already occurred and we're blocking either RALDH or RER and we're able to bring that system back almost to where it was before that remodeling had taken place. We haven't looked at the morphological consequences of remodeling actually I'm talking to Brian quite a bit about this and collaborating on this aspect of the project. So and these two approaches I should say are not mutually exclusive. So not only do inhibitors like this not only are they predicted to improve low vision but those same corrupting influences are going to limit how well these vision restoration technologies work. If you regrow photoreceptors from stem cells and the rest of the downstream circuitry is corrupted and not processing information correctly these same inhibitors might correct that problem and sort of potentiate the effect of vision restoration technology. So just in terms of a sort of a verbal summary of this or written summary of this what I've told you today is that photoreceptor degeneration leads to remodeling changes including hyperactivity of retinal gangs themselves and that corrupts neural information processing. This exacerbates that we think a loss of sight and low vision subjects and will undermine new vision restoration efforts in blind subjects. The degeneration of panoramic activity is caused by overly active retinol acid signaling. Pharmacological or genetic inhibitors of either the synthesis of retinol acid or retinol acid signaling can suppress hyperactivity and restore the normal vision information processing capabilities. Dysulfurum which is an LDH inhibitor by inhibiting the retinal form of LDH is a logical first choice for testing in human patients and we are setting up, not really us, it's our colleagues at University of Washington and University of Rochester are setting up a clinical trial with Dysulfurum on RP patients to see if it actually improves their vision. And that may be beginning as early as this fall. The barriers for getting that to happen are low because this is already an FDA approved drug. And once proof of print, and I guess the most important thing about those studies is that if they work, Dysulfurum itself may be a good drug for improving vision but because of all the baggage that comes along with it, I mean, patients would not be able, they'd have to be completely abstinent from drinking alcohol. And we don't really know what the really long-term consequences of blocking all aldehyde clearance would do to a person. It may not be good for as a long-term treatment but once if we can approve proof of principle that it works in humans, retinog acid inhibitor, receptor inhibitors might be more favorable as a long-term treatment. And that includes small molecules, RNA interference drugs that block the expression of retinog acid receptor. These would be RNA therapies like anti-sensal aglomucleotides or small interfering RNAs. And finally, I mentioned AAV for deliver permanently blocking RAR activity with this dominant negative retinog acid receptor. And I should, this is the most important slide, is crediting the people who did, all the wonderful people who did all this work. Most recently, the retinog acid receptor work in my lab was carried out by Michael Tellius who was a post-doc in the lab and he's now on the faculty at University of Rochester Vision Science program I forget the name of their I Institute. Kevin Cow who is a current post-doc in my lab, Bristol Denglinger and other post-doc and various other people. And our collaborators, including Michael Gord at UC Santa Barbara and his graduates in Kevin's City have been just wonderful. And the work we've done together with Daniel Plonker and this work was supported by the National I Institute, Foundation Fighting Blindness, other foundations and several years ago, we started a small commercial enterprise called Photoswitch Therapeutics that is interested in commercializing some of these technologies. So thank you, I'm happy to take questions and discussion items. Thank you very much. Thank you, Dr. Cramer. Well, anyone you can either ask questions via chat or by raising your hands. I'll just start with a question. And these RD-10 mice you're targeting the 10 to 12-week timeframe for the clinical trials with disulfram, how are you determining that intersection of death of photoreceptor versus upper regulation of retinal died? So what level of vision loss are we talking about? Yeah. You know, this is where it gets into sort of clinical evaluation by our colleagues. I mean, clearly these people still have light perception. I think that, you know, the most precise kind of measurements we can do are micro-perimetry where we actually can map the extent of scatomas and really look very fine grained responsiveness to light even down to the level of individual photoreceptors using adaptive optics. So the exact stage and how sort of numbers of all of this, I'd have to ask my colleagues for the numerical, the quantitative numbers about how degraded their vision would need to be. Paul, go ahead. Okay, thank you. That was very interesting. As you may know, for a Stargardt disease, we're looking even earlier in the pathway with RBP-4 inhibitors that would lower vitamin B levels in the eye. Do you think that's doing a similar effect? Have you looked at that? You know, in terms of its effects on retinal acid, hyper-mediated hyperactivity. So I just, I coughed at exactly the instant you said, but could you repeat that once again? Sure, you know, we're looking for RBP-4 inhibitors for Stargardt disease and even for macular degeneration. Do you think that that would affect retinal, have you looked at whether that affects some of the retinoic acid levels and some of the hyperactivity that you're seeing? We have not. Yeah, so I mean, it very well could be working through that mechanism. I mean, there is sort of a conflict here in some ways because for at least certain disorders, vitamin A has supplemented and as a potential treatment. And so I think that's one of the reasons instead of interfering with the metabolism of retinoids we're probably safer downstream. And I mean, what I really favor is this idea of inhibiting the retinoic acid receptor and not messing with retinoid metabolism. So disulfurum is nice because we can do those experiments immediately and we can sort of do trials on humans immediately. But beyond that, I think the advantages are gonna fall on the retinoic acid receptor side of things. All right, thank you. All right, Dr. Kerber, that takes us to the end of our time with you for clinical faculty. Again, on behalf of our faculty, thank you so much for the talk today. Look forward to your other engagements throughout the day. Yes, I will see you in a week or so. Thank you. All right, take care. Thank you.