 We're going to get started here. I'm very pleased to introduce our basic science speaker this morning, Vladimir Kapilov. I met Vladimir just a little over a year ago when I was a guest speaker at Washington University and saved a list of where he was. And I found out that although I was familiar with some of his work in public literature we shared an awful lot of things in common, particularly with the interest of the visual cycle and cryochemistry of vision, and which people will be talking about this morning. He also will be giving a second talk later today to the basic science faculty when we work them or await them. And that will be at new years. And that would be on a different subject than calcium in the eye. And so I hope that many of you can be able to make it to that talk also. Just in terms of a background, Vladimir is from Bulgaria. He is into motorcycling and actually lived or not, he did drive here from St. Louis over the last three days on motorcycling. Rainstorm in, or rain and snowstorm in Wyoming just to be here. He still has a multiple day trip, a journey back. He's going to take a more southern route, which hopefully will be a little more temperate than that. He did his undergraduate and master's degree in Bulgaria between 1988 and 1993. And he was into physics, hard core solid state physics at the time. And then came here to the U.S. and worked in carnitornals, a laboratory at Boston University, and got his Ph.D. there. Often be into the physiology and early events of visual pigment regeneration. And then he did a postdoctoral fellowship with King Wai-Yau Johns Hopkins, working on the functional differences between retinal rod and photoreceptors. He's been at Blashew for a little over 10 years now. He's moved up to the ranks very rapidly and now is a full professor. So I think I will let him go from there. Thank you Paul for the invitation and for the kind introduction. I'm really excited to be here. Good morning everybody. I'm still on central time, so it's 7 a.m. for me and like Paul said, I'm a basic scientist. So I'm usually up at this time of the day but my brain is still a little slow. So this is a new presentation for me. I haven't given this talk before. The same goes for the noon talk. If I say something that doesn't make sense to you or maybe you have questions, feel free to stop me and ask me anything. So as Paul said, my lab is interested in the physiology of photoreceptors and I thought I'll tell you a story today which essentially the punchline is that we believe that some of the studies that we have been doing have a very fundamental clinical relevance and the story that I hope you remember from this whole presentation is that the mule agliocells actually play a very important role in mediating the function and more importantly the survival of comphoto receptors and of course those are the cells that mediate our daytime vision and without them again I'm sure you all know we are legally blind and cannot function well. So before I start into diving into the science I just want to give credit. This work is mostly done by a couple of people from my lab. I have a small lab, Jin Shan Wang and Yun Lu who is now a student in Konisepko's lab at Harvard and some of the work was actually also done by Sasha Kolesnikov. I'll show some collaborative results with Joe Corbos' lab who is also at Washington University and at the end I'll show you some really neat gene therapies studies which were done in collaboration with John Finnery's lab who makes these just amazing AAV viruses that can drive expression in comphoto receptors and we've been helped with colleagues with reagents and mice and of course we've been fortunate enough to still stay afloat despite how difficult and even more difficult funding in science becomes and I have no conflicts of interest for what it's worth. Alright so you all know this picture of course you know that the retina is in the back of the eye and light travels through all the layers in the retina and eventually it is detected by Rod and Con photoreceptors and for the purposes of this presentation you should be aware that of course the photoreceptors are not functioning in vacuum. They are actually surrounded by two separate layers of supporting or helping cells. Behind the photoreceptors is the monolayer of retina pigment epithelial cells and their processes span between the photoreceptors and of course the RPE cells play a critical role in supporting the function of photoreceptors by providing them with nutrients by removing the shaded outer segment disks and of course by also providing chromophore which is vital for the function of photoreceptors. But in addition to that on the other side of the photoreceptors there is another supporting helper cell which is usually ignored and that's the muller glial cells. So the muller glial cells actually span the whole retina. They're like rubber bands that essentially keep the whole retina together and they have processes that kind of also wrap around the cell bodies of photoreceptors and as I'll tell you today they actually play a very important role in supporting the function specifically of con photoreceptors. So of course we have two photoreceptor types and they have very similar structure but they mediate very different kind of visual experience and I like to use this slide to kind of remind everybody how different rodent cone mediated vision is. So rods of course we use in dim light. They are extremely sensitive. They can detect even a single photon of light. But this high sensitivity comes at a high price as well. So rod responses are relatively slow. In addition our visual system integrates the signal from hundreds and thousands of rod photoreceptors into a single ganglion cell that goes then to the brain and as a result the spatial resolution of our rod mediated to dim light vision is relatively low and that's why in dim light the images that we see are kind of blurry and of course because we only have one type of rod photoreceptor we're not able to detect light colors to discriminate colors and again this is why in dim light everything looks kind of grayish, greenish. You cannot see red and green and blue colors. In addition rods saturate even in moderately bright light so as soon as the sun comes out actually your rods become saturated and they no longer contribute to your visual experience and finally I'm sure all of you have experienced this. Your rods actually are very slow to dark adaptor to reset their high sensitivity after exposure to bright light and the example that I like to give is if you are going to the movies and you are late and you walk into the movie theater after the movie has already started and it's dark in the room and all of a sudden you are blind. You don't see anything so you kind of stumble around and you find a place to sit and if you look around half an hour later you can actually see pretty well and the reason it takes so long before you can see is because your rods which are the cells that you need for dim light vision take about 30 to 40 minutes to fully reset their sensitivity. They are extremely slow and so again this is what happens in dim light and of course the situation in bright light is very different. This is when your com photoreceptors take over and because you have three types of coms red, green and blue you can discriminate colors and in addition coms have very fast responses which mediate very rapid very good temporal resolution of your com mediated vision and this is one of the topics that I will talk about at noon if you get a chance to stop by and critical for the function of coms as our daytime photoreceptors is their ability to remain functional and adapt even in very bright light. So even if you are out in beautiful Utah skiing in the winter and there are billions of photons hitting your photoreceptors your coms are still somehow able to adjust their sensitivity and remain able to respond to light and finally coms darken up very quickly so there is no refractory period as in with your rods and if you go from bright to dimmer environment but still bright enough for your coms to detect light again there is no period where you're blinded and so my lab has been really focused on understanding these particular properties of com photoreceptors, their ability to function in bright light and also their ability to darken up very very rapidly and one of the features that we have been focusing on is the visual pigment and what happens with the visual pigment in coms after it is photo activated and it turns out that what happens in coms is very different from what happens with rods and it helps to a large degree explain this functional difference between rods and coms. So let me tell you what I mean by the visual cycle so in both rods and coms detection of light happens in the outer segment of photoreceptors which again are kind of embedded in the RPE cells and detection happens when a photon of light is absorbed by the visual chromophore which is 11 c-th rate now and this chromophore is bound covalently to opsin, this is a G protein coupled receptor and the two together form the visual pigment and the outer segments contain stacks of lipid disks which are packed with so much visual pigment that a photon of light traveling along the outer segment has about a 50% chance of being detected and activate visual pigment molecule and when this pigment is activated by light it triggers the whole transduction cascade which ultimately result in generation of the light response but then consequently this pigment needs to be recycled or resetted just like a light switch if you turn it on you have to turn it off again before you can turn the light on again and so this happens through a fairly complex set of reactions called the visual cycle so first what happens is that the c-th rate now which light when light absorbs it is switched to all trans-rate now and this is the molecular switch that activates the pigment comes out so there's the covalent bond between opsin and retinal which is broken and opsin is left behind and then all trans-retinal is reduced to all trans-retinal which then is actually exported from the photoreceptor into the RPE cell and there through a set of enzymatic reactions is recycled to 11 c-th retinal and then this 11 c-th retinal comes back into the photoreceptor finds a free opsin molecule binds covalently and the pigment is regenerated again and so there is as you hopefully can appreciate there is a fairly involved process in resetting the visual pigment and the reason why rods take about 40 minutes to darken up is because it takes about 40 minutes for their chromophore which was destroyed essentially by the light to be recycled from the RPE cells and for the pigment to regenerate and this same process happens also in comfort receptors and for the last 100 years the dogma has been that the RPE cells is the only location for recycling of chromophore and the only source for chromophore for both rods and cons are the RPE cells however if you think about it if it takes the rods 40 minutes before the RPE cells can provide them with chromophore then how come the cons can darken up not in 40 minutes but in 2 minutes so something smells fishy something looks like it's not right and if you look through the literature over the last maybe 40-50 years you can see bits and pieces here and there of evidence that cons might use a separate visual cycle that is independent of the RPE and provides chromophore only to them and not to the rods and the idea was again based on biochemistry from multiple groups is that actually the milder glia cells in the neural retina itself might also play a role in recycling chromophore and taking all trans retina, the spent chromophore from photo receptors and then simply converting it back to 11-6 retina which will then come back into photo receptors be oxidized to 11-6 retina and used for a generation of pigment and so this was all kind of the state of knowledge and this pathway had been shown to again there were biochemical reactions mostly than from condominant species like chicken and ground squirrel suggesting that the mule cells might be able to do that but there is really no physiological evidence, no functional evidence that this pathway actually exists and it wasn't known whether it functions in rod dominant species such as humus or mice and it was not known what if any the functional role of this pathway is and so this is at the time when I was setting up my lab and I thought that it looked like an interesting question to tackle and kind of the proof of principle, the basic experiment that we did is conceptually extremely simple, almost embarrassingly simple and it's amazing that people didn't try it before us or maybe people thought it was too easy to work or maybe somebody tried it and didn't believe the results and so the basic idea is that if there is a pathway that allows you to regenerate pigment only in cons that does not involve the rp cells and instead involves the mule cells then all you need to do is basically dissect the retina out from the pigment epithelium so get rid of the rp and then expose this retina to bright light that will bleach the pigment and convert it to opsin and destroy the pigment in both rods and cons and then simply put this retina in darkness for an hour or two and then go back and examine the state of adaptation or the amount of pigment and ask will the pigment come back or not and so if there is another pathway that involves the mule cells the cons should darken up, they should regenerate their pigment and regain their sensitivity but if the rp is the only mechanism for providing chromophore we should see no recovery once we get rid of the rp. Everybody still with me so far? So how do we study this physiologically? It turns out that the sensitivity of photoreceptors is directly correlated with the amount of pigment that they have and this kind of makes sense is the more pigment you have in the other segment the more likely a photon is to be detected and to generate a light response so what we can do is we can indirectly measure the state of adaptation or the state of the visual in the visual cycle by simply measuring the sensitivity of the photoreceptor so if we place the animal or a subject in darkness for a long time and we allow all the pigment to regenerate in both rods and cones they will achieve their highest maximum sensitivity on the other hand if we bleach the pigment and prevent it from regenerating then the cell should be highly desensitized by orders of magnitude so again we can use physiological measurements to detect the sensitivity from there deduce what state of the visual pigment we are looking into so how do we study this we do this mostly in mice we've done some of those experiments actually also in primate and even human retina and what I'm going to tell you holds there as well so the basic approach is again pretty straightforward turns out that photoreceptors are polar cells they're essentially like a double A battery you can think of them as the outer segment as the plus sign and the inner segment as the minus sign and that's probably the closest I can get to a double A battery but so what is that current actually flows into the outer segment of the cell in darkness and then the current leaves through the other end of the cell so essentially there's current that flows along the photoreceptor in darkness and when you shine light this current is reduced or fully blocked and so what we can do is we can make a glass electrode that has just the right diameter so that we can draw the cell inside like that and so now the inner the outer segment of the cell will be inside our recording electrode and the current that flows through the cell will also flow through our suction electrode and then when we stimulate the cell with light and the current changes we can actually record the changes in this current and the beauty of this technique is that you don't have to break into the cell to measure the current that flows through and as a result the cell actually lasts a long time so when we do for instance recordings with amphibian photoreceptors we can fully dissociate the cells we can take a dissociated rod or comphotoreceptor completely detached from the retina get it in our recording electrode and record from the same cell and keep it alive for 5-6 hours so again you can do a lot of experiments on a single cell and so this is demonstrated here and it's kind of hard to see I know but this is an infrared image from a piece of mouse retina and you can see the outer segments mostly rod outer segments sticking out and you may or may not be able to see but you have to trust me on this is that there is one rod outer segment that was drawn in our glass electrode and so again when we do this and when we stimulate this cell with light we can record the changes in the current transient changes when we give a flash brief flash at time zero and of course the brighter the flash the bigger the response until eventually the responses saturate and so using this method we can measure the responses from single rod photoreceptors and if we take now the peak of each of those responses and plot it as a function of the light intensity of the flash that was used to generate it we can get an intensity response curve or a dose response curve for this particular rod and again because this is a mouse rod this is a highly sensitive dark adapted cell you can see that the cell starts to respond to as few as maybe 1 to 2 photons per micron square so extremely sensitive detecting single photons of light okay so this is kind of the baseline so now we can compare this cell with a rod that was taken from a retina that was removed from the RPE and then bleached and then kept in darkness for a few hours so no longer RPE around so no way to regenerate the pigment and when we look from a rod from a retina like this we find that the responses now are significantly smaller and a response that before gave us the maximum maximum amplitude this is the red trace the same flash now barely gives a threshold response at all again you see there is a little red trace here and if we do the intensity response curve for this cell again you see that there is maybe a hundred fault or more than a hundred fault decrease in the sensitivity you need a hundred times more light to get a response compared to the dark adapted so no surprise there the rod once you bleach its pigment stays permanently desensitized even four hours after the bleach so the RPE is really critical for the regeneration of pigment okay so what about the cons so we can do the exact same experiment in cons and this is shown here again those are dark adapted responses from a mouse cone and you can get in test response curve and now you need more light of course because that's a cone it's less sensitive but compare this to a cone that was taken from a retina without RPE bleached and then allowed to dark it up for two hours and the first thing you notice is that the responses actually come back the amplitude is all the way back and if you look at the intensity response curve maybe there is a two three fourth reduction in sensitivity but there is no way near the decrease in sensitivity that we saw in the rod and because this experiment was done without any RPE around the only way you can explain the recovery of sensitivity of this cone is that somehow it was able to regenerate a substantial amount of its pigment without any help from the pigment epithelium and as a way of understanding how this is happening taking place as the next experiment we actually pulled the cone from the retina and then we gave the bleach and in this case as you can see the cone was no longer able to dark it up and now the situation looks much like what we saw in the rods and so together what this shows is that the cones can dark it up in the retina with the help of cells they cannot do this autonomously but they rely on contact with other retinal cells and eventually we were able to show using pharmacology that the mule or glial cells play a critical role and they are the location where the chromophore is recycled and provides the dark adaptation in comfort receptors everybody still with me? okay all right so the kind of the emerging picture then is that whereas rods rely only on their RPE as the source of chromophore you can actually draw chromophore from two separate places one is kind of the canonical RPE visual cycle and the other is this novel neural retina visual cycle where the mule or cells provide the chromophore and through a set of experiments we have been able to show and most of those are published now that this pathway actually is what provides the initial rapid component of con dark adaptation and then the RPE visual cycle comes in later to complete this last default of sensitivity that the cons did not recover so one question or one preissue that we had not been able to resolve until recently is to find a way to genetically manipulate this pathway and the reason we wanted to do this was to understand what is the relative significance of this pathway versus this pathway in mediating the function of cons and also the long term survival of comfort receptors and so we started looking around and trying to figure out again a way to tease apart the two visual cycles and understand the relative contribution of the RPE versus the mule or cells in mediating the rapid regeneration and dark adaptation of cons and also their long term health and when we were doing this we realized that we might be able to do this when we found that one particular protein called CRLBP which stands for retinaldehyde binding protein is expressed in both the RPE cells and in the mule or cells so CRLBP is a chromophore binding protein and it binds specifically to 116 retinoids and so it's believed in both the RPE cells and in the mule or cells that CRLBP speeds up the recycling of chromophore by binding to the product to the 116 product of the reisomerization reaction and by a simple mass action law accelerating the turnover of chromophore in both RPE cells and in mule or cells. Patient there are mutations in CRLBP that have been associated with a whole range of visual disorders in people in humans and also mice where CRLBP has been knocked out has abnormally slow wrought dark adaptation. Again consistent with the notion that CRLBP essentially speeds up the recycling of chromophore in the RPE. So we wanted to know whether we can actually tackle the question what is CRLBP doing in the mule or cells and indirectly from there maybe get a genetic or molecular handle on manipulating individually each of those visual cycles as a way of understanding its function of significance. And so to do this we applied two separate recording techniques because we wanted to record the recovery of course in real time. So again I'm sure all of you are familiar with in vivo ERGs where you basically place an electrode on the cornea and then a reference electrode somewhere else on the body and this allows you to record the voltage changes across the retina when the retina is stimulated with light and you get this kind of prototypic ERG response consisting of a downward A wave which is generated by raw photoreceptors and it's just the initial rising component of the photoreceptor response actually which is very quickly masked by an upward B wave which is generated by bipolar cells and so using this again people this is widely used in the clinic I'm sure by some of you but also it's a very useful research tool in animal studies and you can do ERGs for mice and it's a very informative technique. This technique however has limitations especially when you want to study cone function photopic ERGs because the amount, the fraction of cones in human retinas or mice retinas is very small 3% to 5% and so the photopic A wave generated by cone photoreceptors is usually miniscule very very hard to see and so typically any photopic study that you will see is actually looking at the photopic or cone driven B wave because the B wave is much larger and so the B wave is a wonderful tool but it's an indirect measure of the function of cone photoreceptors because you're looking at the cell downstream from the cone not at the cone itself and so to go around that we've been using another technique kind of a variation of this which is ex vivo ERG so it's the same principle but now you dissect the retina again obviously you cannot do this with humans but with animals you can take the retina out and have an electrode on each side and then again you measure the voltage across but the advantage that you gain by doing this ex vivo is that you can use synaptic blockers to prevent the signal from traveling beyond the photoreceptors and so whereas in vivo you'll get the A wave and then the B wave ex vivo you can essentially block the B wave and what you will get then will be the A wave but the B wave will no longer mask most of the response from cones and so you'll get actually a beautiful response that will look like this which will be generated by the cones and you can study then the kinetics of this response, the amplitude and all that, the sensitivity and so we've used a combination of those two methods to study the role of CRILBP in the function of cones and this is an example of what we did in vivo so these those are in vivo ERGs from wild type mice mice that are deficient in CRILBP and that's just the gene name of the protein and heterozygous mice and again you see that the A wave which is the downward inflection in the response is relatively small but you can see a substantial B wave, the upward wave and if you compare, if you plot the amplitude of the B wave as a function of the flash intensity again you find that the wild type and heterozygous mice have normal ERG responses cone driven ERG responses but the knockouts have severely desensitized cones and also their maximum response amplitude is substantially reduced and so is this coming from the cones or is this coming from the bipolar cells because again the readout here is bipolar cells and so to go into addressing this question then we did the ex vivo ERGs and you see how you can get beautiful responses now those are driven from the cones and again if you think about that this is the A wave inflection that we see here but if we block the B wave pharmacologically then you get the full response that will look like this and that's what I'm showing you here and those are the controls cones and those are the knockout cones and so again in the absence of CRILBP cone sensitivity severely reduced the cones require more light to be activated and their maximum response is also substantially reduced so why is that? Are the cones fewer? Are they sick? Why is their response lower and why are they less sensitive? So to address this question we looked at the morphology of retinas and the first thing we noticed was that cone pigment was mislocalized in our knockouts so whereas in wild type cones, coenopsin is localized exclusively in the cone outer segments in the absence of CRILBP coenopsin was all over the cell and this is consistent with an idea that Rosalie Crouch has proposed maybe 10 years ago namely that coenopsin requires chromophore as a chaperone to be folded properly and to be targeted to the outer segment and when we take out this protein CRILBP we are essentially depriving the retina from chromophore and as a result coenopsin is mislocalized. We also noticed that there was degeneration so the number of cones gradually declined with age in our knockouts so it looks like again taking out CRILBP had both functional and morphological changes in confotoreceptors. So what about dark adaptation? Ultimately again we are interested in how this protein and how the two visual cycles affect dark adaptation of cones. So again we did this both in vivo where we can look at the dark adaptation of cones in the context of the intact system where both the RPE cells and the Mueller cells are still there and providing chromophore and this is shown here. So the basic experiment is you take an anesthetized mouse and you measure the sensitivity with a dim flash first in its highly dark adapted state and so that will be when the cones will be the most sensitive and then you expose the eyes, the retina to very bright light for a minute or two that is designed to bleach over 90% of the pigment in the photoreceptors and then you put the animal back in darkness and you follow how the cone sensitivity is coming back after this bleach and this is shown here again the sensitivity initially goes down more than 15 fold and then with time as the pigment in cones is regenerating their sensitivity is coming back and I should point out that because those are anesthetized animals the kinetics of this sensitivity are somewhat slower than it happens normally. If you do this in an awake animal or in humans again the cone sensitivity is back within 5 minutes so again you see how the time course of cone dark adaptation in the normal condition. What happens when we take out this CRLBP protein? Well what you find is that this dark adaptation of cones is largely suppressed and that's really a striking result for me because that's the first animal model ever that I've seen where cones are unable to darken out quickly. You can do so many things to cones and they always bounce back immediately. It's almost annoying because if you want to study the dark adaptation it's so quick it's difficult to measure but in this case again dark adaptation was highly suppressed in the cause for mice lacking CRLBP so how much of this is because the mule cells are not providing chromophore and how much is because the RP cells are not functioning well. You see that the late phase is compromised and we know that the rods that could up slower so we know that the RP cells are slower but in order to look at the mule cells we can do the same experiment but now in the isolated retina where the only way chromophore is getting to the cones is from the mule cells and so this is the example in the control so now again this is an isolated retina no RP and you see that again right after the bridge this sensitivity goes way down initially there is this initial recovery is actually the response that the cell turning off from the light, from the bleaching light and then there is this late recovery of sensitivity which is driven by the mule cells and if we kill the mule cells essentially we will get the recovery up to here so what happens when we take out CRLBP it's exactly the same thing so this recovery driven by the mule cells now is largely suppressed and so what this result clearly shows is that the mule cells require CRLBP to drive this recovery of cone sensitivity still with me? Okay alright so that's great so based on this we know that CRLBP in the mule cells actually plays a functional role because we can see how when we take it out we suppress the recovery of cones but we still have an answer to the major question which is how can we discriminate between the role of RP cells versus the mule cells so one way we could do this is to now restore the function of either the RP cells or the mule cells by genetically or by using a gene therapy approach to drive expression of CRLBP in one of those two tissues and so the idea is again to take CRLBP knockout mouse and use an IV injection as a way of gene therapy basically rescue one or the other cell that either the RP cell or the mule cell and then ask which one will give us more beneficial effect for the function of cones and so this is just a wild type retina showing the expression of CRLBP and again it's present and abundantly expressed in RP cells and it's also abundantly expressed in the mule cells that span the whole retina and of course in the knockout all of this expression is gone and so we again we worked with John Flannery and they made this really neat virus which allowed us to reintroduce CRLBP selectively in the RP cells by injecting the virus into vitrally and the beauty of an intravitory injection as opposed to subretino injection is that the virus can basically penetrate and give you uniform expression throughout the whole eye and again you see now that the RP layer in the whole eye expresses CRLBP and this is also shown in retina section so now we have normal RP visual cycle that expresses CRLBP but the mule cells still lack this protein so how will that affect the function of cones or not will that rescue cones or not and so what we found was that there was really no rescue at all so this is of course the isolated retina recovery which since we haven't rescued expression in mule cells we didn't expect any change so we are comparing injection of GFP control versus injection of CRLBP driven only in the RP but more importantly when we look at the function of cones and their amplitude of their response and their sensitivity it was not affected even though we restored the RP visual cycle cones were still desensitized and they had small amplitudes just like in our knockout so there was no beneficial effect from the RP visual cycle what about the retina visual cycle so we did the opposite experiment where now are restoring expression of CRLBP only in the mule cells but not in the RP cells and again you can see how the mule cells throughout the retina are expressing CRLBP what will happen with the function of cones now boom so now all of a sudden now we see both the restoration of dark adaptation in the isolated retina because we've rescued the mule cell pathway but more importantly we see a substantial increase in the sensitivity of cones and also their maximum response was increased and so what this result clearly shows is that a functional mule cell visual cycle actually is critical for the normal function and for the health of cone photoreceptors and when we take this pathway specifically out even if we still have the RP pathway still there cones become desensitized and I'm not showing you this but we also saw rescue partial rescue at least on the mislocalization of cone opsin and so what we think is happening essentially is that chromophore coming from the mule cells feeds into the cell bodies of cone photoreceptors where cone opsin is being expressed and folded and by binding to this opsin it helps to fold it properly and then helps to target it normally to the outer segment and ultimately this provides a healthy and functional cone photoreceptors and when the mule cells are no longer able to do that when they lack for instance CRLBP then cone opsin becomes mislocalized because there is not enough chromophore coming in from the mule cells and then cones eventually degenerate because of the lack of opsin and so to summarize what I showed you again we find that this protein CRLBP plays a critical role in supporting cone function and survival it is a key component not only of the RPE visual cycle but also the mule cell pathway and specifically in the mule cells again it plays a role in providing sufficient chromophore to sustain the function of cones and this feeds again into this more broad hypothesis that chronic chromophore deprivation is actually really bad specifically for cones and not so much for rods and if anybody is interested I'm happy to talk to you afterwards we are doing some experiments by switching rod and cone opsins trying to really understand how the differences in the biophysical properties of the rod and cone opsins actually modulate this higher susceptibility of cones to chromophore deficiency versus rods and again I showed you that when we slow down the mule cell pathway by knocking out CRLBP we get a substantial suppression of cone duct adaptation another way of saying the same thing is that the functional mule cell pathway actually is important for the rapid duct adaptation of cones so where we are going with this is that we are trying to understand how chromophore is traffic between the mule cells and cone photoreceptors what determines the specificity of this pathway only to cones and not to rods and finally the last point that I want to touch upon is what are the kind of therapeutic or functional implications as far as clinicians are concerned of the presence of two separate visual cycles and so there are many pharmacological pharmaceutical companies nowadays that are interested in developing drugs that target specifically the visual cycle there are many mutations for instance in rodopsin or in any of several of the enzymes in the RP visual cycle both in the processing of chromophore in rods and in RP cells that slow down the pathway and also lead to the accumulation of toxic byproducts which eventually lead to retinitis pigmentosa for instance and so again there are drug companies that are investing a lot of money trying to target this pathway and find a way to slow it down as trying to slow down basically the accumulation of toxic byproducts and so one cautionary tale into this is that any drug that is targeted has to keep in mind what happens with cones because as I just showed you adequate supply of chromophore to cones actually is critical for their long term survival so you don't want to spite your face and cut your nose because if you get a drug that works so well that it actually fully blocks supply of chromophore to cones you might protect the rods but you will end up losing the cones and so this obviously will not be a useful drug and so I think if you're targeting a drug to a reaction in the RP visual cycle that should work because you will still have the mule cell pathway but if your drug is targeting a reaction that is common to both rods and cones for instance the reduction of retinol to retinol then again you will slow down the overall recycling of chromophore and that might have detrimental effects to the function of cones even if the rods are protected in the process another interesting twist and something that might be clinically relevant is for instance what happens with retinol detachment so if you think about it the retinol detachment is kind of like the experiments that I've been telling you about you tear off the RP cells and so when we started publishing this work I got an email from a Dutch clinician who was really excited because he had this patient who had retinol detachment and he was able to measure scotopic and photopic vision in the detached area of the retina and what he found was that rod vision very rapidly declined but cone function persisted for days and days and days and one reasonable explanation for this would be that of course when you get rid of the RP just as I showed you today rods very quickly ran out of chromophore and stopped mediating vision but cones can still rely on their Mueller cell visual cycle and get chromophore and recycle chromophore and sustain their vision so again I think that there are potential clinical implications and particularly I think pharmaceutical companies that develop drugs targeting the visual cycle have to be absolutely sure that any drug that they test is not killing the cones in the process of rescuing the RP pathway and the rods. So this is what I wanted to tell you today again I hope kind of the message that you will take home from all of this is that in addition to the RP pathway which everybody knows is critical for providing chromophore to both rods and cones there is this other pathway which resides in the Mueller cells which is cone specific and it helps to regenerate the pigment only in cones and not in rods and this pathway has placed a critical role in providing chromophore to cones and helping to express and target cone option and in the long term sustain the function and survival of cone photoreceptors and so again with this I'll stop and I'll be happy to take questions. Thank you. So Fabulous. Thank you. It's great work. It shows even though people say this is an old cycle we've known and so on a lot of others a lot of still information. Exactly. Yeah. So once come out of our laboratories here is out of Robert Mark and Brian Jones lab about the fact that for a long time clinicians assumed that the rental system was kind of like wiring in your house and light bulb goes out all you do is you screw the light bulb in there and they work you fine and their work is shown to wipe out the rods and cones from any process you want to use disease and there's this extensive wiring curves that essentially neural tissue there's evidence down the brain as well if you're not getting normal stimulation those cells want to be stimulated so they don't work. What is your sense how much of the Mueller cells play in this rewiring and this transformation to this very bizarre language. I'm sure you've seen some of this. Sure. Sure of course. Yeah. Very bizarre. I don't know I would imagine so I think one kind of emerging theme is that the Mueller cells actually are much more important than previously appreciated for the function of specifically photoreceptors. I think now there is new data coming from Jim Hurley's lab for instance that metabolism in photoreceptors is closely tied to metabolism in Mueller cells and photoreceptors rely heavily on Mueller cells. I think it will be so kind of the long wind that that's a long wind and as the short wester is I don't really know how the Mueller cells are involved in rewiring but one way we've been thinking about asking the question in this question in the context of the visual cycle is there are diseases where specifically the Mueller cells are affected for instance in response to stress there is an upregulation of genes in Mueller cells they change their metabolism and it will be really interesting to see if some of those diseases there is an effect either on rewiring or in our case in the visual cycle and specifically on the function of cons. Again in some cases it might be beneficial again this upregulation of gene expression in Mueller cells might have a protective role or it might be kind of the early signals that drive the degeneration of photoreceptors. So unfortunately the Mueller cells are really important for the function and maintaining the structure of the retina so you can knock out you know you can kill roles, you can kill cons, you can kill certain ganglia cell types. If you kill Mueller cells the retina falls apart so again it's really tricky to study the function of role of Mueller cells because again they are so important and the moment you take them out the whole retina falls apart. So when we were doing, I didn't show those experiments but the way we showed that the Mueller cells are critical for the function of this dark adaptation of cons is we would take a retina and incubate it for a brief period of time in a gluteoxin basically kills selectively the Mueller cells and if we did this for an hour or so in a mouse retina we could show that the Mueller cells basically vanished, some of them not all of them and then we could see that the recovery of cons was suppressed but if you incubate it with the gluteoxin for 3-4 hours basically the whole retina would fall apart and we would not get anything out of it. So again I think that's a great point and the bottom line is that Mueller cells are clearly again much more important than previously appreciated and taking them out is probably not altogether is not feasible but if you want to study a specific pathway then if you have a genetic way of altering this pathway selectively in Mueller cells that will be very instructive and so that's the direction in which we're going, we're hoping to learn more about the enzymes involved in recycling of chromophore in Mueller cells and then target them and see how this affects the function of cons and the survival of the photorescent. Yes. Thank you. Yeah. Well is that really true though? So there are two issues. I think when you deprive of chromophore by vitamin A deprivation or by knocking out CRWP or any of the other RP, visual cycle proteins, so the cells are no longer able to respond to light as efficiently because they just have less visual pigment but the difference is that the rods don't die. So if you look for instance of the most dramatic at least in mice way of essentially fully blocking chromophore supplies knocking out a key pathway in the RP cycle called RP65 and if you take out this gene basically there is no chromophore in the retina at all but if you then take a mouse that is one year old that never had any chromophore and then you introduce chromophore back the rods will resume their function just fine but the cons will long be gone. So the cons die weeks after birth in these animals whereas the rods can persist for months and months and months. So again we think that there is some fundamental difference between the absence in rods and cons which again help rods to survive even without chromophore but in cons seems like chromophore is really essential for their long term survival. So what we're doing with again I don't have time to go into this but I'm happy to talk to anybody if interested afterwards is we basically made a mouse where we switched a cone option with rod option and the thinking was that we take this unstable cone option which requires chromophore and put the rod option which doesn't seem to care whether there is chromophore or not and this seems to have beneficial effect and enables cons to survive even in the absence of chromophore. So that's the bottom line. So it gives me a follow up question. As I remember in severe vitamin A efficiency, I'm talking about the level of vitamin A efficiency that tends to use people. But in histopathology there is broad loss of the cones but I can't remember. Do the rods survive in this? I know the cones just disappear. This is where people eventually are losing vision. I think the patient is an intern that actually died from the efficiency and had gone functionally blind other than some vision which fits what this does. I don't know. I remember that histopathology that was just broad wipe out seemed to be like maybe it was the cones and not the rods. That seems to fit again with what we see in animal models. It's not so much in the patients that I've had with vitamin A deficiency. There's still some specific delivery to the cones. There's something different because if you talk about it real, I don't think it's here right now. The cone function is actually preserved pretty well at least in humans with vitamin A deficiency. It's the rods that are down. The cones aren't dead yet. Until you get to the total crash with all bodies falling apart. If he's doing a full field of ERG then how much of his A wave is really, I mean his B wave is really cone function and how much of that is just the... You talked a little bit about the isomerase in archaeology. Tell me what you think is the state of knowledge of what's going on in the Mueller cells. Is it this one? Is it something else? So that's a thing still an active area of research. So what Paul is asking about is what is the enzyme that converts all trans retinol into levesis retinol. So Gabe Travis's group at UCLA published a paper a few years ago suggesting that an isomerase called DES1. DES1 is the one responsible for this reaction. We don't know yet. So the only way I guess the most straightforward way you test this is by just knocking it out and asking whether this will affect this pathway or not. And they have been suspiciously quiet for two, three years. I know they've been working on that and every time I see Gabe I said, hey, what's going on? When is this mouse coming out? And we haven't seen any results. So this is why we actually went for the serial because we were waiting for somebody else to knock out a key enzyme from this pathway. So I don't know, again I'm not... I don't have a stake in this fight. There are people that believe it is. There are people that believe it is not. Ultimately, again, somebody has to just do the experiment and find out. So I mean one of the problems with DES1 is it's a very nonspecific isomerase. It just, it equilibrates but it's not very efficient making 11 cis. Well RP65 that's what it has. Sure, sure. So when you knock out C-R-A-L-B-P and you remove the cells, do you see lots of different isomers? That's a great natural question. So we've actually been looking into that. So and our thinking was exactly this, is that DES1, so this isomerase in mule cells, seems to make more nicest retinal than 11 cis retinal. Whereas the isomerase in the RP cells makes exclusively 11 cis. And so we thought maybe some of the chromophore coming from the mule cells would be nicest. And we should be able because nicest can bind to ops and inform a visual pigment. But it will be blue shifted. It will have different spectral absorption properties compared to 11 cis pigment. And so we, by measuring the spectral sensitivity of course we should be, we thought we should be able to discriminate between nicest and 11 cis pigment in course and see which one is coming back from the mule cells. And so we've done this both in amphibias and in mice and we see no evidence for nicest retinal which was kind of bothersome. But then we actually looked at the C-R-A-L-B-P knockout mice. And one feature of C-R-A-L-B-P is that it binds specifically to 11 cis and not so much to 9 cis. So one possibility is that C-R-A-L-B-P actually favors by binding to 11 cis, favors the production of 11 cis over 9 cis. And indeed when we looked at mice that lacked C-R-A-L-B-P we saw evidence for about 50% nicest retinal in those cones. So we think that again the DES1 is still a viable candidate because if you take out C-R-A-L-B-P you seem to get a lot of nicest in the comfort receptor. But that's a very insightful question. Thank you. Okay thank you so much.