 and we are officially live. Hello everybody and welcome back for the third season of our Sussex Vision Seminar Series as always within the Worldwide Neuroinitiative. I'm George Caffèdiz, a former master's student in Thomas Euler's lab and currently a PhD student with Tom Badden. And as your host for today, I would like to once again begin by thanking Tim Vogels and Panos Bozellos for putting forward this very initiative towards the greener and much more accessible seminar work. Okay, I lost you, George. You cannot hear me? I can hear you now, but there was a period of time when you went silent. Okay, okay. As my internet connection is a bit unstable. So let's open. I'm keeping an eye open for the chat and in case people also confirm that they lost me, I assume it might be an issue from my side. But let's see. So yeah, as I was saying, please allow me to get back to the reason we all gathered here for today and introduce our guest from Harvard University, Professor John Dowling. I cannot help but wonder how one summarizes such a successful and influential career in only a couple of minutes, but I will definitely give it a go. So John first became interested in the functional organization of the retina when George Walt suggested he studied the effects of vitamin A deficiency on rats photoreceptors. His undergrad and PhD research on the topic led both him to take an indefinite leave of absence from his medical degree studies and Harvard in appointing him as an assistant professor in 1961. In 1964, he moved to Johns Hopkins University as an associate professor of the Wilmer Institute of Ophthalmology and Biophysics only to return to Harvard as professor of biology in 1971. He has been located there ever since, nowadays being the Gordon and Loura Gunt research professor of neuroscience. This is the main trajectory, but an equally important and parallel one occurred when a call from Francis Carlson took him to Woods Hole and the Marine Biological Laboratory to establish a neurobiology course. That year was 1967 and John has been spending summers there ever since. In his extremely fruitful attempts to understand the organization of the retina, he has employed throughout the years anatomical, biochemical and electrophysiological techniques and has worked on an impressive number of organisms from pure rod or rod dominated retinas of skates and rats to ground squirrels, primates, mud puppies, frogs, rabbits and eventually zebra peas for a good 15 years. And I'm pretty sure this list is by no means exhausted. Since 2015, when he became a research professor, John has focused exclusively, if I'm not mistaken, on the phobia of the primate retina, both in health and disease. And today we will have the pleasure of hearing about their connectomic attempts of reconstructing it. Fellow of the American Academy of Arts and Sciences, member of the National Academy of Sciences and of the American Philosophical Society, recipient of many, many more personal awards and honors and author of a number of books, including the retina, an approachable part of the brain. It is with great pleasure that I'm leaving the stage for him, Professor John Dowling, for a token title, analyzing retinal disease using electron microscopy connectomics. So without any further ado from my side, please all welcome Professor Dowling. John, the stage is officially all yours. And you are muted. I'm just observing that. So please go ahead and unmute. All right. Splendid. Thank you very much, George, for a wonderful introduction. And thank you again for the invitation to present in this series. I'd like to start with a little background on the material that I will be presenting to you today. I'm now retired, have closed my laboratory and now put emeritus after my name. But that doesn't mean that I've stopped doing research. Indeed, I've joined the laboratory of one of my colleagues, Jeff Lickman, who is a pioneer in developing connectomics, serial section electron microscopy. And with Charles Zucker, a colleague, we have been studying then a rare form of age-related macular degeneration. Can you all hear me at this point? George has disappeared. We can hear you crystal clear, John. Excuse me? We can hear you, yes. Oh, okay. I wanna make sure you can hear. And yeah, okay. So we've been looking at a rare form of macular degeneration. It's the disease called maculotelangiectasia or MacTel for short. Why MacTel? Why are we interested in MacTel? Because a number of years ago, I was invited to serve on the scientific advisory board of the Lowy Medical Research Institute, which is in La Jolla, California. And they focus on this disease. And along the way, they gave us two eyes of patients who have the disease. One, a daughter of 48 years old and then her mother of 79 years old. And that material is very valuable because of course it's very hard to get human eyes. And so with Zucker, we decided to look carefully using connectomics to analyze a disease to see if we could get at its etiology. So that was the main purpose of the work that I'll be telling you about today, which of course is still very much in progress as you will see as we go along. Now, MacTel is an interesting disease in that lesions form in the macular of the retina in the central retina. They form in a small area called a MacTel zone, which is approximately three millimeters in length, two millimeters width. It always includes the fovea. And in the MacTel zone, lesions form that cause scatomas, blind spots. And these blind spot scatomas are confined to the MacTel zone. Now further, what has been shown by light microscopy is that there seems to be a lack of Mueller cells in the MacTel zone. Secondly, what has been shown is that in the central retina of patients with MacTel, there is a loss of serine. So what we've been then trying to do is to understand what might be going on. Serine, of course, is a very interesting amino acid. It's not sensitized by neurons anywhere in the nervous system, but by glial cells. And so in the retina, we believe that it's the Mueller cells that are producing serine. So what we've been asking is, what does the lack of serine, if that's primary, how does that affect the retinal cells? And then what is the significance of the MacTel zone? Why the MacTel zone? What does it mean? And those are the two major questions that I'll be addressing this morning. So let me begin by switching over and looking at slides. Okay, okay. And let me begin first with a little bit of methodology that's been developed in Lickman's laboratory for conectomics. So if we go to the first slide, let's see there is my cursor. The first big development that they have provided us is the ability to cut thousands of sections from a fairly large piece of tissue. And this is the apparatus that is used. The tissue is here, like classic electron microscopic tissue embedded in an E-POM plastic. The sections are cut on... And I do believe you most likely lost John and so did I. So let's wait for a couple of seconds as I assume he's trying to get back to us. Internet issues. This is probably the disadvantage of taking it digital. And I'm trying to refrain from incessant mumbling. John, I see you are back, you are muted again. So please do unmute yourself. Great. So what I would suggest given the internet issues that you have is to switch off your camera because it might be a broadband issue. So switch off the camera and start screen sharing. Keep your microphone on. Okay. So what I should do is go to screen sharing. Okay. Yes. Okay. You can hear me now. Yes, absolutely. Okay. And I would just suggest to avoid this issue to maybe switch off your camera by pressing stop video. What would you like me to press? Stop video. Stop video. Yeah, don't worry. Don't worry. Try to go on with your presentation. If it happens again, we will adjust accordingly. Okay. All right. Now what has happened is that I've lost now my cursor. All right. Let's see if I can bring it back. There it is. Okay. So we have these long strips of tape containing the sections. They're then oriented, condensed, and then on way over here on the left, you can see the sections all lined up appropriately. And in this case, we're looking at a piece of cortex and hippocampus. That then allows us to pick out a region of interest in whatever piece of tissue we're looking at and then to scan it, to image it using scanning electron microscopy. Now with scanning electron microscopy, we also have an advance that's important. And that is that we have a multiple beam scanning electron microscope. Let me spend just a minute telling you a bit about multi-beam scanning electron microscope. This is the instrument here that's made by Zeiss and inside, it looks like a refrigerator from the outside but inside is the guts of the machine. Okay. So from a gun on the right hand to electrons are emitted and then through an aperture, a single beam then is brought down to focus on the piece of tissue and the area that we're interested in observing. Of course, some electrons are absorbed by the tissue others are reflected, those that are reflected then go up and are captured by a second tube and then image in that at the end of that tube into an electron micrograph. And it looks something like this, the scanning electron microscope scans the area that you're interested in. And from that an image is formed that it looks approximately like this at a magnification that gives you information about tissue. It can be increased, it can be decreased and so on and so forth. But with a single beam, one gets an image only of about 10 micrometers on a side. The scanning electron microscope divides the beam into 61 separate beams, each separate beam focusing on a slightly different part of the tissue. And you end up then with 61 images similar to what I showed you on the last slide but now we're looking at an area not 10 microns on a side, but closer to 100 microns. And then of course what one can do is move your tissue and when you do that, then you can, all right, then make another 61 images. And with a little bit of overlap, all of which is done automatically, then you can begin to image a huge area, very, very convenient for studying retinal disease where you wanna look at particular areas which are of interest. So okay, now let's go back. The other advantage of what I've been telling you about is that most connectomics is done by the block face method which means that you take an image, a single image of the block face and then cut another section. And that means that you have only one image at one magnification and you lose the material. Here with the sections arranged on tape, it means you can go back over and over and over again looking at a particular area and you can look at it at different resolutions so that it means that you keep your material and you can study it in various ways. Unfortunately, it seems like John lost connection again. So what I will do is I will take the initiative and stop his video when he comes back hoping that this will kind of facilitate his talk. So please bear with us. It wouldn't be a premiere if we didn't have some technical issues, would we? Hello John, you are muted again. So every time you enter the room, you enter us on mute. Do you mind if I switch off your camera and you start screen sharing? Sure. Great. And I hope this will help with the issues. Okay. So please do start screen sharing. Okay, screen sharing. Very good. Got you again. And share. All right. Splendid. And I will stop your video just to make it easier, okay? Okay. So get rid of me, that's perfectly fine. Don't read it all. In fact, I will. So. thumbnail sketch. Fine. So this is a little description of the eye that we've worked on most seriously. 48 year old. We were able to process the eyes within two and a half hours of death. And they were then fixed in classic electron microscopic fixatives, first paraformaldehyde, then osmium, then we embedded them in plastic and so on and so forth. So all of that is quite convenient. Now, let me show you what her retina looked like, her fundus. And here is a fundus picture of the material that we worked on. This of course is the heroptic nerve, blood vessels coming in and out of the optic nerve. And then this is her macular. And of course, characteristic of the macular, and I think you can see this, is that it's filled with a yellow pigment called a macular pigment that excludes blue light. And why it excludes blue light of course is because blue light is refracted more than red and green light, and therefore to improve resolution you have the yellow pigment. Now, this is the 48 year old woman and the mactail zone is approximately this region here. And characteristic of the mactail disease is that there's a loss of pigment in the mactail zone. Here is a lesion that occurred in her retina right here. This represented a fairly large blind spot in her retina. This is the macula, I mean the phobia over here, which of course, as I mentioned, is always included in the mactail zone. Now to show you what we believe is the mactail zone, over here is the same photograph, but now dotted is the mactail zone. Phobia, lesion, depigmented area, and then the dotted line depicts the edges of the mactail zone. Now, so here is the mactail zone and it's somewhat higher magnification, the lesion, the depigmented area, and this being the phobia. Okay, so what we decided to do, first of all, was to study the retina, not in the mactail zone, which we knew would be deceased, but five to three to five millimeters away where vision was quite good, not perfect, but still quite good. And that is characteristic again of mactail. That is that except in the mactail zone, vision remains fairly intact. So we decided to look first out there to see how the fixation was and so on and so forth. And that, oh, one thing I forgot. This is the lesion in her mother. And you can see it's very much larger than her lesion. This happens to be the phobia, which interestingly enough, and fortunately enough was not affected and derived. We'll come back to talking about the 79-year-old briefly, but we're very much in the middle of analyzing it at the moment. But anyway, let's go back to the 48-year-old and look at her retina, three to five millimeters from the mactail zone. And here we are out at the outer limiting membrane. These are the photoreceptor inner segments that extend from the cell nuclei of the photoreceptors. We happen to be in a region where we have all rods. This is the inner segments. Then the ellipsoid regions are somewhat above and filled with mitochondria, thought to be critical for maintaining the outer segment and so on and so forth. Now, what forms the outer limiting membrane, as I'm sure all of you know, is the Mueller cells making junctions with the photoreceptor cells. And if you look carefully, you can see the densities between the inner segments and the Mueller cells. And in the light microscope, there are so many of them. It appears as though it's a membrane, but of course the outer limiting membrane, as I'm sure, most of you know, is not a true membrane but formed by the junctions between the Mueller cells and the inner segments. And here, three to five millimeters away from the Mac-Tel zone, the Mueller cells look quite nice. Somewhat darker cytoplasm than the inner segments but look quite intact. We didn't notice occasionally a little bit of pathology, but in virtually every human retina, you'll see something like this here and there. So we didn't think too much of it. So from the outer limiting membrane region, we went down to the outer plexiform layer. And here, of course, is where the photoreceptors, these being photoreceptor nuclei, forms terminals. These are the rod terminals, the so-called spherules, where processes from the bipolar and horizontal cells penetrate into the terminals to make synaptic contact. And of course, as all of you know, synaptic contact is made at regions where you find these synaptic ribbons surrounded by vesicles. One synaptic ribbon here, a second one here, and so on and so forth. These are the rod terminals, looks quite normal. Here is a cone pedicle, always larger than the rod spherules. Many more processes contact the cones, but again, you see synaptic ribbons as one here and so on and so forth. And if you look in the outer plexiform layer, for the most part, it looks quite normal. Not too much. These little dark areas sort of interested us, but it didn't seem as though there was anything drastically wrong with this region of the retina, which is what we would expect. It's been shown that it's quite functional. Something that did catch our eye was down here. This is a portion of a horizontal cell. And here, in looking inside the horizontal cell, we noticed that a number of the mitochondria had seemed to have lost their Christie and were swollen and in the number of the mitochondria were these dense areas. Now, as we look elsewhere in this region of the retina, and this is an example of probably a displaced ganglion cell because we're at the border of the inner nuclear layer and the inner plexiform layer. And this is a large cell, probably a displaced ganglion cell. This is likely to be an amicron cell because of course, amicron cells typically have invaginated nuclei such as you see here. But what I wanna emphasize here is just as we saw with the horizontal cell near the outer plexiform layer, a number of the mitochondria contain density and loss of Christie. And you see that both in the amicron cell and if we look over here in the ganglion cell. So you can see there are relatively few Christie here. Some of the mitochondria looks swollen and here we see it more prominently. In fact, in this ganglion cell and some of its processes, what's happened is that because the dense material that seems to be primarily in the mitochondria is denser than the rest of the tissue, that the tissue splits at this point causing a little tear in the section. And so you see just the absence of tissue at this point. So clearly even three to five millimeters out, it seemed to us that the mitochondria were somewhat abnormal, although the rest of the cells look pretty darn good. So we then moved in closer to the mactail zone or remember this was three to five millimeters away. But when we move now to one millimeter or so from the border of the mactail zone for the rest of the retina, whereas the tissue still looked fairly good. This is a cone pedicle now. Here's a synaptic ribbon triad complex that's so characteristic of the photoreceptor terminals, lateral horizontal cell processes, central bipolar cell dendrites. When you look in the outer plexiform layer, now you see the changes that we saw in some of the cells in the outer region of the retina, much in the three to five millimeters away, much more prominently. Densities, swollen mitochondria was loss of Christie and so on and so forth. And that's shown again in a higher power here where again, here is a synaptic ribbon in a terminal of pedicle that looks fairly normal, but most of the mitochondria clearly are abnormal. Few Christie, swollen densities, so on and so forth. So we began to suspect that we have a problem in mactail with mitochondria. Look at this group down here in which the mitochondria are quote, quite swollen, quite devoid of Christie and so on and so forth. And if we look elsewhere in the retina, this happens to be a piece of a horizontal cell, you see the same thing. That is the mitochondria have lost their Christie, contain these densities, there's an example here and probably a bipolar cell process and so on and so forth. The point being is that as we move into the area where the mactail zone is, we see more and more dramatic changes, particularly in the mitochondria. So moving on, looking at what's happening with the mitochondria, this is higher power showing you a cell just outside the mactail zone in which many of the mitochondria look quite normal. With many Christie and so on and so forth. But there are densities in some of the mitochondria and some of them are quite swollen with a considerable loss of Christie. And then some of the mitochondria have just developed these very dense internal structures which we think represent an expansion of the densities that we see in the other more normal looking mitochondria. So this was an example here. And here's another example at higher power of the changes that are taking place in the mitochondria. And this is now inside the mactail zone and almost all the mitochondria are affected. Some look fairly normal as these do here, but look at this cell here, which presumably is the beginning stages of the degeneration. And these are more advanced stages and so on and so forth. Don't pay any attention to these dense lines here that you see in mitochondria, which you'll see more of as we go along because clearly the dense material in these mitochondria is denser again than the rest of the tissue. And what happens here is rather than the tissue pulling apart, it folds up and you end up with a dark line. Okay, so where we are and our first observations are that it looks like the mitochondria are considerably altered. And one can, again, with conectomics, begin to look at mitochondria through their entire depth. So for example, and this is an advantage of doing conectomics in that you can do serial section electron microscopy very readily. So if we look here, uh-oh, this is supposed to be a little video clip and I'm disappointed that it's not working because it shows you what one of these mitochondria will look like. Here's the density, here's the fragmentation of the Christy and so on and so forth. And you can look with these techniques all the way through individual subcellular organelles to begin to look at what's going on. Organelles to begin to understand what the changes are. Here are a couple of mitochondria here that look pretty normal. Couple others here, not too bad. This one is showing disruption of the Christy but I don't know why, unfortunately. The film won't play, it's played every time. John, I'm sorry for interrupting you. So if you try to switch your cursor back to mouse mode, like by going again, right click. Okay. And what should I? Point or options. Use what? Go to pointer options. Yeah, okay. I've got the options here. Yeah, so like there's one that says point. Okay. So right click. Yeah. Doesn't seem to want to do anything. Yeah, no, we can try to remove the laser pointer. So go pointer options, do you want below? Do you want below? Right. Not then so, pointer options. There is one option that says pointer options. I've got the options, yeah. And so I should go down, yeah, okay. Yes, pointer options. Choose the pointer options. I see all the options right here. Yeah, and there is one called pointer options. Right. So choose this one. Okay, pointer options. Okay, very good. And then go to arrow. Okay. And now hopefully your video will play. Try again, please. Yes. There it is, very good. That was excellent. Okay, well here we are. This is an example of serial section, connectomix being able to analyze a single subcellular organelle. We were looking at that. Yeah, okay, that's perfect. All right, here's another one up here. Oh, you can't see that though. I lose my pointer, I guess. What's around here that is probably glial cells because we're well inside the mactel zone, probably muller cells that are degenerating. Okay, so here we are. And we'll look at it again, but just to give you a sense of the ability that you have with this connectomix technique of looking even at subcellular organelles and what's going on with them. Okay, okay, well we can leave this now. What's going on? What we would say from what I've shown you so far is that the initial changes that occur in mactel are in the mitochondria. And the closer in and particularly the mactel zone, we saw much more dramatic changes and it then suggested to us that this could very well be a mitochondrial disease. So what might these mitochondrial changes relate to? I mentioned earlier that in the central retina of mactel patients, there's a serine deficiency. And it turns out that serine is critical in forming various lipids, ceramides, finger lipids, et cetera, that are essential for mitochondrial function. So a paper laying out all of this is shown, uh-oh, on the next slide. And we're not gonna spend a lot of time on this and this would sell culture, but what they were able to do, okay, let's see if I can, I'm gonna have to go back and right click again and then get printer pointer options. Okay, here we go, laser pointer. There it is, okay. So this paper, which is a study of cells and culture, what they were able to do is show when you deprive the cells of serine that it had profound effects on the mitochondria. The mitochondria tended to break up and this resulted in the cells losing their ability to proliferate and so on and so forth. What they didn't do, unfortunately, was to look at the cells with electron microscopy. But what they're showing here is that serine is critical for making a variety of lipids, including the ceramides and against finger lipids. So what our guess is, is that the loss of serine that occurs in the retina is responsible for the mitochondrial changes. And because once in the Mac-Tel zone, the mitochondrial changes are so dramatic and cause so much of the generation of the mitochondria that this is what is causing the disease. That's a hypothesis at this point, but do we have any evidence backing it up? Well, we do think we have some evidence backing it up. And let me move on to the next slide. If we look, for example, at the Mac-Tel zone transition, here we are, this is the edge of the Mac-Tel zone. And then we look at the boundary between inside, which is devoid of macular pigment, and outside, which has normal macular pigment. What we see is that there is a very clear-cut transition zone consisting of some degenerating material, which I'll show you in a minute, and cells that usually are not seen in this region. What I wanna focus on is that if you look over on the left-hand side, i.e., inside the Mac-Tel zone, okay, which we know is depigmented, if we look carefully at the higher power of a region, we see that the Mueller cells, the glial cells that usually separate the cone and rod axons, the so-called Henle fibers that are responsible for carrying information in the central retina from the inner segments to the terminals of the photoreceptors, and always in normal tissue, there is Mueller cell, glial cell, that separates the cone and rod, so-called Henle fibers, and I'll show you more about this in just a minute. None of it is there. The cone and rod Henle fibers are chock-a-block up against one another. On the other hand, on the other side, outside of the Mac-Tel zone, if you look with higher power magnification, here's what you see. Here's a cone Henle fiber, here's a rod, and see the Mueller cell cytoplasm separating the two types of Henle fibers. I'll show you first the transition zone. There's an area of degenerating tissue here. We're not sure what all of this is about, but then surrounding it are cells which ordinarily are not seen here. We've been able to identify these by serial section as being displaced aberrant photoreceptors, and we could do that by following in serial section these photoreceptors and showing that they do connect to what look like inner segments and even partial outer segments, as well as terminals. What they're doing here, why they're here, we don't know. That's something that we're still very much involved with analyzing. Now, if going back now, I wanna spend a minute talking about the zone in the area where the Henle fibers are, which is what we're looking at here, and say something about Henle fibers. And again, to begin with, I think this is something that you all know. And that is in the phobia, of course, the centrophobia. The inner layers of the retina are swept aside to allow light to impinge more directly on the photoreceptor, inner and outer segments. But what this means is that then from the cell body, a long axon has to be present to connect the central cones with their terminals, which start out here on the edge of the phobia. And these are the so-called Henle fibers, the photoreceptor axons. And this displacement of the terminals with the cell bodies and where they are continues well out from the macula. Indeed, here we are, a piece of human retina, a millimeter and a quarter from the centrophobia. We can see the rods and cones here, cone here, rods here. And I've drawn what a cone would look like, its inner segment, outer segment. And then here is its long Henle fiber that goes down to its terminal. And it's at the terminal that this cone connects with first bipolar and horizontal cells, of course. And then the bipolar cells carrying the visual information into the inner retina, the bipolar cell connects with the amicron cells and ganglion cells. And of course, as the ganglion cells run along the inner margin of the retina, forming the optic nerve. So if we take an electron micrograph in this region here, in longitudinal view, so that the Henle fibers are elongated rather than in cross-section, this is what we see. The lighter areas being the Henle fibers, the axons coming from the photoreceptor cells, with the Mueller cell being in between. And you can see every Henle fiber is separated by Mueller cell cytoplasm, okay? This is the normal situation. What happens in MacTel? This is what you see. The Mueller cell cytoplasm is pretty much all gone, there may be a little remnant here, but for the most part, the Henle fibers are adjacent to one another. What difference this makes in terms of physiology? I wish we knew, we don't at the present time. And whereas we see some of this outside the MacTel zone and vision is reasonably good, it means that these Henle fibers still must be fairly functional. We see the generating material here probably Mueller cell material is degenerating, but we don't really understand that. We still need to do more analysis, but almost certainly this is Mueller cell that is degenerating and so on and so forth. Okay, so what may be going on here in the MacTel zone? What is different between the MacTel zone and the rest of the retina? Well, something that is now becoming, I think, quite well accepted is that there seem to be two types of Mueller cells in the retina, at least the primate retina. The classic Mueller cell, which is shown here in a reconstruction that was done by Dennis Stacy in a project that Dennis, Rachel Wong and ourselves were involved with. This is the classic Mueller cell. Its cell potty is down in the middle of the inner nuclear layer. It then goes to the outer plexiform layer and then extends a long process up to the external limiting membrane. And of course, it's the junctions between the Mueller cell and the photoreceptors that form the external limiting membrane. And so this is an actual micrograph that was taken from this material, this being the external Mueller cell extending up between the photoreceptor cell nuclei. Okay, and here is a terminal of a cone, a second one here. But then what you see is what we have identified as a second type of Mueller cell, which ends at the level of the cone pedicles. And you can see it's in yellow here and it's covering pretty much all of this cone pedicle. Okay, and here's the reconstruction that Dennis made of this in which the cone pedicle is enveloped by the inner Mueller cell. And this is seen in the Mac-Tel zone and maybe somewhat beyond but that hasn't been determined as yet. But clearly in the Mac-Tel zone, we have a lot of these inner Mueller cells and they end here at the cone pedicle region and envelop the cone pedicle. Now, if we look for example at what's going on here in the Mac-Tel zone, here for example, is a terminal that looks quite normal. It's at the edge of the Mac-Tel zone. So the tissue still looks pretty well. Actually, I'm sorry, let me back up. This actually comes from a normal retina, okay. And what would be the Mac-Tel zone. And what we see is the cone pedicles are surrounded by Mueller cell cytoplasm. In this case, three Mueller cells are surrounding this cone pedicle, okay. And we would identify these as being the inner Mueller cells, okay. Now, what happens in Mac-Tel? And if we look at a low power Mac-Tel, in the Mac-Tel zone, you can see that these inner Mueller cells have greatly swollen. Here's the terminal here and the surrounding inner Mueller cells have enormously expanded. Here's one here, a second one here and a third one and so on and so forth. This is, and then you can see that above there seems to be very little in the way of Mueller cells cytoplasm and so on and so forth, which indicates that both the inner Mueller cells and even to some extent the outer Mueller cells are deficient early on in Mac-Tel. So what we're suggesting, and let me come back to that in a minute, is that it may be that the inner Mueller cells, which we know are concentrated in the Mac-Tel zone, as best we can tell at the present time, are the primary synthesizers of searing in this region. And so that there is a particular deficiency of searing in the Mac-Tel zone. And since we've already related the lack of searing to the changes in the mitochondria, the guess that we're making the hypothesis is that the inner Mueller cells, which are particularly affected early on in Mac-Tel, and they are the primary searing synthesizers. That is why the Mac-Tel zone is so sensitive to the lack of searing and why we see the major changes there. So again, as I said, this is only a hypothesis, but one that is attractive. So I've given you the major changes that are going on in the retina that we've been able to elucidate so far. But there are other things that we've made interesting observations on. So for example, we're very interested in trying to understand what's going on in the lesion area. And here is a micrograph of one of the lesion in the 48-year-old retina. And what you can see is that there are invading blood vessels in the lesion. And we've been able to show that these come from the intra-retinal circulation, which seems to extend small blood vessels into the lesion area. And then there are cells which are just chock-a-block full of degenerating mitochondria. Here's low power, here is higher power. Now in looking at all of this in some detail, not only are these retinal cells showing enormous changes in the mitochondria, but in some cases we can see pigment granules in these cells, suggesting that there are pigment epithelial cells that have broken off from Brooks membrane and are migrating through the retina to contribute to the lesion area and perhaps giving it some of the dark area that the lesions typically show, the darkness that they typically show. And indeed what we do know, of course, is that the pigment epithelial cells tend to break off from pigment epithelial cells when the photoreceptors are lost. So one of the things we've been looking at is the loss of the photoreceptors in the mactail zone. And this gives you an example. Here we are at the edge of the mactail zone where the cells still are fairly intact. You can see the outer segment seems to be more degenerating than the inner segment. And what you can see in the inner segment, the mitochondria show densities, some of them are swollen and so on and so forth. If you look in the very center of the mactail zone, the outer segments are in terrible shape. Here, for example, is one we could recognize as an outer segment because there are stacks of membrane which clearly we think represent stacks of membrane that we find in a normal cone. But you can see that the outer segment disks are breaking up into these small vesicles and so on and so forth. So again, what's going on here is that we're losing than the photoreceptors. Now, I've been telling you so far everything about the 48-year-old. What about the 79-year-old? Is she very different in terms of the changes that are occurring in her retina? And the answer is no, but they're more extreme. So for example, here is some cytoplasm of a retinal neuron deep in her retina. And what you see is the mitochondria are showing exactly the same things that we saw in the 48-year-old. Densities, swollen, loss of Christie, so on and so forth. And if you look at her photoreceptors, again, what you see is, and this is even at the edge of her mactail zone, a retina where most of the outer segment is showing severe changes, this is likely a cone because if you look carefully out here, you can see that the disc membranes are conflict with one another. And this is the border between the outer segment and the inner segment. And notice again how swollen the mitochondria are and so on and so forth. And if you look down into the ganglion cell layer of the 79-year-old retina, you see enormous amount of degeneration spearheaded, we think, by the changes in the mitochondria. So where we now stand, we have very much more work to do. And of course, because of the pandemic, we lost a lot of time this past year is we are going to be focusing more on the lesion areas, the generation of the photoreceptors in both the 48, the 79-year-old retinas and trying to put it together. So our major conclusion to end this talk is that we believe mactail is primarily a mitochondrial disease. And it's caused, we think, by syrinolact. It seems to affect all retinal cells. The mactail zone, which is where it the most degeneration occurs and the lesions that cause the blind areas is because of the presence of the second type of mular cell, the inner mular cell. So this is where we are with the study. More analyses are needed, especially of the lesions and of the 79-year-old retina. But what we're also hoping is that study of the 79-year-old retina will tell us something about aging changes that take place in the retina. So let me leave it there. Be glad to answer questions. And I hope that you, I appreciate the attention that you have paid to the talk. Thank you very much. Bye-bye. Thank you very much, John, for this very interesting presentation. At this point, I would like to ask you to start your video again. I already sent you a request for that. Okay. I can start it. There it is. Okay, I'm back. We cannot... Not quite. All right, now I'm back. Great, now I'm gonna see you. Very good. I hope that was intelligible. Yeah, so as people are already starting to post some questions in the chat, I will also make the Zoom Room link available so they can also join us in this very room if they wish. That's already one question from Simon. So I will start with that. Great talk. Very much appreciated. Do we know why serine reduction is associated with the degeneration? Can you repeat that once more, George, because I was manipulating, yeah. So do we know why serine reduction is associated with degeneration? Well, okay. Serine is required for synthesizing a number of important lipids that are required for retinal, particularly mitochondrial function. These lipids also may be important in the outer segment disc formation. We're not sure about that, but clearly I think the relation between the lack of serine and the loss of mitochondria, which I illustrated by that paper that was in cell reports that showed that in cell culture that with serine deficiency, you lost the ability to synthesize ceramides, sphingolipids, and other lipids that are critical, particularly for mitochondrial function. And in those cell cultures, those fragmentation of the mitochondria and so on and so forth. That's why we think primarily a mitochondrial disease caused by the loss of serine. And what we're proposing, of course, is that the serine is being synthesized mainly by the inner Mueller cells that we know are concentrated in that central macular region, which we call the mactail zone. A lot of that is still hypothesis. We're working on it. Thank you very much. So we'll continue moderating the discussions with questions that appear on chat and that we keep in mind for later on. Karola Jovanovic asks, would you say that alterations beyond mitochondrial damage start earlier in photoreceptors than in other cells? Well, it may very well be the case. I can't say that yet, but it's always been viewed that mactail, like other retinal degeneration, start with the photoreceptors. In this case, if anything, what we see is the changes in the mitochondria occurring postsynaptic to the photoreceptors. We see them most prominently early on in horizontal cells. And then if you look through the rest of the retina, as I showed, you also see changes in ganglion cells and amicron cells and presumably bipolar cells as well. So it's going to be interesting to see. It's always discussed as mactail having a loss of photoreceptor cells. That clearly occurs, but the other cells are compromised very early on as well, which makes it an interesting retinal degeneration. There is another question, I believe asked by Tom, but you have already touched on it. So he says you focused mostly on the outer retina, but presumably you have the whole thing. Can you summarize what, if anything you see on the other side, like retinal ganglion cells? Well, we see changes in the retinal ganglion cells, okay? As well as the amicron cell, I call it. We see everything. And we see these mitochondrial changes in every cell type, which is what you would expect because searing is required to maintain both structurally and functionally mitochondria and all cells. So that's probably not a surprise, although what had long been said was that it was primarily mactail was a disease of the photoreceptors. That may not be the case. It may be that the photoreceptor outer segments are particularly sensitive to a loss of searing. I sort of suggested that. We can't say that unequivocally, but of course, there are a number of unique lipids in outer segments. And of course, in searing is so critical for the synthesis of various kinds of lipids. That might be why the outer segments are particularly sensitive. And if you'll notice in one of the micrographs, whereas the outer segment looked very degenerate, the inner segment looked fairly good. Okay. Yeah, if I'm allowed to add like my naive and definitely not well informed opinion, I think like your assumption slash speculation is quite right in the sense that if it's like a metabolic issue and cones do require like elevated amounts of energy to function than rods, then having it in the mactail region where you have so many cones, kind of pin points to the right direction. And I think there was like a Gordon Fein and Sampath's paper from last year claiming that cones do need way more energy than rods. One question I have like with respect to the medical part of mactail, when is the symptoms onset? Like when do people start noticing the difference? Well, it varies a considerable extent, but it's primarily a late form of age-related macular degeneration. Usually it's people in their 20s and 30s that begin to show the scotomas and the blind areas. And they also show what is called metamorphose. That is that the straight lines look wavy and so on and so forth. And many patients with mactail do very, very well into their 70s or even their 80s, but there are some fast progressors in which even in their 40s become virtually completely blind. As long as a lesion doesn't appear in the phobia itself, you can get along pretty well with mactail. But if you have a lesion in the phobia area, then of course, you lose all of your high acuity vision, which is what you would expect. I mean, it's interesting that the 79 year old, I showed you her lesion, we haven't really been able to study her as yet, her retina as yet, but her phobia looks like it's fairly intact, even though there's lesion all around. So that'll be interesting to look at, how normal her phobia is. So if I understood correctly, the mactail zone kind of coincides with the border of the phobia, right? Like it's not at the center of the phobia itself. No, the phobia usually is on one side. Mactail usually starts prominently on the temporal side of the phobia. This, of course, would be where the center of the mactail zone is. And I assume we do have some rods there, although in really, really small mactails. Yeah, I mean, the very center of the phobia, of course, is all cones, but as we move away, you know, several hundred microns, four or 500 microns, we begin to see rods appear. And do you observe the same, the center of the structures? By a millimeter from the center of the phobia, there are lots of rods. In the micrograph, I showed you of the human retina, which is a millimeter and a quarter from the center of the phobia. There were at least as many rods, if not more than there were cones. On this, the highest rod density in the retina is just adjacent to the phobia itself. And you observe the same damage to mitochondria within the rods, right? Right, yeah. Best we can tell both rods and cones are affected. Interesting. As we are slightly over time, I think I will be stopping the live broadcast any minute now. So I will post once again, the Zoom room link, if you want to follow up on the post talk, informal cheats that we will have, please do make sure you follow the link I just posted. I would like to thank the audience for being here. I would like to remind you that this is the premiere of our third season and we are continuing with the next talk this very Monday with Steve Massey and yeah, John, I would like to thank you once again for honoring us and delivering a talk in our series. Well, thank you, George, for helping me getting everything sorted out. And I think it worked well. Hope people enjoy it. Yeah, so what I failed to communicate to you is that a lot of people were also thanking you for your talk in the chat. I only started with questions, sincere apologies for that. So I will be stopping the live stream now and officially we are offline.