 here is Brian Jones and he's a research associate professor at G. If you think that was cool, wait until you see this slide. I'm really raising the bar high for him here. But he's going to talk about some retinal remodeling and some of the degenerative diseases of the retina. We don't do a lot of translational work per se. So I'm not going to pull up with you any active translational work that's going on in the lab. But the work that is going on has massive implications for a lot of translational work that's actively going on right now around the world. So thank you, buddy. So we study the retina, the back of the eye, or you guys are sort of used to this view. We're interested in what happens in two diseases. A retina is pyrotosa and age-related macular degeneration. This is sort of the classic view of R.P. We're going to sort of look at the histology a bit more. If you sort of zoom in, this is a backlit view from behind the globe sort of illuminating the retina in these different espicules. If you look carefully, you can see a couple of ganglion cells there. This is sort of another view up close. Another patient of R.P. I have a pigmentation. But again, a lot of these pigmented bone espicules. And if you look at these pigmented bone espicules, you have to sort of start wondering about the histology. So there are some tools that we can use clinically. OCT in this case, and it sort of can give you a view, this is an early case, of R.P. of what's going on in the retina. But really the gold standard is histology. So the important thing with histology, we're going to look at photos that are sticking up in these samples. It looks obvious now, but in a minute, it'll get a little more complicated. So the important thing to remember is a million has about 70 kinds of cells. So if you start making changes to the numbers of cells and their connections, it turns out it has a lot of implications into how information is actually processed. So light comes through, it's photoreceptors go back, photoreceptors do detection, and then transduce those signals through a set of circuitries that do ruins detection, contrast detection, movement detection, a lot of the sort of visual primitives before sending those data out to higher structures, cortex, and sub-portex for further analysis. The problem is, in disease like R.P. and AMD, these photoreceptors become compromised. And a lot of the community for a long time presumed that when these died, this structure or these structures romantically hang out in the way to be rescued. And so we've been beating this drum for about a decade and using animal models. And so what we can do with animal models that we can't do very well with humans is identify the gene, give them the disease gene, and then track the disease as it progresses through animal models. This is at an outstreadment, over 700 days, as it's remodeling. So the photoreceptors get stressed, the photoreceptor outer segments shrink, photoreceptor cell bodies start to die. Bipolar cells and amryprin cells start to die and move and do wire ganglion cells, migrate into the amryprin cell layer, and the whole topology of the retina changes. So this happens in every single animal model that we have looked at. Rats, mice, pigs. There's a very expensive transgenic pig that's available from Louisville. The last pig that I handled and took the eyes from was about $100,000 from birth to where we harvest the eyes. We have a rabid model now that's much more affordable, smaller, and still a large-time model. It's about 230 size of human eye. And it turns out it's a perfect gimmick for our somatomic hearty humans. But regardless, all of those animal models took this. And we've been hammering on this for years and are distressed by the number of human clinical trials that are going forward in biologics and biological and gene therapy and optogenetics that aren't taking this real biology into account. So we started collecting human tissues. This is a normal human retina. Actually, it's probably early AMD. It did not have a guide, most of us, when we got it. But the reason I say it's normal, probably early AMD, is this is one of the first things that we see in AMD. So this is the rebelpigment epithelium up here. And in normal, healthy, rebelpigment epithelium, you shouldn't see any color variability in this particular label. So this particular label is actually three separate labels that's taurine, the small molecule taurine, small molecule glutamine, and a small molecule glutathione that are assigned to red, green, and blue color channels. And that gives us this sort of view where we can visualize human cells in gold and the photoreceptors and some of the other cells. But it also allows us to see nicely the RPE here. And I'll show you in a minute, in normal, healthy tissue, you don't see any variability there. And we think that this is actually the first indication of disease and locations in dry AMD. Here's another view where we've changed the labels. This is GABA in the red color channel, for lysine in the green color channel, blue color channel. This allows us to see excitatory and inhibitory neurons in here. So the important thing with this normal human rep notice, even though there are some indications of disease, the overall apology is intact. Photoreceptors out here, you can see cone photoreceptors standing up here. There's got a nuclear layer, interplexiform layer, some inhibitory neurons and cells, and on-cone bipolar cells here and the ganglion cells down there with some vascular organs. Here's a normal, happy primate retina. We can, it turns out, we can get primate tissues, non-human primate tissues, faster than we can get human tissues. This was collected within about 20 minutes post-mortem and a lot of the signals are a little fresher, but the important thing here is to look at the RPE. So this signature, this fingerprint, it's what normal RPE looks like. And you can profile all the RPE cells in your normal human and you'll get absolutely this. So again, so if you keep this in mind, normal healthy retina, ganglion cells down here, here's the optic fiber layer down there. Again, we can switch it. This is the view that allows us to see excitatory inhibitory neurons. But again, normal lamination, everything looks really, really, really happy to healthy. This is central retina from a person with RT, about 74 years old, NLP, no visual process. The interesting thing is the IPL in this particular guy was largely intact. There are almost no, there's no rod photoreceptors, there are a few cone photoreceptors and this is actually important. We'll come back to one minute and this is Galaglice glutamate labeling. We, if we can get the tissue freshener off, it's still a lot. And we can do some experiments, we have a molecule called 1-UFO, or LARGO-UTENANT. It's a non-selective blue-leach channel marker. So we basically put the retina in each of you and stimulate the retina with pharmacologic succonic acid and NDA or with light, in this case, light wouldn't do much. And blue-leach channels open up, HV flows into these channels. And so we can get a visual picture for which neurons have been activated. So we basically recorded from every single neuron in this tissue, the more green in particular cell, the more HV that's flowed in. So we can do this HV labeling in addition to our other small molecular markers. And what we can do is we can generate a classification mask. And so what we can do is we can find all the horizontal cells, we can find all the ganglion cells, and then in an amortority, an amortority and clustering, you can leverage ganglion cells in different bipolar cell classes here. And so the interesting thing here is in a normal amortority and ganglion retina, the bipolar cell class, your on-combed bipolar, your off-by-polar, and your rod-by-polar, you should have about 33% of each. And so there should be equal numbers of, equal ratios of those bipolar cells. The first thing that starts happening in RP, we've seen this in animal models, and now we've confirmed it in humans, is that these ratios change. And what you see is you see the on-combed bipolar cells dramatically reduce the number, and the off-by-polar cells dramatically increase the number. So in this particular case, they've effectively doubled. And that's interesting. Even though the retina, even though the topology of the retina is intact, you can still see some comforters that are up here. And it turns out as long as cones are present, the overall topology of the retina is maintained, this is what's seen in the retina, cones disappear, that sort of restraint disappears. So here's another region of retina, and this is the YGP, or the TQE, just to slide that down. So again, torian glutamate, glutamate, glut-data, glycine glutamate, glutamate. So these are the same cells that you're seeing in both. We're just visualizing them differently so that we can see different things. And the first thing to look at is we're starting to get, so here's a retinopathylium, so it's died in this particular case. And then we can look down here. This is actually very interesting. Brad's doing the lab now. She's chasing this problem aggressively. So normally Mueller cells should all be the same color too. They all have the same small molecular. Interestingly, this is the case across species. So burns, turtles, fish, mammals. This sort of signature of this mammalian Mueller cell signature, the job that Mueller cells are doing in metabolism is very robustly made across evolution. But what starts happening when retinas get stressed is we start seeing this variability in the Mueller cells. So the metabolism in the Mueller cells starts stretching that metabolic envelope, and they start doing some very different things. We can start visualizing that here. So in these particular Mueller cell problems, there's more tooring in them, and so they're going to more red than regular sort of Mueller cells here. This is also true on the ultra-structural level. If you look at the ultra-structural data, but it's not gonna show here, there's lots of protein changes that start happening as well. And we can see that with electron microscopy. If you look at these boxes, so this one is just to show you that there are some cone photoreceptors, even though there's sort of nubbins, the nubbin in the outer segment is still left, but it's helping to maintain the overall topology of the retina. This box we're gonna zoom in on here. And the important thing here is to look at sort of these finding processes that are coming up from glycinergy-gamma cells. So normally glycinergy-gamma cells are here, and the processes come down, to connect in the center plexiform layer down here. In this case, they're starting to project up in the bone direction. We actually saw this in the animal almost first, but what they start doing is when you effectively de-affirmed the retina and you remove photoreceptor input, all the cells downstream have two choices. They can either die, which a lot of them do, or they start sprouting, and they start sprouting these processes, and they start talking to other neurons. And they do this likely to maintain calcium regulation in gene regulation. And there may be some other mechanisms for that, but this is a very common point in this sort of scrubbing. Turns out if you look at the ultrastructure, there are synapses in these spreads, so these are not sort of quiet processes. They're active processes where these cells are funding other partners that they shouldn't be talking to. Things can get really weird. These are amicron cells in the subredimal space. So these are cells that they migrate up from where they normally live, and they're now taking up residence right here. So in addition to sort of the sprouts, the cell bodies can migrate and go in weird places. And then we've taken a punch here, so this is a three millimeter punch from human RT, and so look at the red strip here. We basically took a histologic slice right out of there, and we wanted to look at far periphery retina. So the idea is that the data that I showed you before was sort of more central retina, and because RT is sort of the periphery of the central, the disease, we wanted to see how bad it could get. So we looked at OCT, not a whole lot of OCT signal, not a whole lot to see, but if we look at the YGE, there's basically a lot of vascular components, the vascular components are sort of in hypertrophic. There's almost nothing left of the normal retina. There is nothing to see, which is why you couldn't see anything in OCT. There are some mule glia elements here. Turns out there's some astrocytic elements for some other work that I could play for the grad student to learn about is what we're currently chasing. But almost all the ganglion cells are gone. A lot of the gavagy inhibitory neurons are gone. Collisionage ganglion cells are still left. Most of the bipolar cell population is gone, and all of the bipolar cells are effectively gone. This is, for all intents and purposes, a dead retina. There's no rescue in this one. These are other mappings. This is toward gliobrimary. I'll show you sort of the glial components. They're still there. There's some proteins, G-FAT, C-R-L-V-P, and arginine. We're sort of trying to look at some protein. Which proteins are still left? So small molecules change much more rapidly than the proteins do. And this is a probe for adopsins. So there are no, so normally you should be able to see nice, pretty photoreceptors up here. The only oxins that we can see are sort of trapped in the retina's vascular elements. They're probably sucked down and failed metabolize. So this is another sort of stretch of peripheral retina. Again, just to show you how bad things can get. So this is a little distance away from that red stripe that we showed you earlier. And again, this is what a normal retina and normal retina thing should look like. So if you sort of take this bottom edge, this vitriol edge down here, and sort of go up, this is where the disease retina stops, and that's how much further it should go. So the idea is in IP at least, this is completely true. And this is sort of a, we took a horizontal section through the retina, through the mule or cell. So we sectioned into the screen just a little bit more. We'd be into the vitreous. This is just before we get to the vitreous. So these are all of the mule or cell goofy. In normal, happy, healthy tissue, you should see this nice sort of mosaic tiles, which all be the same signal. This is clearly disrupted. There's a lot of stretching going on. There's some vascular elements that are sort of pushing in. And the signatures are all different. So this is glutamine sympathase toward the glutamine and red-green blue. And again, this is showing that there's effectively metabolic chaos in the mule or cells. And this is sort of another question at what point. So sort of mule or cells are really important for maintaining a normal homeostasis in the retina. They help extremism respond more to the components and recycle a lot of components for their translators. So the question arises, how far can you sort of push the metabolism in the mule or cells and expect retinas to be able to function normally? This is another mapping of chlorine that just to show you again, the diversity gets pretty crazy. So the other piece of paper that we just published was AMD. So this is sort of the classic view that a lot of you guys look at. And I sort of read the introduction on what happened. From this copy, we can sort of zoom in again and we can see these nice sort of pre-indruising form and some sort of pigment structures here. Here's some histology. There's some nice cuts to here, I apologize. But again, it's sort of directing the RPE. Again, it's sort of a tiled appearance, which would be absolutely smooth and influence signatures. There's a small groove right there. This is very early. This is Tori and Lacy, we were finally mapping. And this was just looking at Tori. So this is just looking at the RPE here. So here's the vascular chloride. And here's some putters that are outer segments. This was a cruel image because it shows you the tiling that goes on. So each one of these is separate, retinal pigment that would be in themselves. And normally, they're coupled. So they're gap junctions between them. And that allows the small molecule signals to flow in between them. And that's why the signature is absolutely reforming the normal level of tissue. What's happened is the gap junctions have become uncoupled. And each one of these cells is doing their own thing. And we suspect that leads to dysfunction. And so what we're starting to see, we don't quite know what they are, but these are Tori rich deposits, sub-retinal, sub-fruzine components. Who knows, actually. There's some argument when we published this paper, there was actually hardly a black and port with three of the reviewers, exactly what these were. We don't quite know, but what we think is that it's fail outer segment metabolism. So the RPV is no longer phagocentrism, effectively the outer segments of photoreceptors. We're starting to get debris that builds up underneath the RPV, which is an exciting stress. And I'll list all that. But it's just the arrow that goes across. Here's some other early AMDs. This was OCT, not great. But here's the histology with Tori. Again, we've got some nice bruising deposits. And so the question is, what's going on underneath the sub-fruzine, at least sub-retinal underneath the bruising deposits? Again, you can start to see the RPV. It's becoming uncoupled in and around the cells. And then we can start to see changes in metabolism. So this comb photoreceptor here, did I highlight that? Yes, this comb photoreceptor here has a very different signature than the comb photoreceptors on the other side, the same in this box as well. So we're starting to see the earliest signs of metabolic dysfunction that comes underneath these bruising deposits. So something is happening to the RPV, and it's causing downstream cell stresses in the photoreceptors. Here's another really AMD, again, variations in the RPV coupling. This is looking at oxen, a common oxen. So when the rules start happening is you get a build-up of debris underneath the RPV and an oxen de-localization. So normally, the oxen should be up in the color of the segments. It's de-localizing down around the inner segments and down even to the synaptic input, where it synapses with bipolar cells. This is sort of an indication of the cell stress. And another image of early AMD photoreceptors are starting to get shorter in this particular line. But down here, we're starting to see the same signals in the Mueller cells. So Mueller cells, again, like RP, the Mueller cell signals are starting to diverge, and they're no longer uniform. As you know, we'll look here to sort of show you the signals that are starting to change. We don't know if this is a dynamic process. These are just individual snapshots in time. And we don't know if metabolic cells are ringing, if metabolism is toring or ruining these cells. It's coming and going in waves, and we're just capturing into this particular cell with up-regulation of toring, while in this particular Mueller cell, right next to it, as normal toring. We don't know that yet. This is important because it shows, again, in early AMD, early to mid-AMD, in this stretch, we don't have any drusen, but there are some drusen. Other areas, the glycogenome consuls are starting to spread again. And so in this cell, this patient still had vision, but the visual performance was likely declining. So anal consuls and porzonal cells do a lot of sort of spectral tuning of signals, help you sort of refine the visual performance. And this may be some of the earliest sort of cellular histologic evidence that the circuitry in red is breaking down. Oh, yeah, we can sort of zoom out to show you some of this spreading a little bit better here. This is GABA here. So these are GABA-regulated cells. So GABA-regulated processes are coming from evidence cells going along the road. Here's a case of late AMD. We're starting to get some vascular involvement here. It's going to be a sort of healthy drusen. There's the GABA-blessed glutamate signal. And we're going to zoom in on it. Very here, we can start to see photoreceptors are dying off and being lost. The IPL is getting a little more loose. We're starting to see changes to the retina underneath the same thing in new cell cells. You can't really see with this projector, but the signals are starting to change in the nutrients as well. So the model that sort of holds in AMD, RP, and any other disease that causes photoreceptor cell death is that there's sort of happens in phases. Photoreceptors get stressed. The other segments sort of shrink, and rods are lost. As long as the cones are present, the overall topology of lamination is good. Cell populations seem to be intact. There may be some spreading that's starting to occur, but everything looks pretty good at this point. By phase three, when cones start to die, we get early neurotic modeling from GABA-regulated glycerinic amicron cells. Horizontal cells actually also aggressively spreading this as do ganglion cells. And then by latent phase, there's local remodeling. There's massive cell loss, massive sort of topological destructuring of the retina. We've done a lot of molecular work in these phases. So in phase one, we get a variety of genesis. We produce photoreceptor cell markers in phase two, which is the O and O elation. We get changes in the glutamate channel expression. I'm sure these are some of those data, but we also get changes in retinalic acid signaling. And once the cones start going, a lot of these signals continue. We get changes in glutamate channel expression, or two receptors go up, or five receptors go down, or more than six receptors go down. And then we start getting changes in proteins as well. This is an area that Becca's chasing for her PhD dissertation right now. It turns out there are some massive changes in the glutamate synthetase, and once GS disappears, it never comes back. And so there's a real question in late-stage disease if you can even rescue a retina once the glutamate synthetase, which normally turns over to a receptor, or neurotransmitter molecules, goes away. So the translational component comes because a lot of people are looking in ways that sort of remain. And bionics is one of the big sort of approaches. So there's a couple of ways to do bionics. One, you can sort of put a bionic implant underneath the retina, and you can surgically detach the retina, slip the bionic implant in with the idea that you stimulate the surviving retina. The more diverse, the more successful, in terms of sort of the market, penetration seems to be the under-retinal implants, but they're putting down the ideas to stimulate the ganglion cells. The problem is there's a field effect. And so you have to sort of ask yourself, which cell populations are you stimulating? And you can say, OK, well, we're only verbally stimulating the ganglion cells. It's still a problem. There are also 12 to 20 channels that outflip the ganglion cells to, and it just doesn't go to a visual cortex as opposed to a subcortillary as opposed to a super-collecular cell gene in a lot of different places. So you're sort of stimulating these ganglion cells and discriminating them in a lot of cases. Well, in all cases, we're using bionic implants. There are some other approaches to do optogenetics. You've done some of these studies where you can take a virus and engineer it in an opsin. Basically, if photoreceptors disappear, you can put the opsin in surviving cells. And the idea is the light comes into the retina. It's these cells that have been genetically transduced so that you can express the opsin. And then the thinking is that these cells then stimulate surviving retina. The massive assumption here is that once you do this, that the retina will stop degenerating. And it turns out we did some early experiments and retinal remodeling in this process that we call this sort of plasticity. It's a freight train. And because you put a little plastic one from the freight train, it doesn't stop the freight train. The freight train is still going down the track. So we intervened with optogenetics. And we waited and tracked the animals for a couple more years. And the retinas just wanted to help, even with the optogenetic varieties. But the assumption is that you can stimulate bipolar cells, you can stimulate amycones cells. This can say two amycones cells. Or you can put in optogenetics and ganglion cells. And there's a lot of very cool evidence that this works. In fact, the first guy that did it really got kind of drunk around this guy and went to State University. It was the first to actually show that you can do optogenetic therapy in retinas. And he largely got the award. He presented that at Argo in 2005. And it was the single coolest thing I saw that year at Argo. And Stanford is a little better at promoting their people. There's some very talented people. Actually, Ed Boyden is wonderful. He's now at MIT. And Carl Fisseroth really got most of the fame for actually doing this optogenetically. And there are a lot of reasons why that happened. But Pan was the first guy to actually do this. So the problem with all this is, even if you do successfully transduce these cells, the cells die. The cells rewire. And this circuitry sets great changes. So then the question is, how bad does it change? And the other main mission of our lab, this is sort of Robert Marks, maybe, was that we studied how more wet than it is required. So these are two A2M cells. And it turns out there is a very specific structure. There are very specific synapses and gap junctions that all of these cells make with other populations of cells in the retina. And it's very tightly constrained, the signal, the cell partners that they normally partner with. And so it turns out we can identify these actually, we can go in and pick in our connectable volume, and pick synapses and gap junctions and actually visualize them in the electron microscopy. And we can start this and make these beautiful sort of figures. And then we can sort of make these connection diagrams. So here's one of those A2M cells here. And we can start mapping out broad bipolar so that when we put onto it, there's another gatherage of the energy that we put. And then we can sort of start mapping this out. The trick is it gets really complicated. So this is just two synaptic hops away from that A2M cell. And so if you start asking yourself the retinal degeneration, which one of these connections gets broken, and how many of them can you break and still expect normal retina signal processes to occur, that's kind of what we're at now. We're trying to figure out what goes wrong, where. And then there's a lot of modeling that can be done to try and figure out how bad can we get at what point can we no longer intervene with biologic approaches. Just wrap this up real quick. So we've done some biologic work with people. We've done some optogenetics work. And we've done some cell-based work. Turns out underneath biotics, the modeling is accelerated in a lot of these cases. And this is distressing. Optogenetics, as I told you, the modeling progress just precedes, just keeps going down the track. We've done some cell transplants. Turns out the modeling is accelerated in transplants. So we haven't done a lot of genetics yet. There's some other experiments that we'll be doing both in. And the question's open there. The basic take-home message is that there's a lot of rules that are happening that we need to understand what the rules are. This is the lab, these are some of our collaborators.