 Why don't we go ahead and get started here? I'd like to introduce our distinguished speaker, Dr. Jim Hurley. He hails from the University of Washington. He's had an interesting career. He started out in environmental sciences and forestry. He studied plant metabolism. He earned his PhD from the University of Illinois in biophysics and physiology. He did three postdocs in California, one right after another. And now he is professor of biochemistry, an adjunct professor of ophthalmology at University of Washington. So Jim has been interested in answering fundamental questions in neuron phototransduction. He has over the years identified and characterized a number of different genes that are critical for vision. And more recently, he's been interested in photoreceptor and RPE energy metabolism, and that's what he's going to talk about today. So we know that photoreceptors are incredibly energy consuming cells. And one of the most fundamental questions is how do they wrestle energy away from the RPE? How do they harmonize to efficiently get enough energy to enable vision? So without much further ado, his title is the importance of nutrient supply and energy metabolism in the retina and RPE. Thanks, Lawrence. And thanks for the invitation to speak with you guys and meet with you guys. So what I'm going to try to do this morning is to sort of bring you guys up to date on our current thinking about energy metabolism in the retina and the pigment of the thylian. And so the bottom line is going to be that the, as Laura was saying, that the most, a lot of nutrients come from the coronal blood flow. And to get to the photoreceptors in the retina, they have to go through, in the rest of the retina, they have to go through the pigment of the thylian. So what we've been sort of realizing over the past several years is that these, the energy metabolism in these cells is actually very specialized. So that each cell has a sort of specific function in this ecosystem, if you will. And that if any one of those energy metabolism, energy metabolism in those cells fails, then the whole ecosystem could fail. So that's sort of the bottom line I want to be able to tell you about. And I'm not a clinician, but since this is a basic, a grand rounds, I thought I'd start with a few case studies. And so these are, so what I'm going to tell you about next is several different lines of mice that people have made over the years that over the past 10 years or so, that gave sort of puzzling results. And so I'll go through those and then I'll tell you a little bit about what our model is for this retinal ecosystem and how from that perspective, those results make a little bit more sense. So the first, so each of these mouse models had a, you know, the investigators made a specific defect, a genetic defect in the mice. And so this one, the mitochondria in the pigmentophilium cells were disabled, but the surprising finding was that the photoreceptors suffered as a result of that. And in this mouse, another group enhanced glycolysis in the pigmentophilium cells. I'll review a little bit about energy metabolism in a few minutes, but in the first case, they disabled the mitochondria. In the second case, they enhanced glycolysis so they used more glucose, but it's a result of that, the photoreceptors degenerate. And then in the third example I'm going to show you, the rods degenerate because they had a mutation, there was a mutation in a rod-specific phototransduction gene called a cyclo-GMP, it seemed like causes the cells to degenerate. But in that case, there was a, the glucose didn't actually make, couldn't actually make it through the pigmentophilium, get stuck in the pigmentophilium. And then in another model, the rods degenerated also because of a rod-specific gene. But in that case, even though the defect was in the rods, it's the combs that actually are suffering because the combs are starving and not getting nutrients. And then in the final example, there's a retina where there were fewer than a normal number of photoreceptors. And so, but the photoreceptors that are there, even though they're perfectly genetically normal, they don't grow properly. It seems like they don't get enough nutrients. Let me go through some of these examples. So this one is a mouse that was made in Doug Volras lab at Stanford. And what they did was they knocked out, they inactivated the gene for T-FAM, which is a mitochondria-specific, it's a transcription factor that's required for mitochondria. And so as a result, the mitochondria were dysfunctional. They were small and barely there. And the pigment epithelium. But what you can see is, so as a result of that, the pigment epithelium was abnormal and it de-differentiated somewhat. But the really striking finding was that the photoreceptors were stunted. They didn't have the four-hour segments and they degenerated. But remember the defect, not the photoreceptors, the defect was in the pigment epithelium cells, okay? So that's one example. This just shows the, I think these are the ERG responses and in the normal and in the mutant ones. So these are coming from the photoreceptors again, even though the defect was in the pigment epithelium. Okay, and then another example this was made in Marty Friedlander's lab. And in this case they knocked out a protein called the Vontable Lindow Factor, BHL. And that's a protein that's required for degradation of something called a hypoxidic dismal factor, which is something that enhances glycolysis, enhances transcription that promotes transcription of genes that are involved in glycolysis using glucose. So when they knocked out BHL, the pigment, specifically in the pigment epithelium, the RB cells became more glycolytic, okay? So did not do anything really damage the RP, you just make it consume more glucose. But as a consequence of that, the photoreceptors also suffered. So it resulted in photoreceptor loss, there's fewer hard nuclear layer nuclei for the photoreceptors. And it's just showing the ERG responses also again, indicating the photoreceptors are functioning properly. So the two examples where you altered the metabolism in the pigment epithelium, but the photoreceptors were the ones that suffered. And just as a review, because probably I don't think about this all the time, just want to remind you a little bit about energy metabolism. So of course, glucose gets into a typical cell through a glucose transporter, goes through the steps of glycolysis. One of those is glycerol triphosphate dehydrogenase uses NAD and transfers electrons from the substrate to NAD make NADH. And also it produces a couple of ATP's in glycolysis. And then the final product of glycolysis is pyrophate, under this from biochemistry classes. So the final product is pyrophate and pyrophate can be reduced to lactate by the NADH. That's one path that it can go through. The other path it can enter through a pyrophate carrier protein to get into the mitochondria. And then it can go through this thing that you learned all about called the specific acid cycle. And in the process, all those carbons get oxidized if you have to go around a couple of times. All the carbons from pyrophate, pyrophate is three carbons, get oxidized to CO2. And eventually what that does, it transfers electrons to NADH, that NADH is used to pump protons. Remember you get a proton gradient and proton gradient drives synthesis of ATP. And the electrons from the NADH, electrons from these molecules that are originally on pyrophate that are gone from the CO2, those got transferred to oxygen to make water. Because that's your review of glycolysis and respiration. And so the interesting thing about photoreceptors has been going for a long time is that photoreceptors are extremely glycolytic. There's back in the 1920s, there were a couple of investigators out of Warburg and Pond's Krebs who were studying this. And they found two tissues that were extremely glycolytic. They had to say, most of the carbons from glucose and could convert it to lactic. One was tumors, and the other was retina. And so in the retina, it's the photoreceptors that do all this glycolysis. Because that's a little bit of background. So let's keep going through these examples then. So Doug Dean and Hank Kaplan at the University of Louisville made a mouse. Looked at a mouse that had a mutation in rhodopsin that caused the rod photoreceptors to degenerate into a 23-hiss mouse. Maybe some of you know about that. And what they did was then they injected a fluorescent molecule of glucose into the mouse. And so that fluorescent glucose can get taken up into cells through the glucose transporter and it can get phosphorylated. When glucose gets taken up into cell and gets phosphorylated, it's trapped inside the cell. And if the glucose is fluorescent, then you can see the cells become more and more fluorescent. Okay, so normally when they did this in a wild-type mouse, what they saw is that maybe a little bit of, so the glucose was coming from the quarry here through the pigment epithelium to the retina. So glucose would be flowing this way. It makes pretty well through the pigment epithelium and it gets taken up and trapped in the inner segments of the photoreceptors, like I said, that's what you normally find. But when the photoreceptors die, then it turned out that the glucose didn't just pass through the archaea and get into the retina. It got actually trapped in the pigment epithelium. So that's another piece of evidence that there's some kind of metabolic interaction between the photoreceptors and the pigment epithelium because if the photoreceptor's not there, the pigment epithelium just takes up the glucose and traps it in the pigment epithelium itself. And then another observation, this one was from Claudia Buzzo, who was post-diagonal in Connie Septo's lab, was that in a mouse, this is a Phosodestrius mutation, the RD1 mouse, but they were monitoring phosphorylation of the mTOR protein. And mTOR gets phosphorylated when nutrients are available. And so what they're showing here is that when the red is the phosphorylated mTOR, indicating that the nutrients are available, that the cones were getting glucose. But in the RD1 mouse, where the rods degenerated, that the cones were still there, there's no longer a strong red signal. So the cones, even though they're genetically normal and healthy, they're not getting nutrients. It's probably because the rods aren't there, okay? So all these are evidence of interactions between these cell types and the reds that they're doing. Last example I want to give you is, this is an experiment done by Steve Sang, and here is the reddish-latently radopsin, and red here is lately the rod-specific Phosodestrius. And this is another Phosodestrius mutation, it's not the RD1 mutation, it's just another similar one that degenerates a little bit more slowly. But as you can see in these retinas, all the rod photoreceptors are degenerating. But then he worked out a way to rescue some of the rod cells. And so it used a CRE to activate a CRE enzyme that activates a gene encoding the Phosodestrius. And that CRE is regulated by the amount of tamoxifen that you inject into the animal. So the more tamoxifen that you inject into the animal, the more photoreceptor cells activate that CRE and activate that Phosodestrius gene. So you can rescue either no photoreceptors, a few photoreceptors, or a lot of photoreceptors. And so that's what you see here. So this is 30% of the rods are rescued, 50% of the rods are rescued, 70% of the rods are rescued. And what they saw was not only were there 30, 50, and 70% of the rods rescued, but the more rods are rescued, the healthier and bigger the rods looked. Okay, so the more rods that are there, the more support they're getting, seems the more nutrients they're getting. That's another sort of puzzling observation that indicates that there's these metabolic interactions. So I'm not gonna go through now about how we came up with this idea for this metabolic ecosystem in Redna, and I'll go into that in more detail if you can make it to the seminar later today. But basically what we're proposing, and we think there's pretty good evidence for this, and I don't think it's by any means proven yet, but our working model is that glucose comes from the blood and it has to go through the pigment epithelium. There's tons of glucose transporters on both sides of the pigment epithelium. The glucose gets through, it goes to the Redna, gets taken up by the photoreceptors. As Warburg and Krebs discovered many years ago, the photoreceptors are very, very glycolytic. They convert the glucose to lactic acid. But so now that if the glucose is going through the RPE, what's the RPE gonna do for nutrients? If it's not using any of the glucose? So what it can do is it can use the lactic acid that's produced by the photoreceptors as fuel. But to do that, it needs its mitochondria. So that's why the mitochondria is so important for this metabolic ecosystem. Mitochondria and the RPE are so important for the metabolic ecosystem. There's also another interaction with the viewer cells. I'll talk more about that later in the seminar later today. So from that perspective, then, these kind of make sense if you think about it from that perspective. So when the mitochondria and the RPE cells are disabled, the RPE doesn't have any way to extract energy from the lactate. And so instead, it uses all the glucose before the glucose can get to the photoreceptors. And so the photoreceptors would starve. If glycolysis is enhanced in the pigment epithelium cells, then the pigment epithelium cells are going to use the glucose before the glucose can actually get to the retina. If the rods degenerate because of mutation, then the rods aren't here to make lactate. And so there's no fuel for the RPE. So the RPE desperation is going to use the glucose for itself. If the rods degenerate again, the RPE is going to use the glucose for itself. And the colons, which were genetically normal, will starve because the glucose is being stuck. It's stuck up here in the pigment epithelium. And then finally, if you have fewer rod photoreceptors here, there's fewer rods making lactic acid. So the pigment epithelium cells need energy. They don't have lactic acid. So they're going to use all the glucose for themselves. For my generation, you might say that they're going to bulk-art the glucose. OK, so that's our hypothesis then. It's that the pigment epithelium does best when it minimizes glycolysis and when it's mitochondria are most active, right? You don't want it to use the glucose. If you want its mitochondria to be very healthy so that they can use lactic acid and not use glucose. And rods do best when they maximize glycolysis so they can make as much lactic as they can. And so there's a couple of applications of this. A couple ways of thinking about this in relation to disease. One is what happens in aging. And it turns out that there have been several studies down. This is, I think, one of the best. That was a morphological study back in 2006 of RPE cells. They're looking at the mitochondria in RPE cells from young donors and the aged donors, old age donors. And then if you look carefully, you can see that the mitochondria here are fairly robust and healthy-looking, whereas here they're looking sort of ratty. Something is wrong with them. It's not really clear what. But morphologically, they don't look normal. And they actually did the study very quantitatively. So these are from the donors without donors that are affected by AMD of the macular degeneration. And this is a function of age. And you can see, even in the normal donors, there's a decline in number of mitochondria, the number of chrystapine mitochondria, the overall area of the mitochondria, declines with age. But it declined faster in the AMD and the donors that had AMD. So there's something, you know, mitochondria and the RPE are deteriorating. And by our model, that's a bad thing, right? Because mitochondria have to be functional in order to allow the RPE cells to not use glucose. And then Deb Farrington has done some nice studies where they looked at, they used a method to estimate the number of mutations in mitochondrial DNA. Mitochondria have their own genome. And so it's possible to estimate how many mutations have accumulated in mitochondrial genome. And she saw a correlation between the severity of macular degeneration in the donors that she looked at and the number of mutations in mitochondria. And not so much of a correlation in the mitochondria and the retina. It seems to be something specifically happening in the RPE cells. And again, by our model, it's a bad thing, right? Because the pigment of the million cells that they want functional mitochondria, then they're going to use glucose for the cells and then glucose won't use the retina. And, oh, there's a, the data line isn't showing here. But this is one of our studies. I'm not really showing much of our data in this part of this morning, but I'll show you a lot in the summer of this afternoon. But this is an excerpt from it. What we found was that the, and young mice versus old mice, if you look at the amount of lactate produced by icups, which is muscle metabolism from icups is coming up from the pigment at the phthalium in the way we do the experiments, they generate a lot more lactate from the same amount of glucose compared to the young animals. Okay, so this is consistent with their mitochondria failing. So the RPE cells depend more on glycolysis and consume more glucose. And I'll show you other data later today, also, to kind of that thought. So our theory then, our hypothesis is that as we age, our RPE cells become more glycolytic because the mitochondria start to fail. And when the mitochondria, when they become more glycolytic, they use up the glucose before it has a chance to reach the retina, and then the retina starts. And that's why the photoreceptor, that's why we are vision deteriorates as we age. So another way that this might relate to disease is, might be relevant to treatments for disease is because of the diversity of types of mutations that can cause photoreceptor degeneration. So actually, I've watched one of these videos before. I think John Planner and material showed the same slide. But this is from Steve Degger's compilation of data showing that there's something like 300 different genes, I guess, that have been identified that have mutations that can, when they have mutations, they cause photoreceptors to degenerate. And then if you look at the types of genes, they're very diverse. Anything from photochancex and the visual cycle to outer segment structure, cell-cell interactions, so that the types of mutations that lead to photoreceptor degeneration are very diverse. So if there was a way to just generally make the photoreceptors more robust, that might be a way to slow down the degeneration in each of the, maybe not all of them, but it might be a way to make them more robust and more resistant to the effects of these diverse mutations. And so again, that's what I said. If you make the Roj Du Best one, they maximize glycolysis. So I just want to finish up with an experiment that Steve Sandid we collaborated with him on this. And the idea was, so Steve was sort of, he is more on the genetic side of this, and he was very efficient at making mouse models. And so I've been talking with him a lot over the years, and he got interested in this idea of the metabolic ecosystem. And so he asked, can we make the photoreceptors more robust to stress by enhancing your ability to do glycolysis? You don't want the RPE to do glycolysis, but you want the photoreceptors to do glycolysis so they can make lactate to support the RPE. So Steve came up with one way to do this, which was inactivated gene called CIRT-6, CIRT-2 and CIRT-6. Turns out that when you inactivate CIRT-6 in a cell, it increases this hypoxia inducible factor, which is a transcription factor that can enhance the transcripts in the genes that make proteins that support glycolysis. Increases HIF activity, increases glycolysis. And so if you do that, can you slow the photoreceptor degeneration caused by a PD-6 mutation? Supposed to be a mutation. And so what he did was he used a photoreceptor specific promoter to express a CREE that can be activated by a tomoxepin again. And then if that's there, it will delete the CIRT-16 because it's floxed by these two sites that the CREE can use to excise as part of this DNA. And so when he did that, he found that in the CIRT-6 minus mutants in the retinas, there was no CIRT-6, whereas in the HIF-1 alpha, which we were hoping would be up-regulated, does seem to be up-regulated, and there seem to be more glucose transporters than other enzymes that are involved in glycolysis. So it seemed like it worked. If you knock out CIRT-6, you make the retinas more glycolytic. And so we got involved in this project because we can measure metabolic flux. So we measured flux through glycolysis and flux through the TCA cycle. And flux through glycolysis was enhanced substantially. And the TCA cycle is also enhanced, I think it's because we're making more pyruvate that can feed into the mitochondria. So the more pyruvate to fuel the mitochondria, you get more TCA cycle activity. And what Steve found was... So these are the normal ones. Normal mean that they have a PD-6 mutation that causes them to slowly degenerate. So you can see there's fewer than normal photoreceptors. You don't have... So there's few photoreceptors because the photoreceptors have degenerated because of this PD-6 mutation. But when he knocked out CIRT-6 and enhanced glycolysis, it slowed the rate of degeneration. You can see there's a lot more photoreceptors here. So if you look at the outer nuclear layer density, is an indicator of number of photoreceptors, it declines more slowly when glycolysis enhances. So it doesn't stop the degeneration altogether, but it does seem to make them more able to resist it. So I get a different interpretation of that. It seems that it allows things to survive for about three weeks. And then if you look at the rate of decline, it actually declines more rapidly and comes to the same position at eight weeks. But it looks to me like it's allowing it to survive at a more normal level for about three weeks. And then the decline actually is faster. Look at the rate of the slopes. That's actually a steeper slope. Yeah, I think it's still delaying the degeneration. Yeah, delaying the delaying, but then when it goes, it goes even faster. Yeah, it could be a sort of cooperative effect, too. Like I was showing you before, and another experiment from Siege's lab, where he reduced the number of photoreceptors, and when you reduce the number of photoreceptors, that has a negative effect on remaining photoreceptors, because there's less lactate being produced. I'm not saying that, but I'm just... No, no, no, I'm saying that it could be... I just interpret that data a little differently. I've never said that before, but what you're saying is probably right, because it could be like a cooperative effect. Once they start to go, they bring all the others down with them. Okay, so that's the idea. So it's possible that this could lead to some types of therapy. This idea of a metabolic ecosystem could lead to some types of therapy that may not cure the diseases, but they delay the degeneration. So another investigator, John Asch, has been interested for a long time in the AMP kinase. And metformin treatment. But John had found, in another study that I don't have here, he found that when they treated animals with metformin, like animals where the rods were degenerating, that metformin seemed to delay the degeneration. But metformin is doing is, even though it's been around a long time, people really still, as far as I know, they still won't really understand its mechanism. But one thing that it, something pretty good agreement on a thing is it inhibits complex one. Somehow that affects energy metabolism and enhances glycolysis. It enhances glycolysis and it actually up regulates mitochondrial enzymes as well. And so John found this with some of the mice that he was investigating and published that a year or two ago. And then a student in his lab looked at patients, she went through a database of patients in Florida and found patients that have been treated with metformin or are not treated with metformin and either didn't work diagnosed with AMD or were diagnosed with AMD. Not described at all, but I think you get the idea. And what she found was that it was, the fewer patients with AMD who had been treated with, were treated with metformin. So there were fewer patients with AMD when they were treated with metformin. And so the idea, so there's some statistical data described that really poorly, but they have some statistical data and that in his paper suggesting that metformin can to later reduce or decrease the probability of AMD. So that's pretty much it. Like I said, I'll be describing more details of the stuff that we've done later in the seminar. Later in the seminar, I'll give this afternoon and I'll take questions. So a fascinating lecture and a nice general coverage of a very broad area which keeps expanding all the time. That metformin one's been out now for quite a while. And because this was just a correlation, there was a statistically significant correlation with taking metformin and having less of an odds of macular generation. But correlations never show causality and there are many other reasons. And one of the most intriguing that has never been disproved is, is you take metformin generally in the population almost all the time because you have diabetes might the process of diabetes be protective for macular degeneration? And that would explain the correlation and have nothing to do with the actual metformin effect. So until they do a prospective randomized trial of metformin, which they haven't done yet in patients with macular generation, this one is still out. There is interesting and intriguing. But the data set that they had, they tried to, I just read the paper actually on the plane on the way here, but from the data set, this paper just came out, but from the data set that they had, they did try to extract diabetics and non-diabetics and try to address that. And people, there were 10% on metformin who were not diabetics. I just, I find that hard to read. I'll send, I'll read later. Yeah, we're going to chat a little bit. Yeah, yeah. Evan, right. So the ratio between rods and cones would seem important in your model? Yeah. Yeah. So would you hypothesize or could you comment on rates or incidents of retinal degeneration in more cone-dominated organisms? What are the differences? Yeah, I don't really know. Cone-dominated organisms are more resistant. Or is there a different metabolic mechanism in play? I don't know the answer to that. So we're doing these same kinds of experiments in zebrafish retinas. So mouse retina is about 3% cones and the zebrafish retina is about 50% cones. And actually just recently, we've got a zebrafish where the cones are alive in all the rods of degeneration. So we can do 100% cones. So we can do all these same kinds of experiments to measure how much glycolysis there is versus mitochondrial activity in those. And I think that's the way to start to answer that question. But right now, I can't really answer. I'm sorry. I had one more. Intriguing about even in the non-macid or degeneration, the loss of mitochondrial function overall with age. Clinically, my experience, my other colleagues, they disagree. In the absence of macular disease or glaucoma, I'm amazed at how well high contrast visual acuity is maintained well into the hundreds. In the absence of that, the patients I have still are 2020. But I do think when you talk to them, that they talk about other things, that it's clear that there's some contrast differences, night driving, other types of differences. So we need to study that better. But as far as just our normal looking at visual acuity, in the absence of disease, my experience is with age, there's a very, very little difference. Anybody disagree with me on that, Nick? Aren't you seeing if there's no macid, no macid, these patients are still 2020, well with their 90s and hundreds? Yeah, so my guess is that what would be happening is that the RPE is taking more glucose for itself. And not giving the glucose to the photoreceptors. So it's not killing the photoreceptors, it's not necessarily killing the photoreceptors. So the density of photoreceptors might stay the same, which would be, as far as I know, a major factor in acuity, right? Right, contrast acuity. Yeah, so you might not lose acuity, but you might lose some other ability to darken after something like that. Contrast sensitivity. Contrast sensitivity, things like that. Low light level ability to see stuff like that. We need to study that more. That's an interesting thought. Yeah, and the other thing we've been working on is trying to understand, so I don't know if you remember, but I showed you some data that DeFerrenton had indicating that mitochondrion and RPE are accumulating mutations. And we've been working with somebody at University of Washington actually has a way to identify the specific mutations that are happening. And it seems like mutations that are accumulating from oxidative dams like people originally were thinking, but they're probably just due to replication errors from the polymerase, from the mitochondrial polymerase. And that seems to be what's accumulating with age. And another way of looking at it, too, is how can they possibly last that long? And so, especially with a polymerase that's somewhat error-prone or can't really correct itself very well. And so we're speculating that these RPE cells, and maybe the photos that was also made, other cells in the body have some way to sort of proofread their mitochondrial genome and get rid of the bad genomes. But that's for a few years from now. Thank you.