 There are certain centers, there are certain centers. Our next talk is going to be given by my co-chairman of the Translational Research Day, David Krizai, as a research professor. And he's going to talk about some of the interesting work they've been doing on glaucoma and the treatment of glaucoma. So once we get you all fired up there, we'll go ahead. So co-chair that has been AWOL most of the time. So sorry about that. Go ahead. So I made this slide a couple of hours ago, literally after I saw the title of the preceding talk. And the question is, what do these conditions kind of come in? They look pretty different in a way. But basically, if we look at this, what they do have in common is some kind of a pathological stretching, or swelling, or pushing. So there's some kind of a mechanical thing going on. So what I would like to sort of address in this talk is can we find maybe unified principles in all kinds of diseases that are afflicted in the human bodies? And can we find this through understanding a process that is called mechanical structure? Taking account that pretty much every cell in our body really cares about mechanical forces. If we look at the eye, for example, it is a highly mechanically active environment. So I went to our road this year, and there was a poster showing that just rubbing our eye, or blinking, or getting up from the bed increases our intraocular pressure with hundreds of millimeters per day. So there is swelling going on, activity-dependent swelling. There is pushing, there is stretching, there is the effect of hydrostatic pressure. So what I would like to leave you with is this idea of this being a highly mechanically active environment. Pushing can cause corneal obfuscation, and so on. So there are all kinds of pathological phenomena that have a very, very strong mechanical component. And we are interested in many of them, perhaps most. But what I'll focus on is glaucoma. Basically, when we think about glaucoma, we think about two things. First is, how is IOP regulated in the front of the eye? And the second thing is, what is actually going on in the back of the eye? How do these cells sense pressure? Do they sense pressure, or do they sense stretch, or what is actually going on? This has been a million-dollar question. Thousands of papers have been published. And really, we have gotten no closer to the answer. And this answer is what we are looking for. So how we do this connects the front and the back of the eye. The likelihood of developing glaucoma is exponentially related to the pressure that patients have. So there is something going on with the pressure. Indeed, we can increase pressure artificially in the monkey and look at the retinas. And you see that the retinas look pretty good, except here. There is this huge loss of retinal ganglion cells. So these guys looked at the sensitivity of the monkey. For light of the day, they found that the sensitivity loss was actually pretty minimal until the monkeys lost about 50% of ganglion cells, which is what we see in humans. In other words, by the time we realize we are getting blind, it's already too late. So we really need to understand how this loss caused by the IOP occurs. What is the molecular mechanism? Because that is really the only way for us to diagnose early. And that allows us to treat those patients before this fulcrum fall. So the question here is what is actually going on in the ganglion? And the hypothesis in the field are actually all over the place. Some people believe that retinal ganglion cells are intrinsically sensitive to pressure. Other people say, no, it's actually the glia. And it's the inflammation that kills the ganglion cell. There is a strong strain of thoughts suggesting there is an excitotoxic stress. Too much glutamate is being released for some reason. And some people say, no, it's actually the thiocellus and some kind of metabolic thing that is going on. And this is definitely the case for higher IVBs, which causes ganglion. Another really acrimonious debate that's been going on is whether the first effect on increased pressure is at the optic nerve head or is it at the dendrons? And this is a very important question that still needs to be resolved. So how do we put all of this together? The hypothesis that I'll be talking about today is that all of these things are happening because these cells express ion channels that are sensitive to pressure or to all kinds of mechanical stimuli. We can mimic the glaucoma phenotype simply by activating these channels. And we can protect the retina from hypertension by blocking these channels. Or by genetically eliminating them from ice. So this is what we'll be looking for. We are looking at several types of these channels. What I'll be focusing on today is a channel called Transcent Perceptive Potential Neural Invisal form 4, which is a member of a large superfamily of so-called channel channels. It's a non-selective channel. And what is interesting about it is it's activated by all kinds of mechanical stimuli, from swelling, from stretch. But it's also sensitive to temperature and lipids, such as arachidontic acid. It's expressed all over the body, especially in tissues that are still the load bearing, stretch-sensitive, such as the bladder. It regulates uterine, vascular bladder contractions. And it's been associated with mechanical hyperalgesia and neuropathic pain. So something is going on with this channel and mechanical stress. And the null advice were shown to be less sensitive to painful stimuli and to show less inflammatory responses during mechanical injuries. So it's a very interesting candidate to evaluate in a mechanical force-related disease, such as glaucoma. So when we started, nothing was not about it either. What's wrong? So we looked at it, we find mRNA, we find protein. And when we immunostain, we find that it's very strong especially in the ganglion cells. So the red here is a transgenic marker in retinal ganglion cells, slide one. And you see it very strongly co-localizes with 24 antibodies. And the red here is another ganglion cell marker. And you see it's a transcription factor. So you see the only neuron in dissociated preparations of expressiture before our retinal ganglion cells. You also see this green fuss here. These are the end of the feed of retinal mylargia. So we confirm this by immunolabelling with mylargia marker. So why is this interesting? Because mylarcells and retinal ganglion cells are, in fact, the cells that are the most susceptible to some glaucoma just remotely. So we can take a chemical that selectively activates 24 channels. And we can look at what, and we can load these cells with the calcium dye. And then we look what is going on with calcium signals. And what we see is huge calcium increases in response to this channel. And what is amazing to us and really interesting is that the parent of the calcium response in the neuron, the ganglion cell, in the neuron cell is extremely different. So here we desensitize very quickly. And in the glial cell, the signal is huge. It stays on for as long as it stays. Now, what happens to the light response of these cells when we stimulate this putative mechanical channel adenus? So this we are recording an intact retina home out. This is a cell that responds to light with spiking and turns off during light off. And you see the presence of adenus in the cell, it's crazy. Excitability goes on like it's turbocharged. We also see increasing spiking in a cell type that goes on with the light and that goes off with the light as well. So there are also more spikes. And when we actually look at the excitability, which we test by injecting tiny amounts of currents in the cell and in spikes, we see that we need much less current to invoke induced spikes in the presence of the adenus than in the absence. In other words, what this adenus is doing, it is increasing the excitability, making them much more susceptible to any kind of glutamate or any kind of light signal that these cells are going to experience. So producing an excitotoxic stress. Now, this is the case for all ganglion cells. Because when we take the red, now we plug them to a microelectric array, and we stimulate the agris. We see a huge increase over 100-fold in spiking that shows kind of a similar time course that we see with calcium energy. So we are pretty sure that this cell activates excitability, stimulates excitability. What about swelling, which is another mechanical stimulus? Well, when we induce swelling, there is a huge increase in calcium as well. And we don't see it. So this is an increase in calcium when we swell, and we don't see it in the molecules. Now, our red-nose ganglion cells intrinsically sensitive to pressure. Here, we record to single channels in so-called patches. And we can stimulate with very, very defined calibrated possible pressure. And we can induce spikes. And this is very reliable. But when we do longer pulse stimuli, we see, again, inward currents, which are much smaller in knock-out animals, or knock-out red-nose ganglion cells, or much smaller when in the presence of highly selective tricky work patterns. In other words, tricky four is a pressure sensor in red-nose ganglion cells. And when we take it out, they are less sensitive. Now, can we mimic glaucoma? Or can we mimic sort of pressure-induced red-nose cell death simply by activating a mechanosensitive channel? The answer is yes. So when we inject the agonist, this is the control. This is the eye injected with the agonist. There is a huge loss of red-nose ganglion cells. So we can actually reproduce the mechanical phenotype simply by stimulating a chemically stimulating mechanosensitive channel. So what about this question about dendrites versus the optimal head? I would like to say that this is something that we are still investigating right now. We don't have an answer to this. But when we culture cells, and we can see the dendrites, and we can get localized increase in the dendritic calcium level. So we are pretty sure that this is a way to regulate the synapse formation in the inner plexiform layer. And the newest studies published in Journal of Neuroscience are showing that, indeed, the first changes in response to elevated ILP may, in fact, be synaptic. However, when we look at the optic nerve head, the immunostanding is also extremely strong. So it's very likely that by stretching the laminocerosine primates or the real laminate mice, one would activate this channel. So we stretched the substrate. We have a machine that allows us to do that very specifically. We can see huge calcium increases in retinal ganglion cells that are missing in about 70% of cells. In 30%, we still see some calcium responses. So the knockouts show less. Calcium increases. Trips C1 knockouts, this is another type of mechanosensitive channels, are not susceptible in Trips C1 knockouts, but all of them are susceptible to the selective antagonist of Trips C4 channels. So in other words, we hypothesize, we propose that Trips C4 is actually a mechanosensitive channel that is sensitive to pressure and that is sensitive to stretch, and that might ostensibly mediate the effect of hydrostatic pressure, both in the dendritic and optic nerve head. So this is kind of a very simplistic model of what we're thinking about. Overactivation of the channel leads to overload with calcium. And this act is calcium-dependent proteases, caspases, and so on. And we have proven that as well, and leading to some. So what about the second part? What about the glial? So the Mueller cells, in particular, envelope every single retinal neuron. They are absolutely essential for every aspect of retinal physiology. And what is interesting is that they become activated. The first thing that one sees following IOP increase is glial activation together with dendritic and synaptic So what goes on with Miller cells? I will skip over a long period of time of work, a lot of work. I will just show that how incredibly strongly expressed present are Miller cells developing every single retinal neuron. And if you look from the top, you see these little holes. This is where the cell processes of the dendroids and the synoptysis are. So they strongly express TRIP4. And to just summarize our findings, we nailed down the signaling cascade that is associated with TRIP4 activation. Basically, we think that up to four channels, which are the water channels, serve as detonators for activating TRIP4 channels by increasing the rate of swelling during kind of osmotic shocks. But the TRIP4 channels are intrinsically sensitive to stretch and mechanical stress as well. So we are 100% certain that TRIP4 channels are important stress sensors for everybody. Now, what about glaucoma? When we induce chronic glaucoma in certain genetic mouse models or acutely elevate IOP by injecting microbeads in front of the eye, we can get reactive gliosis of these mirror cells. So the question is, can we reproduce this by activating this putative mechanosensitive channel and the answer is yes. There is injecting this adenis induces huge reactive gliosis. So basically, in other words, we can reproduce key-ass retinal elements of glaucoma simply by stimulating a mechanosensitive branch. Now, what about treatment? Can we protect by systemically treating mice with adenis or by eliminating the gene? And the answer is yes. So this is a normal eye treated with PPS. This is eye that has experienced elevated IOP. You see there was a very significant degeneration, about 20% loss of RGCs, which you see here. In mice that were systemically treated twice a day with a high dose of TRIP4 adenis, there was not degeneration. We've visited this many, many times in dozens of mice. And we think this is a fairly solid result. So somehow, blocking the mechanosensitive channel blocks the degeneration that we see here. Now, when we got this result, we were very thrilled. But we started to worry that maybe the effect is actually not at the granular cell. What if we are regulating pressure itself as well? So we tested this. So this is ejection of micro-V to see elevated IOP in mice. And when we systemically inject the antagonist, you see that the IOP drops like a rock and stays down for as long as we keep injecting the antagonist every couple of times a day. So in other words, not only is TRIP4 expressed in the retina, it is also expressed in the front of the eye and it's regulating IOP. Why is this fascinating? Because this would be a way to treat both at the same time, rather than IOP into neuroprotection. So we can get even a stronger effect when we intraocularly eject the antagonist. And you see this lowering lasts for days. We also designed eye drops here at the University of Utah. And these are also effective lasting for day or two, so better than current eye drops. So the important question now is, where is this channel in front of the eye? Is it in the cellular body which produces the fluid? Or is it in the trabecular meshwork or the cellular muscle where that mediated outflowing? You see that trabecular meshwork is much more important because the higher you go with IOP, the more load is going through the trabecular meshwork. Unfortunately, most of the current glaucoma drugs are targeting the uberoscleric component. So there has been a very strong push of looking at trabecular targets. So we published two papers this year showing that it's expressed in bulk, very strong. And we localized it to non-pigment, to epithelial cells. And so we did a huge amount of work on the molecular physiological genetic methods that I will skip over today, just to make point, it's expressed in bulk. But we, for various reasons, we think the trabecular component is more important. So when we do so-called pressure clamp studies, we see that trabecular meshwork cells are highly mechanosensitive. So the ramps of pressure in both directions induce currents, inherent currents. And when we stretch the cells, we see calcium increases that are blocked by the tricky foreign dagonist. These increases are dose-dependent. This is statistics. But what is really beautiful, and here we were saved by a collaborator who can mimic the conventional outflow in nanofibricated devices that have been populated with trabecular meshwork cells. You can do, you can fuse through that, and this has been shown exclusively by them to be perhaps one of the best in vitro models for conventional outflow. So basically, when we do that, we find that the antagonist is highly effective in increasing the conventional outflow facility. So this would be really one of the first drugs that might be targeted the conventional outflow. If you look at the pressure, perfusive pressure on the other hand, the agonist increases it a lot. So this means that the pores in this nanodevice are much smaller because of activation of this channel. This is exactly what happens in humans when you increase the pressure, the trabecular meshwork cells become stiffer, the cytoskeleton and extracellular matrix become upregulated, this increases the pore contractility which further increases out. So is this the case in terms of 54 channels? The answer is yes, because we can stretch trabecular meshwork and you see the actin cytoskeleton and the focal adhesions get upregulated like recently. So we can mimic what has been shown in human primate nanodermal glaucoma at the molecular level. So we also looked at these nanodevice conventional outflow models and we see that the agonist, 24 agonist itself can reproduce the upregulation of actin and upregulation of exocellular matrix. This is highly suppressed in the presence of these 24 agonist. So the model here is that increasing pressure in front of the eye imposes some kind of a mechanical stress that activates 24 channels leading to increased stiffening of these cells and contractility, which increases outflow resistance and maintains or increases at a later time. So what we're doing currently is we are nailing down the very kind of specific components of the cytoskeleton mechanism that link the internal cell cytoskeleton to the focal adhesions and to exocellular matrix. Some things go up during pressure and stress some things go down. And another fascinating approach that we are using is we use FRET probes. So fluorescent probes that function as force sensors. So basically they are stretch sensitive and by looking at changing fluorescence we can tell how much load is imposed on the cell as we are pressing or as we are doing any kind of manipulation as you can see here. The load or the strain is mainly the focal adhesions. So nobody has done really before that in the eye or the brain. So this is really an exciting way to go forward. So I won't go into this but this is what we're thinking is the mechanism that regulates the outer resistance at the level of trigonometric cells. It involves six or 50 enzymes. It involves calcium and gene expression and we have data for pretty much all of these components. Now to conclude, we think that IOP, the 3D4 channels regulates IOP at both inflow and outflow levels although we think that outflow components is probably more important. We think that they also regulate the remodeling that happens during mechanical stress in glaucoma at the level of ganglion cells, the neurons. Anglia, I didn't talk about microglia, they also have these for channels and they also are very stress sensitive. And combined, these events together function to induce the phenotype that we call the glaucoma to speed. What is very interesting, if you look at this, this has very interesting parallels to the neuropathic pain paradigms that we see and other sort of force or mechanical-dependent paradigms we see across the body but they have different names so we think they're different diseases, so to speak. So if I have just one, a couple more minutes, I will tell you about some of our unpublished work which I think is very fascinating as well because I told you about in the fetal cells that may be involved in glaucoma but they also form the blood venavera and are critical for all kinds of other diseases from diabetes to ischemia and the probability of maternity macular degeneration and so on. So we found the expression before, oh yeah, first of all, just to show you, this is in the fetal cell, it is enveloped by a parasite in case of macovascular chair or smooth muscle cell in arteries and that is shadowed by the end-feet of the fetal cell. So this is called the gliobascular unit that is really responsible for the brain and for the retina to be able to function in any way. And when we immunostain, for check before, we see that the fetal cells very, very strongly expressed in the fetal cells and in fact, what is interesting is that they're expressed in the macrobascular, in the retinal, in the fetal cells but not in cordial capillaries. So different macrobascular bands have very different calcium or ion gel signatures which we have explored, but I don't have the time to obviously talk about. One thing which is interesting is that the calcium increases in these cells are enormous. They are tenfold more sensitive to any other cells that we have ever investigated. When we look at the currents, when we look at the calcium, so they really, really, really care about mechanical stimulation. So we, in collaboration with Dean Lee here at the University of Utah, we can look at now a model of blood-threatening barrier by doing high-profile impedance studies in monolayers of these macrobascular cells. You see when we use the agonist, there's an enormous increase in percolability in bascolar percolability, just like crazy. So, again, suggesting that these cells really care about mechanical stimuli and that should be for channel is a potentially major regulator of permeability. Now, we nailed down, we believe, the molecular mechanism of this, so that the permeability of blood-threatening barrier, or the brain barrier, is mediated by V-catering, beta-catering complexes and occluding complexes. And we found that the 24 agonist triggers a retraction. This is V-catering, this is V-catering, I believe we're acting, beta-catering. A retraction of these complexes towards the interior of the cell, which is directly correlated with increased permeability across these intercellular regions. So, again, this is something we are exploring very actively at the moment. And this is kind of the model of this gliobascular unit, because the interesting thing here is that 24 is very strongly expressed, and in the vasculature, and we believe it's regulating and controlling the flow of metabolites and swells pressure sensitivity within the brain. Now, when we treat any disease, we don't want to treat just any target within it. We want to choose the targets where many signaling pathways converge so as to treat several symptoms at the same time. So, I propose that in terms of treating glaucoma, trip before is a very convenient and reasonable target because, first of all, it's expressed exactly, exactly in cell types that are most effective in glaucoma. It is associated with transduction of mechanical forces and stimuli that are highly relevant for glaucoma. And by treating this or targeting this channel, we believe we could alleviate a lot of the phenotype that is associated with the disease. Here, I would just like to mention that most of these diseases that I plotted here, I plotted because I already knew they are associated with trip before dysfunction. Some of them with trip before mutations which are associated with pathological load bearing with sensory motor neuropathies with neuropathic pain. I don't know about intracranial hypertension, but I will not be surprised, especially because they interact a lot more and it's very strong. So, basically, we think or I'm thinking that if glaucoma is just an ocular version of neuropathic pain, so to speak. It's a kind of a semantic switch that perhaps we could make, especially once we better characterize what is going on in terms of signaling and symptomology. So, Tam, for one, was involved in pretty much all the aspects of the work that I was talking about today and she is really spearheading the endothelial project and driving with her ideas. Everything was started by Dan Riscan who was a grad student in the lab and he kind of started this train rolling. Sarah is working on micro-healt. Oleg and Maxime are electrophysiologists who have done absolutely spectacular work on mechanical introduction. I think this is really, in terms of neuronal stuff, something that nobody has done even in brain neurons, stuff that we're doing right now. Andrea is doing phenomenal work now in steroid glaucoma that I didn't talk about. Andrea, where are you? Here she is, yeah. So, and Andrea has been working in acuporins and swelling and stretch assays and I have to mention here in public Dr. Olson because I am a photoreceptor guy and I wouldn't be talking about glaucoma if it wasn't conversations in support from Randy. So who basically said go ahead to some of the crazier ideas that I had and without that those crazy ideas would go to nothing. So I had no support when I started those studies and with support from Randy this actually started to lose. Sorry for that.