 Hi I'll I have to say I was very impressed with this presentation. It's like a detective story and Every basic scientist should really See as many of these as they can because it actually makes you believe that What you're doing could eventually help somewhere and definitely gives you an extra oomph. I would have to say very impressive So I'm going to Talk about kind of a side project from my lab that has morphed into What we hope is kind of an independent independent way of Conducting a new study and it has to do with glaucoma so As you know, it's one of the leading causes of blindness in the world and The genes are really not that well known So people often talk in terms of risk factors and there are many of them including race age family history and so on But the primary risk factor is an increase in interocular pressure and This is also one of the best ways to diagnose this disease. So just to show you the the relationship an increase in Eye pressure makes it increasingly likely that somebody has in fact developed glaucoma now in contrast to photoreceptor diseases, it's often very hard for the patient to tell that they are going blind The disease is in the periphery and there is a lot of central compensation and Somebody can lose like 30 to 50 percent of axons of ganglion cells before they start thinking that There's something wrong with before they go to see an ophthalmologist. So Basically, it can be difficult to diagnose and The problem is also that there is no treatment for the ganglion cells that have been lost so the best one can do is try to Manage the IOP And so in order to help these people it is really crucial to be able to detect things early and to treat IOP early and You of course know this very well how you know how the increased IOP arises, but I'll just quickly mention that Lens in order for the lens to function it we can have blood vessels. So Basically the way these living cells in around the lens are fed is through the fluid secreted from the cellular body and that fluid fluid has to go somewhere and It drains into the venous system through the Schlimm's canal now if there is a prompt mismatch between the production and the outflow there is an increasing pressure and The the structure that is affected however is Here so it's the optic nerve especially the optic nerve head and the retinal ganglion cells which start to die So it's action at a distance. It's like somehow the retinal cells can tell that there is a mechanical Stimulus going on and it and they don't like it So this is a very simplistic View of the retina and all the layers. These are the ganglion cells which are the neurons that are sending the actions to the brain and some very kind of quick pictures that I made are This is the retina and here are the axons that are collected from the ganglion cells going to work towards optic nerve head This is optic nerve head here. You see it's collecting the axons and Another picture is shown here the axons are going into the optic nerve head and then to the optic nerve and I would just like to quickly Mention that the treatment again is limited to the interior chamber. So there are Drugs that modulate the Sympathic and parasympathic systems and they are also lasering surgery options But apparently they have side effects and one would like to avoid them even if one can now What about the posterior retina? There is really The there has been a lot of controversy and a lot of speculation as to what is killing these cells and a lot of the mechanisms that have been implicated into into this are sort of Gliosis in the production of reactive oxygen species various cytokines then Infestation with macrophages coming in It has been proposed that there the blood vessels are getting constricted. So there is hypoxia happening, but Some people have thought it's purely mechanical constriction of the lamina crebrosa, for example, but really it's not known and what There have been a lot of studies trying to modulate cell death in ganglion cells various Glutamate system blockers neurotrophic factors antioxidants and so on but basically it has been Stunningly ineffective. So it is as if we we don't really understand the mechanism through which pressure is impacting these cells so basically What I'm going to tell you about today is Some thoughts and experiments that we have done or and trying to convince you that maybe we have identified that point of impact between pressure and Intracellular physiology of retinal ganglion cells So and that is I will be talking about the general principle of mechanosensation in retinal new which hasn't really not been addressed before and the point is that Every cell in our body is in kind of isometric tension state. So it has to Feel its neighbors. It's it's that we have specialized mechanoreceptors Which detects mechanical stimuli pressure things like that but in some way every cell in our body is also a mechanoreceptor because it has to Coordinate its activity to its name and and What we know about Kind of real mechanoreceptive cells is that the Process of mechanosensation is always associated with influx of calcium Now calcium I won't go into this deeply but basically calcium is one of those magical Messenger molecules which do that's pretty much everything in the cell Name a cellular reaction calcium has to regulate it at some way And if the calcium goes very high because it's so ubiquitous it basically kills the cells and it's Practically goes without saying that a cell that is undergoing this process of degeneration Is going to have high calcium in some way or another But here we want to Investigate whether the process of mechanosensation itself Is associated with changes in calcium if it is Then when we stimulate the cell in mechanical way, we should see an increase in calcium concentration So do they have stretch sensitive calcium permeable do the retinal ganglion cells have Stretch sensitive calcium permeable channels Now one easy way to look at these channels is to stretch the cell through a hypotonic stimulus so in this way Water comes in it stretches the membrane and If you do if you do too much the cell is going to explode But if you just a little bit then you have kind of a harmless And What we do is we take a ganglion cell from the mouse retina With two types of indicator dies one of them is called calcium and this is basically it's a despite its name It's a calcium independent dye that just measures the volume So if the cell volume is going to increase the calcium fluorescence out of it It should go down because it's diluted Then we put another indicator in this is called furia 2 and it's a calcium indicator die So this die tells us exactly what the calcium concentration is so then To stretch the cell and see what happens to the calcium signal here is an example This is a calcium signal we Stimulate with a hypotonic Stretch and we see and decrease so clearly we know that the cell has has expanded And this is a calcium signal here We stimulate with glutamate which as you know stimulates glutamate receptors which are very many of them are very calcium permeable So we know this is a ganglion cell because we see this huge calcium Increase in glutamate and then when we stretch we see this very significant response now know that this This is a saturated concentration of glutamate, which is one of the most powerful excitatory molecules in our brain and look how big the signal is here with a relatively small stretch so So we said well we have something there these cells have stretch sensitive channels and They are in the plasma membrane because when we do this stretch Enormous saline this is what we see and when we repeat it in the calcium-free saline This is what we get so we don't see anything This means that this is a channel which is in the membrane And Now we were able to block it with an ion called gadolinium and why is this important? this is important because gadolinium is a blocker of a particular superfamily of calcium permeable channels called trip channels and There is another blocker called ruthenium red and it is just as good and inhibitor as Gadolinium so we were pretty sure we have one of these channels But there are a lot of them and I mean a lot and they're several super families and each family has many isoforms and All of them are affected by these two non-selective inhibitors so we made a few bets and we Chased several Pathways and I'll just focus on one So when we look at these channels, we see that pretty much all of them are expressed in the retina and I have to tell you that basically Nothing is known about this It's a very hot top in neuroscience in general, but but retina really lags behind and If you look into the literature, it's it's it's a pretty much virgin field. There is nothing going on or very little so we have Focused on one particular Element there isoform it's called trip v4 for Transcent receptor potential vandaloid 4 and the reason why this channel is so interesting is That it acts as an integrator of many different kinds of stimuli So it's not only mechanical sensitive and pressure sensitive. It is temperature sensitive So if you increase the temperature, you will hugely regulate its ability to conduct calcium It is it is activated by arachidonic acid which directly can connect it to the inflammation mechanisms anywhere and It's it is localized to kidney and Non-excitable cells, but it is very strongly expressed in sensory neurons Like send neurons that sense that regulate our osmotic balance in the body The spinal cord door does the root ganglion neurons and also hippocampal neurons so We look for this channel in the retina and we find the mRNA and we also find the protein Now we tried 10 different antibodies and they all gave us the same standing in the knockout until we finally Stumbled upon an antibody that works really well. So this is the Western blood This is in the knockout. This is the wild type and then we expressed the gene in Hexels and and we see a signal with this antibody versus controls And we know the antibody is good because when we look at the tissue that has This channel characterized and we know it's there We get nice staining and we don't see anything in the knockout. So we have so now we have a way to look at this Where is it localized in the red? now Okay, it's not doesn't Translate perfectly, but basically if you look at this picture you see is so close to ganglion cells these are ganglion cell markers called brin 3a and they label the cell body of a ganglion cell and These are the plasma membrane around them. So as you can see you can can see here So we have now identified a pressure-sensitive channel that seems to be very strongly expressed in retinal ganglion cells Now this is a transgenic retina expressing GFP in ganglion cells and with this is double labeling and you see there is a pretty strong color equalization and The green things here are Miller cells and I'll be talking about them if I have time later now we expressed trip before in A construct which has gfp behind it engineered to have gfp because so when we stain for gfp We know where trip before is expressed and it's very strongly expressing ganglion cells again And nothing is in the non-expressing retinas. So so now again additional confirmation When we look at isolated cells we see the ones that's labeled for ganglion cell markers But not for other markers co-localized with trip before This is just another example here you here. It's difficult to see but they are literally like hundred cells or more but only the ganglion cells label with the Antibody and now finally we are looking from the top and we are looking a whole bunch of ganglion cells With with a marker here This is a ganglion cell marker and we look at superposition of the green dots which is trip before Where the ganglion cell markers we see this puncta, which are pretty much focused on the ganglion and The puncta are very interesting because they suggest there is a lot of trafficking going on of this protein So it may be a very dynamic thing, which is something we want to look at in the future now I just wanted to it's not showing very well But basically what this shows is that this the trip before is not expressed in GABA urgent amicron cells Which is something that retinal physiologists care a lot and it's very it's it's very interesting because it shows that the modulatory mechanisms are not expressing expressing this pressure sensitivity and Now going back to the optic nerve head Which is where the major impact of pressure in in the eye is supposed to occur. This is the this these are Ubiquitous stain hematoxilin stain. I think and so on this is a staining for Astrocytes which are feeding the ox axons as they are acting exiting the retina and When we look at the optic nerve head So here here are the axons Exiting the retina you see it's very strongly Expressing this trip before channel. It's perhaps the strongest signal anywhere. So this region, which is Exquisitely sensitive to pressure. It's is also Strongly expressing this pressure sensitive It's not it's the channel is not expressing astrocytes themselves These are two different pictures one of them is from the retina the red is gfap Which is an astrocyte marker the green is the trip before and this is from the optic nerve itself And you see the red and the green don't call localized. So we know it's not expressing the astrocytes It's not expressed in microglia So again, these are the ganglion cells doesn't come through very well These are the microglial cells and there is no Colocalization at all. So this is important because these are the immune cells that are kind of driving a lot of Pathological remodeling in glaucoma now How about the physiology when we isolate these cells and we stimulate them with very specific Highly selective antagonists. We see that the huge calcium increases So and when we measure the diameters of cells that respond to these agonists and Compare them to diameters of identified retinal ganglion cells through very specific markers We see that they're pretty much perfectly superimposed so we now have reason to believe that Activation of this channel is in has It's almost as important as glutamate as far as the calcium levels are concerned. This is how it looks like This is a photoreceptor. This is a ganglion cell. This is a glutamate stimulation and this is Selective agonist and this is nanomolar levels You see there there is a calcium increase and not only that the calcium increase Desensitizes so the response to glutamate is sustained But in the presence of this agonist or membrane stretch calcium comes down So this will be important Presently we can block it again with ruthenium red And also with gadolinium. So we know again. We confirm it's a tripp channel And it doesn't occur in zero calcium just like with a membrane stretch So we know we confirm it's a It's a membrane channel and then Daniel the grad student in the lab did a very clever experiment He the question of course was okay We have a channel that Responses to stretch and we have a channel that responds to very selective agonists, but are they the same? So what he did was he used the ability of the channel to desensitize? To then stimulate with GSK to include the response and there was nothing and when he waited a long time 45 minutes or something of half an hour. He was able to get a GSK response So we know now that The principal channel that is making these cells sensitive to pressure is trip before now This channel is not there to kill the cells right it has to do something in normal physiology of the retina and Wouldn't it be interesting if the retinal cells were actually? Putting the information about retinal pressure into the spiking pattern that they are communicating To the brain wouldn't it be interesting if what we see actually has a pressure component to it So what we did was we took the entire retina Put it on to a multi-electrode array. That's a ray of like 64 electrodes So we can record from 60 ganglion cells at the same time and then we stimulated with this selective agonist And what you see one of this is one is called for alpha PDD and you see when we applied There is an increase in spiking. This is like spiking rate. So this will be like 40 spikes per second 60 spikes per second in a huge proportion of cells and in fact Just with this agonist we see about 60 to 90 percent of cells are responding so Pretty much consistent with our physiology showing that every ganglion cells has these these channels This is the GSK another one Showing that the firing rate increases rapidly after application of this Agonist and it also Desensitize is just like calcium does in our imaging studies and the the change in the firing rate is more than for a hundred fold so what that means is That pressure is capable of Producing an excitatory component that is an integral part of our visual signal in the brain Now I mentioned this is also temperature sensitive channel We haven't looked into this very much But we just did one simple experiment where we raise temperature to 37 degrees from 25 37 is the optimal range of activation for trip before and we see an increase in calcium So it's something we're interested in pursuing further Now what does this do for survival of the cells if you stimulate for a long time? One way of looking at this is to do a so-called tunnel assay which labels dying cells Basically, so if you stimulate them with a glutamate agonist for a long time the number of green cells which are the tunnel positive cells will increase and This will be a sign of cells entering the apoptotic process What happens if you do this with this trip before agonist what happens is you strongly increase the number of green cells so this is under This is now GSK with small diameter cells, which would be like photoreceptors and Amicron cells and there is pretty much no effect very little effect on survival And we looked at large diameter cells which are ganglion cells mainly and there is a huge effect of those dependent effect of this trip before agonist what this means is that Mechanical sensation or just pure trip before stimulation is Going to kill ganglion cells if it happens Strongly or if it happens for a sustained period of time and we think this happens because calcium is increased And we would like to test this in many ways We have just received a trip before knockout mice And we want to see if we can prevent like a cutochronic local With by deleting this particular channel So basically we find that memory stretching juices enter of calcium into ganglion cells. They're expressed these channels They the channels control spiking And if you stimulate them for a long time You you can kill them now the story is so it basically this channel would integrate a lot of different stimuli not just pressure so There are a cadonic acid temperature is osmotic stimuli especially and You can imagine that if somebody has a mutation in this channel They it could conduct calcium at normal pressure, right? So this would account for like low-tension glaucoma, for example, and it turns out that just last year They have been a flurry of papers in nature and nature neuroscience things like that showing that trip before mutations cause very very severe skeletal dysplagias and Hearing loss visual dysfunction so We are now thinking about doing the sequencing from glaucoma patient and see if we can target this channel we just looked at visual acuity and Acuity itself Doesn't seem to be different now now Basically there is another thing that I would like to talk about so I talked about these Channels expressed in ganglion cells and what I would like now to talk about a little more is they are also expressed in Miller cells and the Miller cell is an incredibly Important cell in the retina some ignored but basically this cell spans the entire radius and feeds and integrate signals across these layers very important cell and if we Label it for Miller cells. This is what we see you see that it's this radial organization And when we label it double label it with three before we see it's mostly in Ganglion cells the green but there is some yellow here and this yellow is Collocalization with the Miller cell And but the Miller cell responds very differently from the ganglion cells Yeah, I told you that the ganglion cell Desensitizes in response to three before activation, right Miller cell on the other hand responds with huge calcium which are sustained as long as the Stimulus is present this would mean that This is really the cell that is going to be much even more sensitive to pressure So how does this look like here is a Gang here So this is you will see now we are added three before agonist and if This resolution would be better You would see that not only cat serum goes up But there is a whole bunch of waves going back and forth and another thing you would have seen Thank you is that this photoreceptor here starts to respond Together with a ganglion cell so when this guy goes up sometimes this guy lights up That would suggest that some factors are being released from the glial cell That are impacting the the photoreceptor So Yeah, here are the waves Okay, so thank you now We can block these waves by Blocking calcium release from intracellular stores, and I won't go into this very deeply today, but basically It's every cell has ability to store calcium in Endoplasm Criticulum and it can store huge amount, right? So basically then it can release them upon stimulation in it releases them through a receptor called rionidin receptor So the idea is calcium comes in bansion receptor in it gets this it acts as an amplifier to release calcium out of the cell and what we are what we Thinking then is that not the reason one reason why this response is sustained is that calcium comes in through the trip before channel it activates this release and then Explodes the concentration in the side of soul and basically we find this by by blocking it with a particular blocker of the calcium stores and This is Unseeable, but basically I wanted this would show that these waves are blocked when we antagonize the calcium release from stores now How about the glaucoma? This is a mouse model with severe glaucoma, and you see it's Lacking the ganglion cells which are the Brin 3a positive signals here and the mRNA for Brin 3a goes down and We were surprised to find that the trip before MRA and Renee goes up So because we know these retinas have very few ganglion cells This means that the Miller cells have not regulated the trip before signal that even more sensitive to pressure than they were before and When we look at the staining we see now the trip before staining is Way increased in the retina and these are the Miller cell signals here And so we have evidence to believe that now the signal is not only in the Miller cells It's also in the astrocytes So somehow the regulation is going high what haywire and We are not the first to think about this. This is a paper From sensory neurons, which they've stimulated Chronically with a mechanical stimulation and they found that the mRNA and protein go up when during chronic following chronic stimulation So what about other mechanisms? We find that in glaucoma the this ryanidine receptor which Provides a boost. It's like a rocket fuel for the calcium signaling. It's hugely increased One particular isoform This is this is the chronic glaucoma these these are controls and these are isoforms don't change at all So and the increase is more Significant in severe glaucoma compared to early glaucoma so somehow The cells are interpreting an increase in pressure as as stimulus for hypertrophy and augmented signaling and We find that So this is how of this ryanidine signal looks in the wild type You see that it's mostly in neurons. It doesn't co-localize with Miller cells. It doesn't co-localize with astrocytes It's very strongly expressed in the optic nerve head again. No co-localization with glial cells and This is now in glaucoma. This is control. This is moderate glaucoma This is severe glaucoma and you see it's very strongly expressed in Miller cells And what is very interesting now is we see that Tripp v.4 Collocalizes with a molecule called aquaporin 4. This is a water channel in That Regulates osmotic osmotic function basically so these again the red unorganic cells expression to be for the green Is aquaporin 4 which is expressed in Miller cells? So basically what this says is that this pressure sensitive channel is right next to a water pore so What the Miller cells are doing a major function in the retina is to control the osmotic flux People have been looking for years to actually identify how they do it and we are pretty sure that we know what's going on and in glaucoma the Ganglion cells are mostly gone, but now there is a product huge Gliosis and huge upregulation of these aquaporin channels as well as 3p4. So We are start starting to kind of put together some very qualitative models about what is going on We are thinking that both ganglion cells and Miller cells are intrinsically pressure sensitive They both respond to calcium And but these guys respond in a sustained way and may be able to release all kinds of Inflammatory and neurotrophic and Toxic factors that impact And impact the remodeling of ganglion cells. So it's something that we are considering now Now I would just like to mention something else and and finish with that so we just like Dr. Hageman here is Thinking about AMD and systemic kind of effects that we have started thinking in the same way now a Lot of glaucoma patients our systemic hypertensives so In fact 72% so how about should be for signaling in these in these patients we just look in the mouse of course and We find in Tremendous up regulation of of this trip before channel in the kidney of A mouse with glaucoma minus with glaucoma. So maybe there is something just like Greg is saying With respect to AMD and maybe something similar is happening glaucoma in these trip before channels And we don't really know how to think about that, but a kind of open to suggestions so basically we find that Miller cells but not not astrocytes or microglia express this channel it It's working by activating calcium waves through The Miller cell and what that means is now the whole retina can see what's going on because these waves go up and down nothing down and This this mechanism is operating like up regulated During glaucoma and may have a role in Gliosis that is a hallmark of glaucoma in the mouse model that we study the Miller hypertrophy have the cell happens months before the ganglion cells start to die and This may be part of some kind of a systemic dysregulation although that is not clear at all So this is my group here at Utah and we have very good collaborators here around I mean around the country and I would like to thank John Moran Tiger Award, which was basically Responsible for us doing this study. My lab is a photoreceptor lab What we are funded by NIH and so on and we were not experts on ganglion cells at all but after a conversation with Randy He said this is interesting and he gave us some money and this is how we came to this project So thank you Well, these mice are hypertension. Oh, these mice have increased IOP But we don't have a way to measure the blood pressure. So that would be interesting Yeah, if we have them we would measure