 for you. So our next speaker is going to be Dr. Wolfgang Baer and he is our, he's a professor here, but he's also director of research here at Moran Eye Center. And so it's a pleasure to have you talk and he's going to update us on some of the important research that he's doing. So we change topics quite a bit. I'm a biochemist, molecular biologist, and I think I was invited to present here because I do something translational. My topic is stereopathies and I, my protein or my gene is called IQ CD1 or MPHP5. Sounds very complicated. It stands for IQ domain containing protein B1 or nephrocystin 5. This protein, when mutated, causes senior local syndrome in patients, in human patients, and we are producing mouse models to replicate or see differences in mouse, in mutant mice with mutations in the same gene. So senior local syndrome is an autosomal recessive disorder characterized by nephron of thesis and progressive retinote generation. The worldwide prevalence is estimated to be one in a million, so it's relatively rare. The disease was discovered, like more than 50 years ago, in papers published in 1961 by Dr. Seymour and Dr. Logan, who gave them the name of the senior local syndrome. There are only six genes involved in senior local syndrome. They're called MPHP1, 4, 5, 6, and 10. One is still unknown and I have two students working on those genes. One is Christine Hunker Gokokia, a graduate student from Germany and Michelle Reed, a graduate student of the neuroscience program at the University of Utah. Nico was an empty PhD student in my lab starting the project several years ago. So what is senior local syndrome? In human patients you see diffuse interstitial cell infiltration, dysphiprosis, tubular epiphy, cyst formation, and end stage kidney disease is around 13 years of age. In mice you see mutant kidneys become smaller, shown here. The middle is reduced and cyst formation can also be seen. So it's a mouse, nephronophtesis, not generated by one of my favorite genes. It's another gene which does not cause retina degeneration, just nephronophtesis. So the high disease is retinitis pigmentosa, or LCA, labor congenital amoresis, and it's pretty well known to most of you. I'll compromise peripheral vision, tunnel vision, abnormalities in RPE and cones, and eventual lead in life blindness. So I just made this picture to show you the complexity of the nephrocystine genes going from 1 to 19. In all those different colors are different domains. In proteins you see there's virtually no relationship among all of those proteins. There's no sequence similarity and the function of all of those proteins is unknown. The proteins causing nephronophtesis, causing senorocrancy storms in yellow, and the other proteins, the other mutations in those genes, cause mechal syndrome, butaspeedle syndrome, and churir syndrome, which are synoptesies, syndromic synoptesies. So I'll just give you a very short introduction to the photoreceptor nomenclatures, so they are all on the same page in the rest of my presentation. Our photoreceptors are having outer segments and inner segments. They are connected by what was previously called a connecting selium, and it's now named the transition zone. The transition zone between inner and outer segments. You see there are lots of disks containing the photoreceptor cascade. There's an axon name, which is basically the backbone of the outer segment. I made an enlargement of the axon name to give you all the nomenclature we use in the next few slides. The axon name is a singlet microtubule arrangement. The transition zone is a tuplet microtubule, and there's a basal baldy, which is one of two centrals in the cell. The basal baldy, which is important for generating transitions on an axon name, and then there's a daughter centriole. So also important is photoreceptor outer segment development after birth from about three weeks of age. You see that at the neonatal baldy, the basal baldy comes to the cortex of the cell, and at P3, it pushes a little bit of a membranous bubble here, which will be the transition zone shown here, and eventually you form an axon name shown in blue. So this takes about three weeks from birth in mouse, and we are mostly interested in mutations that change this arrangement or prevent outer segment formation. So the basal baldy, as I said, is very important, basal baldy and axon name are very important for the outer segments' cavity. Without them, the outer segment would be very instinctive. So this shows you, it's taken from an independent paper, shows you that all those nephrocystins are closely connected, either directly or indirectly, forming an elaborate dethrone in the proximal cell here. This would be the basal baldy transition zone in bursing compartment and the axon name. So now we get to my protean, and you see it has several domains. One is called an IQative, which is a thermo-related binding site. SCC is a colloquial region for protein interaction, and ACCP is a site for interaction with another nephrocystin. So multiple non-mutations are associated with senior local syndrome as shown here, throughout the molecule. So what, since the disease is recessive, we were planning to make a knockout mouse, and for a knockout mouse, we would insert a gene type in one of the introns. The gene type basically prevents translation of the molecule shown here. So it stops like this. A gene type is a functional protein cannot be made, and evidence that we have knocked out, the gene is shown here in the control. You see the nephrocystin-5 accumulating at the transition zone, and in the knockout mouse, the protein is absent. So look to electrometanography at P14, when mouse eyes are opening, you see that the mouse is blind, has no response, same as P18. This is a scotopic for rats, and photopic for combs. Again, combs are not active, and the mouse basically is blind at birth. OCT can be used to look directly at retina, and you see that in the knockout mouse at P28, one mouse of age, we see a LCA type, or retinine-specific dosage type, phenotype, vestige generation of rats. Now this gives you an idea how odopsin is developed, and how other segments are developing with odopsin as a marker. Odopsin is the visual pigment in the photovisible odor segments. At P6 and P10, which is before eye-opening, you see slowly developing odor segments, and after eye-opening at P15, odopsin and odor segments are forming very strong. In the knockout, in contrast, you see no odor segments developing, odopsin mis-lokalizing in the inner segments, and eventually a rapid degeneration, and you see essentially one wall of nuclei still present at P30, which most likely is combs only. So degeneration can be assessed by looking at the thickness of an outer nuclear layer, which is only for the receptors, and you see there is a relatively little degeneration in the first two weeks of age, but after eye-opening at P14, there's a rapid degeneration at one month. Degeneration is mostly complete. So to our surprise, kidneys are completely normal. This is a kidney section at one year of age, and control and the knockout, you see there is no difference. So uterine kidneys are completely normal. This was assessed by Dr. Patricia Avello at the ARUP here at the University of Utah. And so there's no default of thesis in our mouse model, so it is not a is not a syndromic synopsis. It's just a non-syndromic synopsis, affecting only eyes. And kidney cilia, as you see here, are completely normal in the mouse. So cilia and kidneys are not affected, explaining why we don't have any form of thesis. And this is a picture of mouse and fibroblasts, and these are very important for development during embryonic development, and you see they have cilia in the controls, and they have cilia in the knockout, basically unchanged, consisting of patient body, daughter, son, and cilia. If this cilia would not be formed in the control, the mouse probably would not survive and would be happy on the glissom. So this is a reason why we have life mice. Now, looking at mechanisms, why do we have degeneration? We decided to force in sections and assay the retina. And you see a force in section here, O and L in a segment, the transition zone, and the outer segment, the base of the proximal part of the outer segment. And you see something green in here. This is a marker, each EFP-centered tool, calcium binding proteins, interacting very strongly with the transition zone and centrioles. So to close it up, we made an enlargement, and you see a transition zone is formed, here's the basal body and the daughter centriole, and these are the cells. In the knockout, we don't see, we see the basal body and the daughter centriole, but we don't see a transition zone explaining why we don't see an outer segment. And this is a second experiment with a protein called Parminium-1. Parminium-1 is located at the base of an outer segment, and it's responsible for formation of discs. And you see in the knockout that this protein mislocalizes, it doesn't form the disc, the proximal disc, and again consists of this lack of outer segment formation. On that ultra-structural level, we confirm that the control of retinal forms, here's the basal body, forms a very nice transition zone and a little bag of membranes, which eventually develops into another segment. And in the control, in the knockout, we see a basal body and a little bit of an extension which resembles a slanted transition zone that is not a normal transition zone. And at a later age, at p10, we see outer segment formation in the control basal body transition zone, axonene outer segment, and there's an outer segment in the NPHP5 knockout mobs. So what we have learned is that the NPHP5 knockout was made, the granular phenotype is LCA, a non-syntromic pseudopathy, degeneration of mutants, what photoreceptors is complete at one month. Mutant kidneys do not develop nephronophysis, nodal selia of NPHP5, photoreceptor transition zones, axonemes, and outer segments are absent. So we would like to know more details about cones. Cones are important for daylight vision. If we can figure out a way to rescue cones in those mice, maybe vision can be restored. So the way to go for cones is to make a double knockout mouse with an NML transcription factor. NML is neural retinal lucency transcription factor. It is required for what development. If we get rid of this gene by a knockout, we would make a mouse that develops only cones. So that's of course a very nice experiment because in the wild type mouse, we have only 3% cones and 97% rods. So we can now with this mouse focus on cones. And you see here a development of cones from P15 to 3 months into controlled mouse. And you see again our centrioles and transition zone and the outer segment, the cone outer segment forming. And in the on the NRL level and the ONL level, you see a beautiful stick ONL. This is only cones not rods are eliminated. And in the control, you see our two centrioles, no transition zone. And surprisingly at three months, the essentially low teach generation of the cones. So we have a stable situation of at least three months where the cone teach generation is basically very, very slow. And we have a window of three months or longer for gene therapy, for gene replacement therapy and rescue cones. So this is shown here again. Looking at ONL sickness, it doesn't change for a period of at least three months. Not significantly at least. So in these are the experiments to come up with a virus for gene replacement therapy. On the left is a vector for transfection of tissue cultural experiments. And this would be a shuttle vector for producing a virus that can be injected into the sub wetland space, expressing nephrocystin 5 and hopefully initiating cellulogenesis and auto second formation. And we have a window of six months to do this. So I mentioned at the beginning, we are trying to do a similar set of experiments with MPHB10. And MPHB10 is known to cause senior open syndrome in human patients. And so far we have produced a knockout. And you see the knockout produces a body beetle syndrome like phenotype. It has six digits and unfortunately the mouse does not survive. It's a neonatal mouse just born but not alive. So the future of those kind of experiments is to make a tissue wetland specific knockout and maybe design a second set of replacement therapies with MPHB10 mice. So this is the summary slide. MPHB5 nullally ill senior open cause senior open syndrome. Nullally ill same mouse effect only the retina. Kidneys are normal in the knockout and little rats degenerate within one month but cones are very stable and survive up to three months or maybe possibly even six months allowing for chain therapy. Here's my thank you slide. Grants from, I have two or one grants from the National Institute. Right now the research department Linus has an unrestricted grant for the department foundation finding Linus and that research foundation gave me small grants to support our grant students. Thank you. So Wolfgang obviously fascinating and indeed if we could figure out how to decouple rod degeneration from cone if we make that disease. I think the changes are very good. The chance are very good in mouse but I can't guarantee it will work in humans the same way because for mouse to humans a large step we would include a large animal model in between maybe a dog model. There is a dog model for senior open syndrome with MPHB5. So if we can do it in a dog model and we have mismodifications of the shuttle vector and the virus we may be able to get them. So I don't know if you mentioned but everyone, Wolfgang is our director of research as well as all of the other work that he's doing and we're just very proud of the outstanding work he has and Nico who he's talking about is one of our residents. Where are you Nico? Raise your hand. Nico here. I think he's on a call. He's not on a call. He said he's on a call. It shows you have this interesting combination of the people working together do interesting things. I actually wanted Nico to present his but he said he's on a call. We do that tomorrow. It's very exciting and you know it's how you've gone through this and the potential to change the cone side with in retrospect it's a huge blessing. So your mouse model of MPH5 is mostly LCA type but at least in humans it's not quite that bad. How do you kind of reconcile that? You know differences from human to mouse are very common. I have many genes which cause LCA in human and when knocked out there is no phenotype in mouse. So and there's a dog model with the same mutation. It's a frame shift mutation in MPH5 and this dog has a lid on it. So there is an axon in form. There's a transition zone and all this and another segment but then in each animal. So what came with the MPH5 mouse knock out why do you think that that gene is not required for kidney development or do you think that there's another gene that rescues the phenotype? The short answer is redundancy of nephrosistins. We've done this in so many nephrosistins and some of them can replace the function or substitute for the function that has been lost. So it's really depending on the tissue and on the cilia in brain, kidney, liver, but whatever. So it's a different situation. So another possibility in human therapy would be to stimulate another one of those proteins that kind of... Possibly if that would be a fantastic idea if that can be done, yes. Well thank you Wolfgang and thank you to all of our morning speakers. We'll break for lunch now.