 Alright, guess we'll get started. We have a few different presentations today from our talented medical students that are rotating with us. Nico Ronquillo is going to start talking about some research that he's done. Good morning. Good morning everyone. My name is Nico Ronquillo and I'm a fourth year medical student here at the U. And today I'll talk about my PhD work under Wolfgang Bär here at the Moran Ice Center and the topic of my talk is a new animal model for a human syndrome called senior locan syndrome. So senior locan syndrome is a syndrome which causes both retinitis pigmentosa and medullary cystic kidney disease in patients. The medullary cystic kidney disease is also called nephrinopthesis. This was first described in 1961 and is inherited in an autosomal recessive manner. This is an ultra rare disease affecting one in one million people worldwide. With these estimates we would predict that there's around two or three individuals here in Utah that have this disease and we have identified three families with senior locan syndrome here in Utah. The classic findings as most of you know of retinitis pigmentosa is photoreceptor degeneration. Patients with senior locan syndrome can have clinical symptoms of RP including night blindness, first appearing as early as eight years old. This is one of our patients with senior locan syndrome and here I'm showing just the hallmarks of RP including the bone spiculus around the periphery of the retina. Now similarly nephrinopthesis the other component of the syndrome also presents early and the median age to end stage kidney disease is 13 years old. There's three hallmarks for nephrinopthesis that I'd like you to know. The first is that these kidneys are small in in in patients. Second is the presence of cortical medullary cysts. And the third is the presence of interstitial fibrosis. And here are just pictures showing the cysts in a grossly in ultrasound and through histology. So the question is what causes senior locan syndrome? So far there have been seven genes when mutated are associated with development of this disease named NPHP145 and so on. NPHP stands for nephrinopthesis or nephrosistine genes. And the pictures on the right just correspond to the proteins that they encode. And I just wanted to emphasize that there's there's no similarities with how the proteins look like or their or their function. Out of these seven genes, NPHP5 are the most frequent cause of senior locan syndrome. And shown in this pie graph up close to 50% of all known mutations of senior locan syndrome. NPHP5 has mutations. And it's also called, NPHP5 is also called IQCB1. However, I wanted to point out that over 60% of senior locan syndrome cases in the world, these, we still don't, do not know the cause of mutations for these genes. And this includes the three families that we are studying here in Utah. We do not know the mutations causing the disease. So I proposed to study NPHP5 because I thought that this was an important protein study for understanding retinal disease specific to senior locan syndrome. I knew it was going to be important because when I looked at all the patients in the world with senior locan syndrome, with NPHP5 mutations, I saw that there was 100% penetrance when you have mutations in NPHP5 progressing to retinal degeneration and senior locan syndrome. So it was, I was very surprised at that. And on more practical reasons, there was no good animal model for mimicking the human disease. And also what was known, when very little was known about about NPHP5, the only things that were known on NPHP5 when I started this project was first that the mutations in the human disease caused a non-functional protein, meaning the protein's gone, it's just doesn't work. Second, we know that the NPHP5 was in the photoreceptors. Specifically, it was localized in mouse and human photoreceptor connecting cilium transition zone or transition zone and outer segment. This is just an immunocytochemistry from one study. The ONO or the photoreceptor cell layer here in blue, and this is just a blow-up, is in here. And the outer segment of photoreceptors is labeled green here by NPHP5, suggesting that NPHP5 localized in the outer segments. Now EM studies or electron micrographs show that these little dots in here correspond to NPHP5, suggesting that NPHP5 is located in the connecting cilium, which is a bridge from the inner segment of the photoreceptors to the outer segment of photoreceptors. And protein trafficking goes from inner segment to the outer segment of cells. So in humans, we know that loss of function mutations in NPHP5 caused a disease. And to model this, we made a global knockout of NPHP5. And just briefly, we basically inserted a gene trap early in the gene so that we have early termination of protein translation having a non-functional protein. So we confirmed absence of the NPHP5 wild-type allele in the knockout mice through PCR. And we also confirmed absence of the NPHP5 protein through Western block, shown here in kidney lysates. In photoreceptor cells, we also showed at an early time point that the knockout mice do not express NPHP5. The blue here is just the photoreceptor cell bodies, the outer nuclear layer. And the red is the NPHP5 here in a control animal. The green is just a marker for the connecting cilium called centrin2, and it's labeled here as green. In the knockout animals, as we can see, the structure early on is a pretty normal, except that NPHP5, we just can't see red, confirming that we do have knockout animals. So with this, the animal is viable. The first question we ask is whether the absence of NPHP5, like in humans, sufficient to cause retinal degeneration in the mouse. And the answer is yes. So these are control retinas at different time points at postnatal day 6, 10, and 14. And here I'm just showing, again, the outer nuclear layer at different time points. And we label the outer segments here in red with rhodopsin. And as we can see, as the retina develops, we see increased rhodopsin or the outer segments are maturing. In the knockout animals, what I'd like you to focus on in this figure is just the thickness of the outer nuclear layer. In early animals, before eye opening, we see that the outer nuclear layer is pretty normal. But once the mouse opens its eyes around P12, so this is postnatal day 14, we see significant degeneration. And these results become even more significant as time lapses, especially at one month of age. And this is a depiction of the fundus photos at one month of age. So these are control animals, both wild type and heterozygote fundus photos, and this is an OCT. And these are normal. But in the knockout animals, we see a hyper fluorescent signal here suggesting pan-retinal thinning. And this is confirmed by OCT as well. So this is at one month of age. And when you look histologically, there is maybe one cell layer left in the photoreceptor cell bodies. So what I've shown you is structurally, there is retinal degeneration. But how about functionally, do we see any functional deficits? And the answer is yes. First, there's absence-cotopic ERG response in the knockout animals. These are just representative traces of ERGs from the wild type and heterozygote animals at different light intensities. And in the knockout animals, we see a complete absence of the rod response at one month of age. We actually never see a rod response, even at the very earliest time point that I've done it at P14, suggesting that functionally, the rods are not working. The cones also at one month of age show the same results. And they have absent-photopic ERG responses shown here by my representative traces. So we see just a flat line at one month of age. So what I've shown you so far is that the loss of NPHP5 in the mouse causes retinal degeneration similar to the human disease. But how about the kidneys? And so just briefly, I just want to remind you that nephronithesis has three hallmarks, small kidneys, presence of corticomedulary cysts, and the presence of interstitial fibrosis. And the first thing that I've shown is that the knockout animals do are smaller and exhibit degeneration. So I've labeled cells undergoing apoptosis or cell death through tunnel staining, so the green stain. And in the knockout animals, we see significant increase of cell death in the kidney. So these are just cross-sections of a kidney. The second hallmark was presence of cysts. And this is just one example of just the cysts that form in these mouse. And this is most likely a ruptured cyst in the knockout mouse. And then finally, the third hallmark is fibrosis. So we looked at the animals, and at one month of age, we do see significant staining with a trichrome stain, which stains for collagen fibers in the knockout animals. And this is minimal fibrosis, but if you quantify it, it is significant compared to wild type. What I will tell you, though, is that there seems to be an intermediary phenotype in the heterozygote animals. So at this point, we think that we have a good model for senior-locan syndrome. So we wanted to explore further the mechanism of NPHP5. And our initial hypothesis was that NPHP5 was important in connecting cellular formation, which is that little zone, that little bridge that connects the inner segment to the outer segment of photoreceptors. And first, so to be able to see this, we've done EM studies. And these are wild type and heterozygote animals at a very early time point at P10. And here, what I'm showing with the IROs is a normal connecting psyllium. So this is the basal body. This is the connecting psyllium. These are outer segment of photoreceptor cells. And the heterozygote animals, it seems to be normal as well. But in knockout animals, the first finding is that we never see an outer segment. We never see these stacks of disks forming. And the connecting psyllium, it's very difficult to say, but it seems that it's abnormal as well, having a smaller lumen compared to both the wild type and the knockout animals. Now, this is a static picture. We still do not understand, really, the function of NPHP5. But we think it's important for protein trafficking, trafficking protein synthesized in the inner segments up to the outer segments. And we have several evidence for this hypothesis, including mislocalization of rhodopsin very early on. I've shown this figure before, but now I'd like to highlight that at early time points of P6, we see that rhodopsin already traffics normally to the outer segments of these cells. But with the knockout animals, we see rhodopsin mislocalizing. And it's more significant here at P10, we see that it's around the perinuclear region of these cells. Suggesting that rhodopsin and we've seen for other proteins are having a difficult time trafficking to the outer segment of cells. Now, it doesn't seem to be a global phenomenon, meaning it may not be causing all proteins to mislocalize. There seems to be a specificity for this because in cone cells, and this may be important clinically as well, so in cone cells, so this is a control animal, and we've labeled the cone opsin with S opsin here shown in red now. We see that these are the cone outer segments. In the knockout animal, so we still see that at a stage where we don't see rhodopsin anymore in the outer segments in the cone cells, we still see evidence of normal trafficking of cone pigments. So this, we can infer from this that the human disease or in the mouse model and maybe extended the human disease that this is that the cell target of NPHP5 mutations are rod cells. And cone cells may be preserved and degenerate early on once all the rod cells have died already. And this may be very important for designing strategies for therapeutics. So just as a summary of what I've told you, the NPHP5 knockout mouse recapitulate the pathologic hallmarks of senior locan syndrome, including the progressive retinal degeneration and cystic kidney disease. Second, we think that NPHP5 is important for completion of siliogenesis or outer segment formation. I haven't shown you some of our in vitro data that suggests that it is important for siliogenesis as well. And then finally we think that NPHP5 plays an important role in protein trafficking in photosynthesis. And I just wanted to end my talk that really the ultimate goal for this project, which started for identification of NPHP5 as an important gene for retinal disease in humans and then modeling the disease using in vitro and in in vivo and in vitro tools, the ultimate goal really is that we want to use these models as a platform for gene therapy and drug discovery for this disease. And some of our ongoing studies that we have in this effect is that we already have an adeno-associated virus that carries a wild-type copy of NPHP5 and is ready for basically subretinal injection in the NPHP5 knockout mouse to be able to show slow down retinal progression in our animal model. We have also have a new collaboration with the National Eye Institute to do a high throughput drug screening using stem cell lines from our mouse and they have also identified a family in Chicago with NPHP5 mutations and to do high drug screening in the induced pluripotent stem cells from that family. And ultimately we want to have those drug targets to be validated in the mouse model itself. And then, as I've alluded earlier, we also have to go full circle, we have human genetic studies as well. We've identified three families in Utah with Senior Locan Syndrome, very rare disease, and we have done for the one family, we have finished completed exome sequencing and we have identified 10 new candidate genes and so we may be able to find new genes and repeat this process again for these very few patients. With that said, I thank my mentor Wolfgang for allowing me to start this project and his mentorship at the lab. We have many collaborators here at the Moran Eye Center and I am hoping for many questions. Thank you. Yeah, that's a good question. So the in vitro assay that we first will probably test first is an RNAI knockdown. So this is, I didn't show this, but these are kidney cell lines. So we think that the primary cilium is a similar structure to the connecting cilium or photoreceptor cells. So in here the kidney cell lines express NPHP5 and the no marker acetylated tubulin. When you knock down NPHP5 we see absence or significant decrease of NPHP5, not a 20%. And so the goal is to be able to do high drug to rescue this effect, to be able to see whether we can rescue the phenotypic or the primary cilium formation again. So and I think it's a pretty robust assay. Acetylated tubulin is good. The assay is straightforward. So I think we have a good readout. Yes, they can. Thank you, Nico. So there's a little bit of a switch from what's on the schedule just in the order. Next up we're going to have Mike Taggart from here at the University of Utah speak about cilium biomarker.