 We're gonna try and get started since we have two presenters this morning. And our first presenter is Nico Runquillo, and he is an MD-PhD graduate student in the Bayer lab. We'll be speaking about the New York-Lokin syndrome. Hello. Can everybody hear me? Thanks, Leah. So good morning, everyone. Again, I'm Nico, and I'm an MD-PhD student in the lab of Wolfgang Bayer, just up in the sixth floor here at the Moran. And I'll talk about today my thesis. Project on a retina renal dystrophy in humans called senior-Lokin syndrome. So first, I wanted to introduce to you to an organelle called the primary cilium. This organelle is expressed in most cell types. And as shown in this cartoon, this organelle protrudes out of cells. As the name implies, there's only one primary cilium per cell. And in contrast to the motile cilia that we usually think about lining the organs, the lumens of several organs, the primary cilium is now, is non-motile. We now know that it is involved in several sensory functions, including photosensation. It's now well-accepted, the photoreceptor-connecting cilium, and outer segment is a specialized primary cilium because it shares the same fundamental structures as that of the primary cilium. Shown here is a cartoon of the structure of a primary cilium and classic is the nine pairs of microtubules that form as a tube that forms the axonim, which is basically the skeleton of a primary cilium. This axonim is anchored by the basal body. This is an EM of a photoreceptor cell showing also similar structure, which includes the basal body as well as the classic microtubule axonimal structure in the connecting cilium. Again, this is just a cross-section through the connecting cilium. And really, the connecting cilium is thought to be a gatekeeper of protein transport from the inner segment to the outer segment, and this is a very small structure. Not surprisingly, mutations of genes that affect cilogenesis or primary cilium function can manifest in virtually any organ. To mention a few, Bardet-Biedel syndrome causes obesity and polydactyly, and Juber syndrome causes cerebellar-vermus hypoplasia. Now, senior-locan syndrome is a psyliopathy that causes a retinal degeneration as well as a medullary cystic kidney disease called nephrinophthesis. So more on senior-locan syndrome. This was first described in 1961 as an autosomal recessive disease that causes, again, RP and nephrinophthesis. Symptoms usually start in the first decade of life, and this is usually polydipsia and polyurea. The median age of NSAID kidney disease is around 13 years old. This is a very rare disease worldwide with a prevalence of one in one million. The retinitis pigmentosa in senior-locan syndrome, I won't talk a lot about this, but usually present several years after the initial renal symptoms begin or several years after a patient has had kidney transplant. The RP in this syndrome is rapidly progressing. Nephrinophthesis means disintegration of the kidneys. Classic findings in nephrinophthesis include corticomedular cysts as well that can be seen actually in ultrasound here and smaller to normal-sized kidneys. This is important clinically because most of the classic autosomal dominant cystic kidney diseases are usually larger diseases. And nephrinophthesis kidneys are smaller. Histological findings include interstitial fibrosis as well as the tubular cysts shown here. And there's also a lot of basement membrane destruction. So three years ago when I, after joining Wolfgang's lab, so he really asked me to come up with a project that involved a disease gene, which of course naturally interested me. And I found that these NPHP genes or nephrosistine genes and they're all called because all mutations in these genes all cause the kidney phenotype or nephrinophthesis. But what interested me the most was some of them caused retinal pathologies as well. And this includes NPHP one to six, eight and 10. I focused on NPHP five in here for three reasons. First, I was struck that NPHP five, 100% of the mutations NPHP five caused the retina and renal pathologies. The second reason I got interested because NPHP five mutations only cause retina and renal phenotype. There were no other organ involvement in contrast to the other genes. For example, NPHP six or Cept 290, it had a retinal renal disease but also affected the brain. So since and I guess practically speaking, there was really no functional studies on NPHP five. We don't know how it was working and there was no animal models of senior local syndrome. So I was able to capitalize on Wolfgang's expertise in making knockout mice models. And since then it's now considered, it's now well known that mutations in NPHP five are actually the most common cause of senior local syndrome in humans. Again, there's several mutations that has been mapped that have found in families. And the main point that I wanted to say in this slide is that all the mutations cause a truncated and non-functional protein which is classic of autosomal recessive disease. So more on the protein and PHP five or nephrosystine five. It's also called IQ-calmodulin binding one protein or IQ CB one. And it contains two IQ domains for calmodulin binding. Calmodulin is a calcium binding protein. And it's interesting or tempting to speculate right now that NPHP five is involved in calcium signaling. It interacts with RPGR and NPHP six or sub two 90 which are known genes mutations that cause retinal degeneration as well. So besides that there were really nothing else known about NPHP five. And so we came up with two hypothesis that deletion of NPHP five in the mouse will lead to the development of the retinal degeneration and cystic kidney disease mimicking the pathology seen in humans. And the second hypothesis was the function of NPHP five which is that it is necessary for cilogenesis or primary cilium function in photoreceptors and kidney cells. So to study NPHP five, I first confirmed localization of the protein in photoreceptor cells and kidney cells. So this is from a polyclonal antibody from a group in the University of Michigan. And we've confirmed that NPHP five is indeed localized to the connecting cilium of photoreceptor cells. Acetylated tubulin is a known marker of a connecting cilium shown here which I'll also use this marker in several of my other assays later. So I've also confirmed NPHP five localization in a kidney cell line called IMCD three cells here shown in green co-labeling with acetylated tubulin. So basically these cells are grown in a dish and at specific conditions, these cells will grow up primary cilium which I can label. Finally, we've also tested in basically all organs of the mouse and tested for NPHP five mRNA and shown that NPHP five is expressed ubiquitously in all organs except notably probably the lungs is it's not expressed. So this result was really not surprising as other NPHP genes were expressed ubiquitously but this also was worrisome because knocking out in the mouse may cause other problems in other organs where it's expressed that may lead to not viable mice. We went ahead and made knockout mice using an embryonic stem cell line with a gene trap in intron four of NPHP five. So basically what this gene trap did was introduce a stop codon that would prevent protein translation basically in early termination and truncation. This is just a representative genotyping results showing absence of the wild type allele in my knockout mice. And now I also confirmed the knockout by Western by Western by checking for protein from kidney lysates. So also in this construct going back to this figure we have a reporter called lack Z basically what so I can basically check where endogenous NPHP five is expressed developmentally. So I took a wild type, heterozygote and knockout animals at E13.5 or at E13.5 embryonic stage and stained with X gal for lack Z expression. So there should be no expression in the wild type and the knockout mice since they have two copies of the lack Z reporter should have stronger expression. And as expected NPHP five is expressed ubiquitously with a prominent expression in the eye and other organs including the limb buds, the spinal cord which I won't talk about too much. But if you're wondering what is NPHP five doing in these organs we do not know the answer to that but it seems like at least in the adult mice they seem normal except in the organs that I'll be talking more about. So again despite ubiquitous expression of NPHP five in the mouse global knockout mice are viable. But the first science that I saw was at postnatal P14 and postnatal day 28 the knockout mice seem to have decreased body weight compared to the wild type and heterozygote animals. Besides this I also noticed that the knockout mice were dying randomly. So I applauded survival knockout mice in black versus wild type and hetes using a Kaplan-Meier curve and shows that it is a significant. So in the human disease the kidney disease is the one that causes death in the patients and we suspect that it's also the kidney disease in the knockout mice. We started characterizing the retina at P28 which or one month of age which is considered young animals. So with the help of ballast lab we took fundus and OCT photos of the wild type, hetes and knockout animals shown here and in knockout animals we noticed thinning of the retina which is more better seen at OCT. However since this is a global knockout mouse it's really hard to say from this picture which layer of the retina is involved. We next checked for rod and cone function by measuring the animals scotopic and photopic ERG responses. So with increasing single flashlight intensities of dark adapted mice so increasing from top to bottom a heterozygote mice at the one month of age seem to have normal responses. However knockout mice have completely absence scotopic ERG responses which suggests complete loss of rod function. So after light adaptation we measured photopic ERG responses at increasing light intensities again and heterozygote mice also seem to have normal responses. However knockout mice had completely absent photopic responses suggesting loss of cone function at one month of age. The mice were blind at one month or at P28 which is just a very rapid retinal degeneration especially in the mouse. So we checked for retinal function at an earlier time point at P14 just right after eye opening because we really don't know whether this was a retinal degeneration problem or retinal development problem. So even at this early time point we see this is the scotopic measurements and this is the representative photopic measurements that there were absent ERG responses from the knockout animals. So we then sectioned the retina to really see what was going on and look at the morphology. So at P10 which is right before eye opening we see already thinning of the outer nuclear layer which is more pronounced at P14 and this is the outer nuclear layer of the knockout mice and the heterozygote seem to have normal outer nuclear layer and this is just quantification of P14. At P28 I've also done ICC on the retina sections green labels for dopsin and blue labels for the cell bodies and at P28 there were complete absence of the outer nuclear layer which makes sense because we did not have any ERG responses at P28. However it didn't make sense why we didn't have any P14 signal because there were still some photoreceptor cell bodies left at P14. It's hard to see in this picture whether there's outer segments involved and so the hypothesis was that at P14 outer segments really don't develop and the hypothesis that NK35 is important in connecting cellium structure and function one can easily look at rhodopsin for proper localization of rhodopsin at early time points. So at P10 heterozygote mice have normal localization of rhodopsin in the developing outer segments shown here in green. So in knockout mice however it doesn't seem that rhodopsin get transported normally. So there's two things happening here. First accumulation of rhodopsin in green in the near the cell bodies of the photoreceptor layers. And it's harder to see here but the green signal in here seem to be in the inner segment of the outer segment. If you look at in the wild type in het there seems to be a black line which is in the inner segment of these sections and rhodopsin is I guess more distally from that. And in here it's harder to see but there's no black line which suggests that it's accumulating in the inner segment. We're trying to confirm these results now we're just waiting for our EM experiments to check with the ultra structure of the connecting cellium as well as to see whether the outer segments actually develop even initially. At P14 we also check for cone outer segments shown here with cone arrestin in red and shown here are beautiful cone outer segments but as you already know at P14 the photoreceptor layer is degenerated significantly. It seems that there are still cone arrestin maybe in the inner segment accumulation as well. So we also know now that the cells are dying through apoptosis as measured by tunnel staining. So at P10 I can detect some dying cells at the photoreceptor cell layer. Again this at P10 there were already rapid accumulation of rhodopsin but cell death seemed to have a lag before or cell death has a lag after a rhodopsin accumulation and I can't detect any tunnel staining in wild type of heresiotic animals. Now at P14 I do see the occasional tunnel staining in wild type but as you can see here there's massive apoptosis happening in these animals which suggests a specific pathway for cell death for these animals. So we now know that the global NPHP5 knockout mouse have an extremely fast retinal degeneration phenotype. So how about the kidneys? So I took sections of one month old animal kidneys and noticed that the kidneys in the knockout animals are significantly thinner all throughout both in the cortex which is the peripheral region of the kidney as well as in the medulla in the middle part. Also there seems to be lots of holes or cysts particularly in the cortex of these animals. So again presence of cysts and atrophied kidneys are important clinically. So I wanted to explore more why the kidneys are smaller in the human disease it's not really well understood why the kidneys are smaller. So again I thought it was reasonable to check whether the cells were dying. So again I did tunnel staining at kidneys at P28 or one month age labeled in green and the wild type and heterozygote seem to at least in this magnification don't show a lot of cells dying. Shown here are two pictures to knock out mouse kidneys and shown as just increased apoptosis primarily in the cortex or the metanethros region. Also shown here is a cyst that must have ruptured. These are just higher magnifications of wild type and knockout animals showing that there are cells undergoing apoptosis in wild type animals which is expected because this is a highly proliferative or active cell turnover layer in the cortex. But as you can see in the knockout animals it's just increased. Now the cell death is not just in the cortex but it's all throughout the kidneys as well including the medulla which I'm not showing here. So I've shown that my knockout mice have smaller kidneys which is probably due to apoptosis and that there's a presence of cysts. Now there's the third clinical finding in humans which is presence of fibrosis in nephrinopthesis. And so I checked for fibrosis using trichome staining. This is a wild type and heterozygote animals and trichome stains fibrosis for blue and it's harder to see here but after quantification there are there's lots of blue in the knockout mice. Again it's harder to see in this picture but it is significant. This is the blue arrow points to an area of high fibrosis and this is just another example of a cyst. So we've confirmed both retina and kidney phenotypes reminiscent of senior locan syndrome in our mouse model. For the second point which asks what is the function of NPHP5? We think that it's involved in early cellogenesis in photoreceptors and kidney cells. And so as I've mentioned we're just waiting for our EM data to come out to look at the connecting cellulium ultra structure. But I did do an in vitro experiment to test this hypothesis in a kidney cell line, again IMCD3 cells. Again at conditions to grow primary cell we can see that the cells grow a lot. Each cell will grow one primary cellium and PHP5 is labeled green and co-labeling with a known primary cell marker acetylated tubulin. After knocking down NPHP5 with RNAi we see that the cells are still there but have significantly decreased primary cellium which really suggests that it's involved in early stages of primary cell formation. I've quantified this and in my hands 70% normally 70% of cells usually develop a primary cellium but after knocking down NPHP5 the primary cell number decreases to less than 10%. So in summary a high suspicion of senior locan syndrome should happen when there's a history of inherited renal disease or a history of renal transplant with retinitis pigmentosa. Now extra organ involvement, particularly the brain by an MRI scan should rule out other overlapping syndromes particularly Juber syndrome that causes both retina and renal phenotypes as well. Mutations in NPHP5 is actually the most common cause of senior locan syndrome and that knocking out NPHP5 in the mouse which is my thesis project is recapitulate all clinical findings as far as we know in humans which is retina pathology as well as kidney pathology. So we now have a senior locan syndrome mouse model. So finally we think that NPHP5 is important information of the connecting cellium and primary cellium and we should know the EM results and basically by the end of this month. So I'd like to end by describing a genetic study that we are also doing in collaboration with McDiangeles. So although most of the mutations causing senior locan syndrome are known there are so more genes to be found and we have individuals that have been diagnosed actually with senior locan syndrome here at the Moran by Dr. Bernstein. This is the family that Dr. Bernstein is taking care of and also Dr. Katz and we have already entered them in this study and we've collected blood and we are going to do whole exome sequencing in this family and this is just historically speaking. I found, when I started this project I didn't know that we had senior locan syndrome patients here since because it's a very rare disease but fortunately for me Dr. Bernstein is in my committee and when I proposed this project at the end he mentioned to me that he has patients with senior locan syndrome and since then I've found out Dr. Katz is another patient and in the Department of Nephrology there's also I believe she's nine years old now that also has senior locan syndrome. So there's three families which should saturate Utah because we have around three million people in Utah so expecting around three individuals here in the state. So we're doing whole exome sequencing right now to find out excitingly probably more causative mutations of senior locan syndrome or at least existing mutations. Of course I've genotyped NPHP5 and I didn't find any mutations in NPHP5 in these families. So finally I'd like to thank Wolfgang for really letting me do this project. This is not his project because I started this and he was a really great mentor for me. My committee members helping me with a project and I'm funded by an F31 NRSA from the NEI. So with that I'd like to take any questions. So throughout my graduate career that's a question that I've always thought about and the underlying in all of these maybe studies that I make is my I guess interest in understanding more fundamental molecular mechanisms of retinitis pigmentosa specifically or in the syndrome. So I alluded in the beginning of the slide that NPHP5 may, it's tempting to speculate right now but that it may be involved in calcium regulation and that there may be some calcium problems within the vicinity of the connecting sodium with these. And so if that's the case then maybe hitting it with downstream targets is important. Although that's purely speculation there has been clinical trials at least on the kidney disease for nephrinopthesis with basically calcium mimetics as well as downstream map kinase inhibitors. Basically it's downstream of calcium pathways that's shown decreased cyst progression and increased survival at least in animal models. And so I think that there's still I guess there's still so much to learn about the molecular mechanisms of these basic diseases that is I guess also to my surprise are not untapped until now. I'm looking for another model organism actually to test my hypothesis for NPHP5 in calcium homeostasis. And so I'm expressing NPHP5 right now in C. elegans to test my hypothesis. So going back to your question, yes, definitely orphan drugs. This is a very rare disease. More rare than usually were the clinical trials for orphan drugs. And I think it's a combination of understanding molecular mechanisms on being better at gene therapy. I'm making also a viral vector for to rescue this with AAV viruses. So I guess it's a combination of a lot of things for at least for the retina disease, for the kidney disease, for gene therapy it's very hard. We don't know a lot of AAV viruses that can transducent give back to these patients. There's problems with renal transplantation. But again, the molecular mechanisms are I think lagging behind and that we, for nephrinoptesis as well, that there's still a lot to be explored. And I don't know the clinical trial now of the two calcium and the map kinase inhibitor that they've used for nephrinoptesis mouse models. So I can't remember what company they were, but. So just one of the comments about that, the National Institute had a big contest, I'm sure, I don't know if you've all heard about it, where they've asked, they've accepted it in a while, she wouldn't have even, and they go, very good job, unless you had it in some way. So these are autosomal recessive. And these are, at least for these pedigrees, they're not consanguinous. So there are diagenic recessive mutations that have been reported for this disease as well. So it may not be just from one allele, but maybe for two, that maybe it is. So are other families unused? This is. Be a truth.