 So our final talk, Jun Yang is a research associate professor and she's going to talk about understanding the mechanism underlying cone rod dystrophy and you get extra credit points for being the last person. We'll see if Randy can get that hooked up. That's Spiro's Trail, mid-mountain in Park City. Okay, so good afternoon. So I'm Jun Yang. So first I would like to thank David Cuisart and Nick Mamelos for giving me this opportunity to present our research here. So the topic of my presentation today is understanding the mechanism underlying cone rod dystrophy. So the long-term recent interest in my life is to understand the mechanisms of inherent development in generations and eventually hopefully we can find some effective treatments for these diseases. So during the past 80 years we have been focusing on Archeo-syndrome type 2. We use mouse mono to study this disease. Archeo-syndrome is syndromic disease, so it's RP, it's a syndromic type of RP together with hearing loss and severe dysfunction. So we found that the genes that encode in that proteins, the positive genes encode in proteins form a complex. So we studied this complex in both photoreceptors and also here cells. So today I will not talk about our research on Archeo-syndrome. We already published a series of papers, so if you're interested you can just read our papers. So now we just want to talk about our research on cone rod dystrophy. So about four years ago we became interested in cone rod dystrophy and started a new research project. Compared with RP, cone rod dystrophy, CRD is a rare disease, so in fact, why in 30 to 40,000 people worldwide. However, compared to RP, CRD had more severe problems with CRD. So unlike RP, RP affects the rod photoreceptors first and then cone photoreceptors, and CRD affects cone photoreceptors first and then rod, and sometimes CRD can affect both rod and cone photoreceptors at the same time. And also CRD can be inherited with all three material patterns of inheritance. So although right now up to 30 cause genes has been identified for CRD, and the genetic causes of the more than half of CRD cases are unknown, and for the identified genes, quite a few, we don't really know their biological functions or very limited information about their biological functions and all. So in my life, we're interested in C8 of 37 gene, which was identified by Astrolado Cosconal in 2012. And since then, more than eight more papers has been published to report more C8 of 37 mutations and also patients in different ethnic groups. So here I just show you the mutations identified right now in C8 of 37 gene. We can see that the mutations distributed throughout the gene and they include spyside mutations, nonsensic mutations, nonsensic mutations, and also fission mutations. And these mutations can cause, you can see CRD and RP, and also RP together with early macropathy. And also you can see it can cause barbidosyndrome. So barbidosyndrome is another syndromic form of RP, which is characterized by RP together with obesity, renal problems, polydactyly, and positive deficiencies. So at the protein level, this protein has 207 amino acids in humans. However, the challenge to understand the function of this protein is that there is no known functional domains and also there is no known similar proteins. So that's not, this protein is just named by the position of the gene in human chromosome age. So, but when we aligned the C8 of 37 protein sequence from mammals to lower eukaryotes, we found that the C-terminal region of this protein is really conserved. So it indicates that this protein probably play a role in a very important process. Okay, so what's the function of this protein and why mutations in this gene can cause retinal degeneration? So first, we just set up an animal model to study this question. So we set up to generate the C8 of 37 knockout mice using Christopher Cassini technology. So by using two small-guide RNAs targeting X1 and X5 of the gene, we generate three mutant mouse mice. So the first one has six base pair deletion in X1 that also includes these translations that call the ATG. And the second has a seven base pair deletion in X5 and third has a large deletion between X1 and X2 of X5. And then we did the immunoblot analysis. We found that all three mutant mouse mice, they don't express C8 of 37. All truncated proteins indicate that these three lines are complete KO mice. Okay, so then we assess the retinal function of this knockout mice using electroretinogram BR gene at five weeks of age. So we can see that the O3 mice, so the red color is the red line, green color is the red line, the third line is blue color, and also the dash line is mutant mice. So we can see that all three mutant knockout mice have about 50% reduction for scotopic A wave amplitude, and also 50% reduction for topical B wave amplitude, indicating both rod and the comb dysfunction. And then we just focus on one mouse line because these three look the same, the same retina phenotypes. So we focus on one mouse line, we test ERG at different ages, and we found that both rod and the comb dysfunction show a progressive trend. And then we asked whether these mice have retina degeneration, because sometimes the retina function has a problem, but the photoreceptors, the number is okay, it's still alive. So first we use this non-invasive approach, the OCT approach, to study the retina. So compared to the control retina, we found that in the knockout retina at both two months and six months of age, this out-segment and inner-segment junction line is missing. And also at the six months of age, this photoreceptor nuclear layer is much thinner compared to the control, indicating retina degeneration. Okay. And then we did the standard histological analysis using like microscopy, and the results just confirmed our OCT finding. So you can see that the outer-segment layer and this other nuclear layer get thinner with age, so at beginning they are fine compared to control. And eventually the outer-segment is disappeared. Okay. However, we found that both female and male mice, they don't show obesity up to six months of age. And also they don't show these polydactylic things, which are characterized as a bit of syndrome patient symptoms. So in summary, for this part of our study, so we successfully generated the C8 officer 7 knockout mouse models. And these mice show both rodent upon dysfunction and progressive retina degeneration. However, they don't show other symptoms outside the retina. So these models, good animal model to study retina degeneration in C8 officer 7 deficient patients. Okay. So with these mice, we asked the question, so why C8 officer 7 mutations or knockout cause retina degeneration? So we first look at example photoreceptor morphology by scanning electron microscopy. So at both postnatal, they 10 and 30. So compared to the control photoreceptors, we saw this knockout photoreceptors show abnormal auto segments, which is here. So these auto segments show a thicker and less uniform in diameter and also less densely packed. And then we wanted to know what's happening inside this auto segment. So we did the transmission electron microscopy. So this is just the control of the segment. So as expected, we see membrane disks are tightly stacked and horizontally aligned. And in the knockout of the segment, we see some membrane, this is probably not this, just the membrane disk, the very long and the vertically aligned. And apparently, we do see these membrane walls in this auto segment region and also this multi-mascular body light structure. And however, the other several structures and also the compartments are normal. So such as this connecting ceiling, phasal body, mitochondria and scenery ruler. And even ER, power geobriders and also the synaptic termos we can refine. So when we zoom out to look at the membrane disks in the knockout of the segment, we see that this is horizontal membrane, they kind of grow over, overgrow as the membrane region and then turn vertically. And also we try to look at horizontal disks and vertical disks and we don't see plasma membrane in between. So this suggests that the horizontal and vertical disks, they are wrapped by plasma membrane inside the same auto segment. So this can explain the phenotype we saw in scanning electron microscopy. The outer segment is thicker and less uniform. Okay. I just want to clarify that the heterozygous knockouts look like a wild type. Yes. Okay. Because this disease is, for C-8 officer cell immunosomal is autosomal recessive. Okay. So for the height mice, we see the, not really phenotype, we see the structures is similar as well. So we consider this control. Yes. Okay. So and then we did a series of your immuno-blocking analysis. And then we found that many auto segments, membrane proteins and associated proteins, their expression level decreased, which I marked here in red. And other proteins, they okay, they has the same, the normal expression level in the non-caught photoreceptors. And also I marked these proteins. I saw these proteins in green. So these proteins are encoded by non-CRD cost genes. So this indicate that the mechanisms of CRD pathogenesis in patients with C-8 officer cell mutations probably shared among patients with mutations in these genes. Okay. So and also in the field it's known that the RDS gap two and the CMGP one are involved in organizing all the segment disks in photoreceptors. So the association between the RDS and the gamma one oligomers with this CMGP one can link the membrane disks to the plasma membrane. And also the self-interaction of gap two is responsible for disk stacking. So we found that the reduction of gap two, CMGP one and RDS, so that can explain why we see that the membrane disk misalignment in the non-caught of the segment. So now we know why this C-8 of 37 non-caught can cause retinal degeneration. However, we want to know now why C-8 of 37 non-caught can cause off-segment membrane protein reduction. So this is, we still in the process try to understand this question. So there's no clear answer to that. I just share with you our preliminary data. So first we want to know where C-8 of 37 protein localized in photoreceptors. We see the immunostaining of C-8 of 37 and we found that the green signal is from C-8 of 37 staining. So we found that this protein is localized throughout photoreceptors, but it's not in outer segment. However, in the non-caught mice we also see this kind of signal pattern. So in that case like this indicate that the signal we see in this control retina is probably from a specific C-8 of 37 signal, but also mixed with non-specific and non-specific signals. So to verify the localization of C-8 of 37 photoreceptors, we collaborate with our Shavisk slide. So we did a tangential section of the retina and then we examined where C-8 of 37 is localized in every consecutive retina sections. And here is the result. So basically these RDS labels were out of segment. And then for stucing is localized where photoreceptor is exactly out of segment. So we see that the C-8 of 37 is in the inner segment, not out of segment. Because like we know, non-caught of C-8 of 37 can reduce outer segment memory protein. So what's going on? So it's possible that we know that the outer segment memory protein is synthesized in the inner segment and it's transferred to the outer segment. So that means something went wrong doing this process, synthesized, and then transferred to the outer segment in the KOIs. Okay, so we did even staining of all, not all, just like several of these outer segment memory proteins we know they are reduced in the KOIs. However, we found that most of them are just localized normally in the outer segment. So this indicated that the C-8 of 37 probably not involved in their trafficking to the outer segment. And also we find that the outer segment memory protein reduction occurs at P5, postnatal B5 during development. And at this time point, photoreceptor still differentiating. So the connective scenario is forming that the outer segment has not been formed. So at this stage there's no outer segment, but the outer segment memory protein already reduced especially. This also suggests the idea that C-8 of 37 is not involved in outer segment memory trafficking. So now we think it's probably something wrong with protein synthesis or degradation. So we still try to do the experiment to detect, like to diagnose which part is really a problem. So in our lab we also try to identify C-8 of 37 interacting proteins. That will tell us what cell process the C-8 of 37 plays a role. So we did a series of proteomic analysis using mass spectrometry. So we did this mass-back study on proteins that even are precipitated by C-8 of 37 antibody from mouse retinas. And also we did the pull-down using GST tag C-8 of 37 protein from bone-mine retinas. So and then we did this five times. So each time we definitely have electric controls. So compared to the electric controls we have these unique proteins identified by mass spectrometry. So that's a lot of proteins. And then we just think if there's a real interaction it should be show up multiple times. So in fact we don't see one protein that show up all five times. So but we do see a protein here that show up four times. Even across different approaches from mouse and bone-mine retinas. So this protein is interesting. So it's encoded by a known home rod discipline gene. However this protein, the function of this protein is not really well studied. So if this interaction is true we still don't know what's the function of C-8 of 37. But these three proteins is interesting. So the KTN1 is on ER. So it's a membrane protein. It can encurse the protein translation factor when complex to the ER. And in culture cells the knockdown of this protein can cause membrane protein synthesis reduced. And these two proteins, they both have the ubiquitin relation activity. So it suggests that this protein is involving ubiquitin proteins on degradation pathway. So that from this study is also kind of suggest two direction, protein synthesis or degradation. So we need to further to at least confirm this interaction and then have some proof what really went wrong in C-8 of 37 knockout device. Okay, so in summary of my study hopefully this is just last part of life, just two more slides. So first we successfully generated the C-8 of 37 knockout mouse model. This model, a good mouse model for future magnetic and therapeutic studies for the future generation caused by C-8 of 37 mutations. And second, C-8 of 37 knockout can cause the outside of membrane protein reduction, which leads to outside of membrane misalignment and eventually graduate generation. And third, C-8 of 37 is in photoreceptor in a segment. So it may be involving all the second membrane protein homeostasis, but it's probably not involving trafficking. And finally, the mechanism of retinal degeneration in C-8 of 37 deficient patients probably is shared among other CRD patients with mutations in other genes. Such as rock 28, which is one RTIS, APAC4, etc. So finally, I'd like to thank the people involved in this study. So first, the people in my lab, they really worked hard and especially Arish Sarif, he is a white student in my lab. And the work I presented here was done by him. Of course, with the help of other people in the lab, especially Dong-ha-yu also did a lot of work here. And Robert Mark and Brian Jones lab helped us to do the TBM study as a whole facility for the department. Wolfgang Beer and Boasim shared their preliminary data on rock 28 studies. And Susan, so this is our campus, not in the lab, this is in the lab. So Susan and the team, they are just like a writing hall of the facility, helped us to gather the C-8 of 37 non-powerful mice. And Chris Kiyo, J.B. Belknight and Dennis Winch, they helped us to study the biological properties of this protein. So I didn't present the data for this part of the research. Also at the university, but then I just led at Dong University, helped us to localize the photoreceptors. And Bill Houseworth at the University of Utah, helped us to generate the AUB vector currently in this C-8 of 37 and C-8. So we're going to use it to study therapeutics, to just go to that direction. Okay, so thank you for listening.