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Robert S. Molday - One of the best experts on this subject based on the ideXlab platform.
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cog wheel octameric structure of rs1 the discoidin domain containing retinal protein associated with x linked Retinoschisis
PLOS ONE, 2016Co-Authors: Martin Bush, Dheva Setiaputra, Robert S. MoldayAbstract:RS1, also known as retinoschisin, is a disulphide-linked, discoidin domain containing homo-oligomeric protein that plays a crucial role in maintaining the cellular and synaptic organization of the retina. This is highlighted by the finding that over 130 mutations in RS1 cause X-linked Retinoschisis, a retinal degenerative disease characterized by the splitting of the retinal cell layers, disruption of the photoreceptor–bipolar synapses, degeneration of photoreceptors, and severe loss in central vision. In this study, we investigated the arrangement of the RS1 subunits within the oligomer complex using single particle electron microscopy. RS1 was seen as two stacked rings with each ring displaying a symmetrical cog wheel-like structure with eight teeth or projections corresponding to the RS1 subunits. Three dimensional reconstruction and molecular modelling indicated that the discoidin domain, the principal functional unit of RS1, projects outward, and the Rs1 domain and C-terminal segment containing intermolecular disulphide bonds are present in the inner ring to form the core octameric structure. These studies provide a basis for further understanding the role of the novel core RS1 octameric complex in retinal cell biology and X-linked Retinoschisis.
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x linked juvenile Retinoschisis clinical diagnosis genetic analysis and molecular mechanisms
Progress in Retinal and Eye Research, 2012Co-Authors: Robert S. Molday, Ulrich Kellner, Bernhard H F WeberAbstract:X-linked juvenile Retinoschisis (XLRS, MIM 312700) is a common early onset macular degeneration in males characterized by mild to severe loss in visual acuity, splitting of retinal layers, and a reduction in the b-wave of the electroretinogram (ERG). The RS1 gene (MIM 300839) associated with the disease encodes retinoschisin, a 224 amino acid protein containing a discoidin domain as the major structural unit, an N-terminal cleavable signal sequence, and regions responsible for subunit oligomerization. Retinoschisin is secreted from retinal cells as a disulphide-linked homo-octameric complex which binds to the surface of photoreceptors and bipolar cells to help maintain the integrity of the retina. Over 190 disease-causing mutations in the RS1 gene are known with most mutations occurring as non-synonymous changes in the discoidin domain. Cell expression studies have shown that disease-associated missense mutations in the discoidin domain cause severe protein misfolding and retention in the endoplasmic reticulum, mutations in the signal sequence result in aberrant protein synthesis, and mutations in regions flanking the discoidin domain cause defective disulphide-linked subunit assembly, all of which produce a non-functional protein. Knockout mice deficient in retinoschisin have been generated and shown to display most of the characteristic features found in XLRS patients. Recombinant adeno-associated virus (rAAV) mediated delivery of the normal RS1 gene to the retina of young knockout mice result in long-term retinoschisin expression and rescue of retinal structure and function providing a ‘proof of concept’ that gene therapy may be an effective treatment for XLRS.
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relation of response to treatment with dorzolamide in x linked Retinoschisis to the mechanism of functional loss in retinoschisin
American Journal of Ophthalmology, 2009Co-Authors: Saloni Walia, Robert S. Molday, Gerald A Fishman, Frank M Dyka, Nalin M Kumar, Mary A Ehlinger, Edwin M StoneAbstract:Purpose To determine if a positive response of macular cysts to treatment with dorzolamide eye drops in patients with juvenile X-linked Retinoschisis (XLRS) can occur with mutations that result in different types of retinoschisin protein dysfunction. Design Retrospective case series. Methods Thirteen eyes of seven patients seen at the University of Illinois at Chicago with a known diagnosis of XLRS were included. Each patient had received or currently was receiving treatment with topical dorzolamide. One patient from each family was screened for a genetic mutation. Using the method of cell transfection and protein preparation, the mutation in each patient was analyzed further and was categorized into one of three groups: 1) total absence of retinoschisin protein secretion, 2) decreased expression of the secreted protein, or 3) secretion of a nonfunctional protein. The response to dorzolamide was observed using optical coherence tomography. Results Significant improvement in the foveal zone thickness was observed with the use of dorzolamide in three of four patients with absence of protein secretion, in two patients with a lack of protein expression, and in one patient with a nonfunctional protein secretion. Conclusions This study showed that the response of macular cysts to dorzolamide in patients with XLRS may be observed independent of the mechanism responsible for retinoschisin protein dysfunction. Hence, treatment with dorzolamide may be effective in patients with different mechanisms of dysfunction in retinoschisin.
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retinoschisin rs1 the protein encoded by the x linked Retinoschisis gene is anchored to the surface of retinal photoreceptor and bipolar cells through its interactions with a na k atpase sarm1 complex
Journal of Biological Chemistry, 2007Co-Authors: Laurie L Molday, Winco W H Wu, Robert S. MoldayAbstract:Abstract Retinoschisin or RS1 is a discoidin domain-containing protein encoded by the gene responsible for X-linked Retinoschisis (XLRS), an early onset macular degeneration characterized by a splitting of the retina. Retinoschisin, expressed and secreted from photoreceptors and bipolar cells as a homo-octameric complex, associates with the surface of these cells where it serves to maintain the cellular organization of the retina and the photoreceptor-bipolar synaptic structure. To gain insight into the role of retinoschisin in retinal cell adhesion and the pathogenesis of XLRS, we have investigated membrane components in retinal extracts that interact with retinoschisin. Unlike the discoidin domain-containing blood coagulation proteins Factor V and Factor VIII, retinoschisin did not bind to phospholipids or retinal lipids reconstituted into unilamellar vesicles or immobilized on microtiter plates. Instead, co-immunoprecipitation studies together with mass spectrometric-based proteomics and Western blotting showed that retinoschisin is associated with a complex consisting of Na/K ATPase (α3, β2 isoforms) and the sterile alpha and TIR motif-containing protein SARM1. Double labeling studies for immunofluorescence microscopy confirmed the co-localization of retinoschisin with Na/K ATPase and SARM1 in photoreceptors and bipolar cells of retina tissue. We conclude that retinoschisin binds to Na/K ATPase on photoreceptor and bipolar cells. This interaction may be part of a novel SARM1-mediated cell signaling pathway required for the maintenance of retinal cell organization and photoreceptor-bipolar synaptic structure.
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retinoschisin rs1 the protein encoded by the x linked Retinoschisis gene is anchored to the surface of retinal photoreceptor and bipolar cells through its interactions with a na k atpase sarm1 complex
Journal of Biological Chemistry, 2007Co-Authors: Laurie L Molday, Robert S. MoldayAbstract:Retinoschisin or RS1 is a discoidin domain-containing protein encoded by the gene responsible for X-linked Retinoschisis (XLRS), an early onset macular degeneration characterized by a splitting of the retina. Retinoschisin, expressed and secreted from photoreceptors and bipolar cells as a homo-octameric complex, associates with the surface of these cells where it serves to maintain the cellular organization of the retina and the photoreceptor-bipolar synaptic structure. To gain insight into the role of retinoschisin in retinal cell adhesion and the pathogenesis of XLRS, we have investigated membrane components in retinal extracts that interact with retinoschisin. Unlike the discoidin domain-containing blood coagulation proteins Factor V and Factor VIII, retinoschisin did not bind to phospholipids or retinal lipids reconstituted into unilamellar vesicles or immobilized on microtiter plates. Instead, co-immunoprecipitation studies together with mass spectrometric-based proteomics and Western blotting showed that retinoschisin is associated with a complex consisting of Na/K ATPase (alpha3, beta2 isoforms) and the sterile alpha and TIR motif-containing protein SARM1. Double labeling studies for immunofluorescence microscopy confirmed the co-localization of retinoschisin with Na/K ATPase and SARM1 in photoreceptors and bipolar cells of retina tissue. We conclude that retinoschisin binds to Na/K ATPase on photoreceptor and bipolar cells. This interaction may be part of a novel SARM1-mediated cell signaling pathway required for the maintenance of retinal cell organization and photoreceptor-bipolar synaptic structure.
Paul A Sieving - One of the best experts on this subject based on the ideXlab platform.
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Cryo-EM of retinoschisin branched networks suggests an intercellular adhesive scaffold in the retina.
Journal of Cell Biology, 2019Co-Authors: J. Bernard Heymann, Paul A Sieving, Camasamudram Vijayasarathy, Rick K. Huang, Altaira D. Dearborn, Alasdair C StevenAbstract:: Mutations in the retinal protein retinoschisin (RS1) cause progressive loss of vision in young males, a form of macular degeneration called X-linked Retinoschisis (XLRS). We previously solved the structure of RS1, a 16-mer composed of paired back-to-back octameric rings. Here, we show by cryo-electron microscopy that RS1 16-mers can assemble into extensive branched networks. We classified the different configurations, finding four types of interaction between the RS1 molecules. The predominant configuration is a linear strand with a wavy appearance. Three less frequent types constitute the branch points of the network. In all cases, the "spikes" around the periphery of the double rings are involved in these interactions. In the linear strand, a loop (usually referred to as spike 1) occurs on both sides of the interface between neighboring molecules. Mutations in this loop suppress secretion, indicating the possibility of intracellular higher-order assembly. These observations suggest that branched networks of RS1 may play a stabilizing role in maintaining the integrity of the retina.
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paired octamer rings of retinoschisin suggest a junctional model for cell cell adhesion in the retina
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Gokhan Tolun, Paul A Sieving, Yong Zeng, Camasamudram Vijayasarathy, Rick Huang, Alasdair C Steven, Bernard J HeymannAbstract:Retinoschisin (RS1) is involved in cell–cell junctions in the retina, but is unique among known cell-adhesion proteins in that it is a soluble secreted protein. Loss-of-function mutations in RS1 lead to early vision impairment in young males, called X-linked Retinoschisis. The disease is characterized by separation of inner retinal layers and disruption of synaptic signaling. Using cryo-electron microscopy, we report the structure at 4.1 A, revealing double octamer rings not observed before. Each subunit is composed of a discoidin domain and a small N-terminal (RS1) domain. The RS1 domains occupy the centers of the rings, but are not required for ring formation and are less clearly defined, suggesting mobility. We determined the structure of the discoidin rings, consistent with known intramolecular and intermolecular disulfides. The interfaces internal to and between rings feature residues implicated in X-linked Retinoschisis, indicating the importance of correct assembly. Based on this structure, we propose that RS1 couples neighboring membranes together through octamer–octamer contacts, perhaps modulated by interactions with other membrane components.
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preclinical dose escalation study of intravitreal aav rs1 gene therapy in a mouse model of x linked Retinoschisis dose dependent expression and improved retinal structure and function
Human Gene Therapy, 2016Co-Authors: Ronald A. Bush, Yong Zeng, Camasamudram Vijayasarathy, Suja Hiriyanna, Peter Colosi, Sten Kjellstrom, Maria Santos, Paul A SievingAbstract:Gene therapy for inherited retinal diseases has been shown to ameliorate functional and structural defects in both animal models and in human clinical trials. X-linked Retinoschisis (XLRS) is an early-age onset macular dystrophy resulting from loss of an extracellular matrix protein (RS1). In preparation for a human clinical gene therapy trial, we conducted a dose-range efficacy study of the clinical vector, a self-complementary AAV delivering a human retinoschisin (RS1) gene under control of the RS1 promoter and an interphotoreceptor binding protein enhancer (AAV8-scRS/IRBPhRS), in the retinoschisin knockout (Rs1-KO) mouse. The therapeutic vector at 1 × 10(6) to 2.5 × 10(9) (1E6-2.5E9) vector genomes (vg)/eye or vehicle was administered to one eye of 229 male Rs1-KO mice by intravitreal injection at 22 ± 3 days postnatal age (PN). Analysis of retinal function (dark-adapted electroretinogram, ERG), structure (cavities and outer nuclear layer thickness) by in vivo retinal imaging using optical coherence tomography, and retinal immunohistochemistry (IHC) for RS1 was done 3-4 months and/or 6-9 months postinjection (PI). RS1 IHC staining was dose dependent across doses ≥1E7 vg/eye, and the threshold for significant improvement in all measures of retinal structure and function was 1E8 vg/eye. Higher doses, however, did not produce additional improvement. At all doses showing efficacy, RS1 staining in Rs1-KO mouse was less than that in wild-type mice. Improvement in the ERG and RS1 staining was unchanged or greater at 6-9 months than at 3-4 months PI. This study demonstrates that vitreal administration of AAV8 scRS/IRBPhRS produces significant improvement in retinal structure and function in the mouse model of XLRS over a vector dose range that can be extended to a human trial. It indicates that a fully normal level of RS1 expression is not necessary for a therapeutic effect.
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convergence of human genetics and animal studies gene therapy for x linked Retinoschisis
Cold Spring Harbor Perspectives in Medicine, 2015Co-Authors: Ronald A. Bush, Lisa L. Wei, Paul A SievingAbstract:Retinoschisis is an X-linked recessive genetic disease that leads to vision loss in males. X-linked Retinoschisis (XLRS) typically affects young males; however, progressive vision loss continues throughout life. Although discovered in 1898 by Haas in two brothers, the underlying biology leading to blindness has become apparent only in the last 15 years with the advancement of human genetic analyses, generation of XLRS animal models, and the development of ocular monitoring methods such as the electroretinogram and optical coherence tomography. It is now recognized that Retinoschisis results from cyst formations within the retinal layers that interrupt normal visual neurosignaling and compromise structural integrity. Mutations in the human retinoschisin gene have been correlated with disease severity of the human XLRS phenotype. Introduction of a normal human retinoschisin cDNA into retinoschisin knockout mice restores retinal structure and improves neural function, providing proof-of-concept that gene replacement therapy is a plausible treatment for XLRS.
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x linked Retinoschisis rs1 mutation severity and age affect the erg phenotype in a cohort of 68 affected male subjects
Investigative Ophthalmology & Visual Science, 2011Co-Authors: Kristen E Bowles, Yuri V Sergeev, Ronald A. Bush, Catherine A Cukras, Amy Turriff, Susan Vitale, Paul A SievingAbstract:A monogenic condition, X-linked Retinoschisis (XLRS) is a leading genetic cause of macular degeneration in young males and affects 1:5000 to 1:25,000.1 It is caused by mutations in the retinoschisin gene (RS1) at Xp22.2 Nearly 150 disease-causing RS1 mutations have been identified.3 RS1 encodes a 24-kDa protein, retinoschisin (RS), which contains a discoidin domain and is found in the retina and pineal gland.4 Retinoschisin is thought to be important for cell adhesion and cell signaling because the protein has been demonstrated in the photoreceptor synapse onto bipolar cells.5 RS is associated with the Na+/K+ ATPase in the photoreceptor inner segment membrane.6 Atomic force microscopy implicates a structural role for retinoschisin in organizing lipid membranes, which may help to stabilize retinal cell integrity.7 XLRS subjects exhibit considerable heterogeneity on clinical examination and functional vision testing.8 Although most XLRS-affected males first come to attention for reduced acuity during school screening, some XLRS experience retinal detachments in infancy. In addition, our clinical cohort suggests that XLRS subjects rarely retain sufficient visual function to obtain an unrestricted driver's license by the fourth and fifth decades of life. The consequence of abnormal retinoschisin protein or complete absence of RS on retinal function can be evaluated clinically using the full-field electroretinogram (ERG). The characteristic dark-adapted bright-flash ERG response in XLRS is an “electronegative waveform” in which the b-wave amplitude is disproportionately smaller than the normal or near-normal a-wave. ERG a-wave modeling has shown that phototransduction remains normal in some XLRS subjects despite reduced b-waves,9 indicating that one site of dysfunction lies beyond the photoreceptors. Abnormal timing of the light-adapted 30-Hz cone flicker ERG response is also reported, further suggesting dysfunction of the inner retina when associated with normal a-wave responses.10 In this genotype-phenotype study, we used the full-field ERG to evaluate retinal function of 68 XLRS subjects who were examined and genotyped at the National Eye Institute (NEI). The RS1 gene contains six exons that encode for a signal sequence (exons 1 and 2), a retinoschisin domain (exon 3), and the discoidin domain (exons 4–6).2 Most RS1 mutations analyzed to date have implicated primarily a loss function rather than an abnormal function from the mutant protein.7 Signal sequence domain mutations impair protein transport to the endoplasmic reticulum and consequently yield a functional null-protein effect. Some discoidin domain mutations allow the production of retinoschisin mutant protein, but it is rapidly degraded and lost.11 The diversity and complexity of RS1 mutations precluded our incorporating all molecular nuances in a phenotype analysis. Instead, we simplified the approach by segregating the RS1 mutations into two groups based on the putative severity of effect on protein function. We included molecular modeling12 and, in some cases, biochemical analysis11 in this approach to classify our subjects as having either a less or a more severe mutation and a putative effect on the resultant protein. We then examined for an association of disease phenotype (ERG measurements) with genotype severity.
Dorothy Trump - One of the best experts on this subject based on the ideXlab platform.
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Wild-type and missense mutants of retinoschisin co-assemble resulting in either intra-cellular retention or incorrect assembly of the functionally active octomer
Biochemical Journal, 2009Co-Authors: Lindsay J. Gleghorn, Dorothy Trump, Neil J. BulleidAbstract:The X-linked disease Retinoschisis is caused by mutations in the RS1 gene encoding retinoschisin, most commonly missense mutations leading to a lack of secretion of functional protein. One potential approach to treat this disease would be the introduction of the wild-type protein by gene therapy in affected individuals. Retinoschisin normally forms homooctamers so co-expression of the wild-type protein with the mutant could result in their co-assembly. Here we show that retinoschisin assembles into an octamer prior to transport from the endoplasmic reticulum and that co-assembly of wild-type and mutant protein can occur when they are co-expressed in the same cell. This co-assembly results in the retention of some but not all expressed wild-type retinoschisin. Moreover, when the wild-type protein is expressed with a missense mutant that is normally secreted co-assembly occurs resulting in the secretion of a heterogeneous mixture of oligomers. Missense mutations of retinoschisin which cause intracellular retention also lead to an unfolded protein response. However, this is not sufficient to decrease cell viability suggesting that the pathology of the disease is not likely to be linked to programmed cell death.
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X-Linked Retinoschisis: An Update
Journal of medical genetics, 2006Co-Authors: Stephen K Sikkink, Susmito Biswas, Neil R. A. Parry, Paulo E. Stanga, Dorothy TrumpAbstract:X-linked Retinoschisis is the leading cause of macular degeneration in males and leads to splitting within the inner retinal layers leading to visual deterioration. Many missense and protein truncating mutations have now been identified in the causative Retinoschisis gene (RS1) which encodes a 224 amino acid secreting retinal protein, retinoschisin. Retinoschisin octamerises is implicated in cell–cell interactions and cell adhesion perhaps by interacting with β2 laminin. Mutations cause loss of retinoschisin function by one of the three mechanisms: by interfering with protein secretion, by preventing its octamerisation or by reducing function in the secreted octamerised protein. The development of Retinoschisis mouse models have provided a model system that closely resembles the human disease. Recent reports of RS1 gene transfer to these models and the sustained restoration of some retinal function and morphology suggest gene replacement may be a possible future therapy for patients.
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molecular pathology of x linked Retinoschisis mutations interfere with retinoschisin secretion and oligomerisation
British Journal of Ophthalmology, 2006Co-Authors: Tao Wang, A Zhou, C T Waters, E Oconnor, Randy J Read, Dorothy TrumpAbstract:Background/aim: X linked Retinoschisis (XLRS) is caused by mutations in RS1 which encodes the discoidin domain protein retinoschisin, secreted by photoreceptors and bipolar cells. Missense mutations occur throughout the gene and some of these are known to interfere with protein secretion. This study was designed to investigate the functional consequences of missense mutations at different locations in retinoschisin. Methods and results: The authors developed a structural model of the retinoschisin discoidin domain and used this to predict the effects of missense mutations. They expressed disease associated mutations and found that those affecting conserved residues prevented retinoschisin secretion. Most of the remaining mutations cluster within a series of loops on the surface of the β barrel structure and do not interfere with secretion, suggesting this region may be a ligand binding site. They also demonstrated that wild type retinoschisin octamerises and associates with the cell surface. A subgroup of secreted mutations reduce oligomerisation (C59S, C219G, C223R). Conclusions: It is suggested that there are three different molecular mechanisms which lead to XLRS: mutations interfering with secretion, mutations interfering with oligomerisation, and mutations that allow secretion and oligomerisation but interfere with retinoschisin function. The authors conclude that binding of oligomerised retinoschisin at the cell surface is important in its presumed role in cell adhesion.
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intracellular retention of mutant retinoschisin is the pathological mechanism underlying x linked Retinoschisis
Human Molecular Genetics, 2002Co-Authors: Tao Wang, Caroline T Waters, Alexander M K Rothman, T Jakins, Karin Romisch, Dorothy TrumpAbstract:X-linked Retinoschisis results in visual loss in early life with splitting within the inner retinal layers. Many missense and protein truncating mutations of the causative gene RS1 (encoding retinoschisin) have been identified but disease severity is not mutation-dependent. Retinoschisin is a soluble secretory protein predicted to have a globular conformation. Missense mutations would be expected to interfere with protein folding leading to an abnormal conformation and intracellular retention and elimination. To test this hypothesis we have expressed seven pathological RS1 mutations (L12H, C59S, G70S, R102W, G109R, R141G and R213W) in COS-7 cells and investigated their intracellular processing and transport. Using immunoblotting and confocal fluorescent immunocytochemistry we show normal secretion of WT RS1, but either reduced (C59S and R141G) or absent (L12H, G70S, R102W, G109R and R213W) secretion of mutant RS1 and intracellular retention. In addition, we show that L12H RS1 is degraded by proteasomes and in vitro transcription/translation revealed the defects in both cleavage of its signal peptide and translocation into the endoplasmic reticulum. Our results indicate the pathological basis of RS1 is intracellular retention of the majority of mutant proteins, which may explain why disease severity is not mutation-specific. Furthermore, we have shown that in vitro expression of RS1 may be a useful functional assay to investigate the pathogenicity of sequence changes within the RS1 gene.
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retinoschisin the x linked Retinoschisis protein is a secreted photoreceptor protein and is expressed and released by weri rb1 cells
Human Molecular Genetics, 2000Co-Authors: Celene Grayson, Silvia N M Reid, Adam Rutherford, John R.w. Yates, Debora B Farber, Juliet A Ellis, Jane C. Sowden, Dorothy TrumpAbstract:X-linked Retinoschisis is characterized by microcystic-like changes of the macular region and schisis within the inner retinal layers, leading to visual deterioration in males. Many missense and protein-truncating mutations of the causative gene RS1 have now been identified and are thought to be inactivating. RS1 encodes a 224 amino acid protein, retinoschisin, which contains a discoidin domain but is of unknown function. We have generated a polyclonal antibody against a peptide from a unique region within retinoschisin, which detects a protein of similar to 28 kDa in retinal samples reduced with dithiothreitol, but multimers sized >40 kDa under non-reducing conditions. A screen of human tissues with this antibody reveals retinoschisin to be retina specific and the antibody detects a protein of similar size in bovine and murine retinae. We investigated the expression pattern in the retina of both RSI mRNA (using in situ hybridization with riboprobes) and retinoschisin (using immunohistochemistry), The antisense riboprobe detected RSI mRNA only in the photoreceptor layer but the protein product of the gene was present both in the photoreceptors and within the inner portions of the retina. Furthermore, differentiated retinoblastoma cells (Weri-Rb1 cells) were found to express RSI mRNA and to release retinoschisin, These results suggest that retinoschisin is released by photoreceptors and has functions within the inner retinal layers. Thus, X-linked Retinoschisis is caused by abnormalities in a putative secreted photoreceptor protein and is the first example of a secreted photoreceptor protein associated with a retinal dystrophy.
Bernhard H F Weber - One of the best experts on this subject based on the ideXlab platform.
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Identification of the retinoschisin-binding site on the retinal Na/K-ATPase
2019Co-Authors: Karolina Plössl, Bernhard H F Weber, Kristina Straub, Verena Schmid, Franziska Strunz, Jens Wild, Rainer Merkl, Ulrike FriedrichAbstract:X-linked juvenile Retinoschisis (XLRS) is a hereditary retinal dystrophy, caused by mutations in the RS1 gene which encodes the secreted protein retinoschisin. In recent years, several molecules have been proposed to interact with retinoschisin, including the retinal Na/K-ATPase, L-voltage gated Ca2+ channels, and specific sugars. We recently showed that the retinal Na/K-ATPase consisting of subunits ATP1A3 and ATP1B2 is essential for anchoring retinoschisin to plasma membranes and identified the glycosylated ATP1B2 subunit as the direct interaction partner for retinoschisin. We now aimed to precisely map the retinoschisin binding domain(s) in ATP1B2. In general, retinoschisin binding was not affected after selective elimination of individual glycosylation sites via site-directed mutagenesis as well as after full enzymatic deglycosylation of ATP1B2. Applying the interface prediction tool PresCont, two putative protein-protein interaction patches (“patch I” and “patch II”) consisting each of four hydrophobic amino acid stretches on the ATP1B2 surface were identified. These were consecutively altered by site-directed mutagenesis. Functional assays with the ATP1B2 patch mutants identified patch II and, specifically, the associated amino acid at position 240 (harboring a threonine in ATP1B2) as crucial for retinoschisin binding to ATP1B2. These and previous results led us to suggest an induced-fit binding mechanism for the interaction between retinoschisin and the Na/K-ATPase, which is dependent on threonine 240 in ATP1B2 allowing the accommodation of hyperflexible retinoschisin spikes by the associated protein-protein interaction patch on ATP1B2.
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x linked juvenile Retinoschisis clinical diagnosis genetic analysis and molecular mechanisms
Progress in Retinal and Eye Research, 2012Co-Authors: Robert S. Molday, Ulrich Kellner, Bernhard H F WeberAbstract:X-linked juvenile Retinoschisis (XLRS, MIM 312700) is a common early onset macular degeneration in males characterized by mild to severe loss in visual acuity, splitting of retinal layers, and a reduction in the b-wave of the electroretinogram (ERG). The RS1 gene (MIM 300839) associated with the disease encodes retinoschisin, a 224 amino acid protein containing a discoidin domain as the major structural unit, an N-terminal cleavable signal sequence, and regions responsible for subunit oligomerization. Retinoschisin is secreted from retinal cells as a disulphide-linked homo-octameric complex which binds to the surface of photoreceptors and bipolar cells to help maintain the integrity of the retina. Over 190 disease-causing mutations in the RS1 gene are known with most mutations occurring as non-synonymous changes in the discoidin domain. Cell expression studies have shown that disease-associated missense mutations in the discoidin domain cause severe protein misfolding and retention in the endoplasmic reticulum, mutations in the signal sequence result in aberrant protein synthesis, and mutations in regions flanking the discoidin domain cause defective disulphide-linked subunit assembly, all of which produce a non-functional protein. Knockout mice deficient in retinoschisin have been generated and shown to display most of the characteristic features found in XLRS patients. Recombinant adeno-associated virus (rAAV) mediated delivery of the normal RS1 gene to the retina of young knockout mice result in long-term retinoschisin expression and rescue of retinal structure and function providing a ‘proof of concept’ that gene therapy may be an effective treatment for XLRS.
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dextran and protamine based solid lipid nanoparticles as potential vectors for the treatment of x linked juvenile Retinoschisis
Human Gene Therapy, 2012Co-Authors: Diego Delgado, Bernhard H F Weber, Ana Del Pozorodriguez, Maria Angeles Solinis, Marcelino Avilestriqueros, Eduardo Fernandez, A R GasconAbstract:Abstract The goal of the present study was to analyze the potential application of nonviral vectors based on solid lipid nanoparticles (SLN) for the treatment of ocular diseases by gene therapy, specifically X-linked juvenile Retinoschisis (XLRS). Vectors were prepared with SLN, dextran, protamine, and a plasmid (pCMS-EGFP or pCEP4-RS1). Formulations were characterized and the in vitro transfection capacity as well as the cellular uptake and the intracellular trafficking were studied in ARPE-19 cells. Formulations were also tested in vivo in Wistar rat eyes, and the efficacy was studied by monitoring the expression of enhanced green fluorescent protein (EGFP) after intravitreal, subretinal, and topical administration. The presence of dextran and protamine in the SLN improved greatly the expression of retinoschisin and EGFP in ARPE-19 cells. The nuclear localization signals of protamine, its ability to protect the DNA, and a shift in the entry mechanism from caveola-mediated to clathrin-mediated endocytosi...
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the na k atpase is obligatory for membrane anchorage of retinoschisin the protein involved in the pathogenesis of x linked juvenile Retinoschisis
Human Molecular Genetics, 2011Co-Authors: Ulrike Friedrich, Heidi Stohr, Daniela Hilfinger, Thomas Loenhardt, Melitta Schachner, Thomas Langmann, Bernhard H F WeberAbstract:Mutations in the RS1 gene that encodes the discoidin domain containing retinoschisin cause X-linked juvenile Retinoschisis (XLRS), a common macular degeneration in males. Disorganization of retinal layers and electroretinogram abnormalities are hallmarks of the disease and are also found in mice deficient for the orthologous murine protein, indicating that retinoschisin is important for the maintenance of retinal cell integrity. Upon secretion, retinoschisin associates with plasma membranes of photoreceptor and bipolar cells, although the components by which the protein is linked to membranes in vivo are still unclear. Here, we show that retinoschisin fails to bind to phospholipids or unilamellar lipid vesicles. A recent proteomic approach identified the Na/K-ATPase subunits ATP1A3 and ATP1B2 as binding partners of retinoschisin. We analyzed mice deficient for retinoschisin (Rs1h(-/Y)) and ATP1B2 (Atp1b2(-/-)) to characterize the role of Na/K-ATPase interaction in the organization of retinoschisin on cellular membranes. We demonstrate that both the Na/K-ATPase and retinoschisin are significantly reduced in Atp1b2(-/-) retinas, suggesting that retinoschisin membrane association is severely impaired in the absence of ATP1A3 and ATP1B2 subunits. Conversely, the presence of ATP1A3 and ATP1B2 are obligatory for binding of exogenously applied retinoschisin to crude membranes. Also, co-expression of ATP1A3 and ATP1B2 is required for retinoschisin binding to intact Hek293 cells. Taken together, our data support a predominant role of Na/K-ATPase in anchoring retinoschisin to retinal cell surfaces. Furthermore, altered localization of ATP1A3 and ATP1B2 is a notable consequence of retinoschisin deficiency and thus may be an important downstream aspect of cellular pathology in XLRS.
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prolonged recovery of retinal structure function after gene therapy in an rs1h deficient mouse model of x linked juvenile Retinoschisis
Molecular Therapy, 2005Co-Authors: Seok Hong Min, Laurie L Molday, Bernhard H F Weber, Mathias W Seeliger, Astra Dinculescu, Adrian M Timmers, Andreas Janssen, Felix Tonagel, Naoyuki Tanimoto, Robert S. MoldayAbstract:X-linked juvenile Retinoschisis (RS) is a common cause of juvenile macular degeneration in males. RS is characterized by cystic spoke-wheel-like maculopathy, peripheral schisis, and a negative (b-wave more reduced than a-wave) electroretinogram (ERG). These symptoms are due to mutations in the RS1 gene in Xp22.2 leading to loss of functional protein. No medical treatment is currently available. We show here that in an Rs1h-deficient mouse model of human RS, delivery of the human RS1 cDNA with an AAV vector restored expression of retinoschisin to both photoreceptors and the inner retina essentially identical to that seen in wild-type mice. More importantly, unlike an earlier study with a different AAV vector and promoter, this work shows for the first time that therapeutic gene delivery using a highly specific AAV5–opsin promoter vector leads to progressive and significant improvement in both retinal function (ERG) and morphology, with preservation of photoreceptor cells that, without treatment, progressively degenerate.
Camasamudram Vijayasarathy - One of the best experts on this subject based on the ideXlab platform.
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Cryo-EM of retinoschisin branched networks suggests an intercellular adhesive scaffold in the retina.
Journal of Cell Biology, 2019Co-Authors: J. Bernard Heymann, Paul A Sieving, Camasamudram Vijayasarathy, Rick K. Huang, Altaira D. Dearborn, Alasdair C StevenAbstract:: Mutations in the retinal protein retinoschisin (RS1) cause progressive loss of vision in young males, a form of macular degeneration called X-linked Retinoschisis (XLRS). We previously solved the structure of RS1, a 16-mer composed of paired back-to-back octameric rings. Here, we show by cryo-electron microscopy that RS1 16-mers can assemble into extensive branched networks. We classified the different configurations, finding four types of interaction between the RS1 molecules. The predominant configuration is a linear strand with a wavy appearance. Three less frequent types constitute the branch points of the network. In all cases, the "spikes" around the periphery of the double rings are involved in these interactions. In the linear strand, a loop (usually referred to as spike 1) occurs on both sides of the interface between neighboring molecules. Mutations in this loop suppress secretion, indicating the possibility of intracellular higher-order assembly. These observations suggest that branched networks of RS1 may play a stabilizing role in maintaining the integrity of the retina.
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retinal aav8 rs1 gene therapy for x linked Retinoschisis initial findings from a phase i iia trial by intravitreal delivery
Molecular Therapy, 2018Co-Authors: Catherine A Cukras, Yong Zeng, Dario Marangoni, Henry E. Wiley, Camasamudram Vijayasarathy, Amy Turriff, Brett G Jeffrey, Nida H Sen, Lucia Ziccardi, Sten KjellstromAbstract:This study evaluated the safety and tolerability of ocular RS1 adeno-associated virus (AAV8-RS1) gene augmentation therapy to the retina of participants with X-linked Retinoschisis (XLRS). XLRS is a monogenic trait affecting only males, caused by mutations in the RS1 gene. Retinoschisin protein is secreted principally in the outer retina, and its absence results in retinal cavities, synaptic dysfunction, reduced visual acuity, and susceptibility to retinal detachment. This phase I/IIa single-center, prospective, open-label, three-dose-escalation clinical trial administered vector to nine participants with pathogenic RS1 mutations. The eye of each participant with worse acuity (≤63 letters; Snellen 20/63) received the AAV8-RS1 gene vector by intravitreal injection. Three participants were assigned to each of three dosage groups: 1e9 vector genomes (vg)/eye, 1e10 vg/eye, and 1e11 vg/eye. The investigational product was generally well tolerated in all but one individual. Ocular events included dose-related inflammation that resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in a dose-related fashion, but no antibodies against RS1 were observed. Retinal cavities closed transiently in one participant. Additional doses and immunosuppressive regimens are being explored to pursue evidence of safety and efficacy (ClinicalTrials.gov: NCT02317887).
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paired octamer rings of retinoschisin suggest a junctional model for cell cell adhesion in the retina
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Gokhan Tolun, Paul A Sieving, Yong Zeng, Camasamudram Vijayasarathy, Rick Huang, Alasdair C Steven, Bernard J HeymannAbstract:Retinoschisin (RS1) is involved in cell–cell junctions in the retina, but is unique among known cell-adhesion proteins in that it is a soluble secreted protein. Loss-of-function mutations in RS1 lead to early vision impairment in young males, called X-linked Retinoschisis. The disease is characterized by separation of inner retinal layers and disruption of synaptic signaling. Using cryo-electron microscopy, we report the structure at 4.1 A, revealing double octamer rings not observed before. Each subunit is composed of a discoidin domain and a small N-terminal (RS1) domain. The RS1 domains occupy the centers of the rings, but are not required for ring formation and are less clearly defined, suggesting mobility. We determined the structure of the discoidin rings, consistent with known intramolecular and intermolecular disulfides. The interfaces internal to and between rings feature residues implicated in X-linked Retinoschisis, indicating the importance of correct assembly. Based on this structure, we propose that RS1 couples neighboring membranes together through octamer–octamer contacts, perhaps modulated by interactions with other membrane components.
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preclinical dose escalation study of intravitreal aav rs1 gene therapy in a mouse model of x linked Retinoschisis dose dependent expression and improved retinal structure and function
Human Gene Therapy, 2016Co-Authors: Ronald A. Bush, Yong Zeng, Camasamudram Vijayasarathy, Suja Hiriyanna, Peter Colosi, Sten Kjellstrom, Maria Santos, Paul A SievingAbstract:Gene therapy for inherited retinal diseases has been shown to ameliorate functional and structural defects in both animal models and in human clinical trials. X-linked Retinoschisis (XLRS) is an early-age onset macular dystrophy resulting from loss of an extracellular matrix protein (RS1). In preparation for a human clinical gene therapy trial, we conducted a dose-range efficacy study of the clinical vector, a self-complementary AAV delivering a human retinoschisin (RS1) gene under control of the RS1 promoter and an interphotoreceptor binding protein enhancer (AAV8-scRS/IRBPhRS), in the retinoschisin knockout (Rs1-KO) mouse. The therapeutic vector at 1 × 10(6) to 2.5 × 10(9) (1E6-2.5E9) vector genomes (vg)/eye or vehicle was administered to one eye of 229 male Rs1-KO mice by intravitreal injection at 22 ± 3 days postnatal age (PN). Analysis of retinal function (dark-adapted electroretinogram, ERG), structure (cavities and outer nuclear layer thickness) by in vivo retinal imaging using optical coherence tomography, and retinal immunohistochemistry (IHC) for RS1 was done 3-4 months and/or 6-9 months postinjection (PI). RS1 IHC staining was dose dependent across doses ≥1E7 vg/eye, and the threshold for significant improvement in all measures of retinal structure and function was 1E8 vg/eye. Higher doses, however, did not produce additional improvement. At all doses showing efficacy, RS1 staining in Rs1-KO mouse was less than that in wild-type mice. Improvement in the ERG and RS1 staining was unchanged or greater at 6-9 months than at 3-4 months PI. This study demonstrates that vitreal administration of AAV8 scRS/IRBPhRS produces significant improvement in retinal structure and function in the mouse model of XLRS over a vector dose range that can be extended to a human trial. It indicates that a fully normal level of RS1 expression is not necessary for a therapeutic effect.
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Preclinical safety evaluation of a recombinant AAV8 vector for X-linked Retinoschisis after intravitreal administration in rabbits.
Human Gene Therapy Clinical Development, 2014Co-Authors: Dario Marangoni, Yong Zeng, Ronald A. Bush, Henry E. Wiley, Caroline J. Zeiss, Camasamudram Vijayasarathy, Suja Hiriyanna, Lisa L. Wei, Peter ColosiAbstract:Abstract X-linked Retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and one of the most common causes of macular degeneration in young men. Currently, no FDA-approved treatments are available for XLRS and a replacement gene therapy could provide a promising strategy. We have developed a novel gene therapy approach for XLRS, based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal route. On the basis of our prior study in an Rs1-KO mouse, this construct transduces efficiently all the retinal layers, resulting in an RS1 expression similar to that observed in the wild-type and improving retinal structure and function. In support of a clinical trial, we carried out a study to evaluate the ocular safety of intravitreal administration of AAV8-scRS/IRBPhRS into 39 New Zealand White rabbits. Two dose levels of vector, 2e10 and 2e11 vector genomes per eye (vg/eye), were test...
