The Experts below are selected from a list of 447 Experts worldwide ranked by ideXlab platform
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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Coexpression and Interaction of Wild-type and Missense RS1 Mutants Associated with X-Linked Retinoschisis: Its Relevance to Gene Therapy
2013Co-Authors: Frank M Dyka, Robert S. MoldayAbstract:PURPOSE. X-linked retinoschisis (XLRS) is an early-onset retinal disease caused by mutations in Retinoschisin (RS1), a multisubunit, extracellular protein implicated in retinal cell adhesion. Delivery of the normal RS1 gene to photoreceptors of Retinoschisin-deficient mice results in prolonged protein expression and rescue of retinal structure and function. However, most persons with XLRS harbor a missense mutation in the RS1 gene leading to expression of a nonfunctional protein. The purpose of this study was to examine the effect that coexpression of wild-type RS1 with disease-causing mutants has on RS1 expression, oligomerization, and secretion to further evaluate gene therapy as a possible treatment for XLRS. METHODS. RS1 mutants (C59S, D158N, C142W, C142S, T185K, R141H, R141G) were individually expressed or coexpressed with myc-tagged wild-type RS1 (myc-RS1) in EBNA293 cells
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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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Progress in Retinal and Eye Research
2012Co-Authors: Robert S. Molday, Ulrich KellnerAbstract: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 diseaseassociated 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.
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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RS-1 gene delivery to an adult Rs1h knockout mouse model restores ERG b-wave with reversal of the electronegative waveform of X-linked retinoschisis. Invest Ophthalmol Vis Sci
2013Co-Authors: Yong Zeng, Nizar Smaoui, Ronald A. Bush, Yuichiro Takada, Sten Kjellstrom, Kelaginamane Hiriyanna, A Tanikawa, Eric F Wawrousek, Rafael Caruso, Paul A SievingAbstract:PURPOSE. To create and evaluate a mouse model of human X-linked juvenile retinoschisis (XLRS) and then investigate whether supplementing with the Retinoschisin protein by gene delivery can reverse the abnormal “electronegative ” electroretinogram (ERG) retinal response. METHODS. An X-linked retinoschisis mouse (Rs1h-KO) model was created by substituting a neomycin resistance cassette for exon 1 and 1.6 kb of intron 1 of Rs1h, the murine orthologue of the human RS-1 gene. RS protein was evaluated by immunohistochemistry and Western blot analysis with a polyclonal RS N-terminus antibody. Retinal function was evaluated by conventional, full-field flash ERG recordings. RS protein supplementation therapy was evaluated by gene transfer with an AAV(2/2)-CMV-Rs1h vector containing C57BL/6J Rs1h cDNA under the regulation of a CMV promoter, and ERG functiona
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 intracellular retention or incorrect assembly of the functionally active octamer.
The 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 homo-octamers, so co-expression of the wild-type protein with the mutant could result in their co-assembly. In the present study, we show that Retinoschisin assembles into an octamer before 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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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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Retinoschisin, a New Binding Partner for L-type Voltage-gated Calcium Channels in the Retina
The Journal of biological chemistry, 2008Co-Authors: Liheng Shi, Kuihuan Jian, Dorothy TrumpAbstract:Abstract The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that Retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCCα1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding Retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that Retinoschisin and the N-terminal fragment of the L-VGCCα1 subunit physically interacted with one another. The expression and secretion of Retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound Retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of Retinoschisin secretion, whereas secreted Retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCCα1 subunits and Retinoschisin play an important role in the membrane retention of L-VGCCα1 subunits and photoreceptor-bipolar synaptic transmission.
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Circadian regulation of Retinoschisin in the chick retina.
Investigative ophthalmology & visual science, 2008Co-Authors: Yilin Liu, Liheng Shi, Dorothy TrumpAbstract:Retinoschisin is a 224-amino acid protein secreted mainly from retina photoreceptors.1,2 Mutations in genes encoding Retinoschisin cause X-linked retinoschisis (XLRS), which is a leading cause of macular degeneration in juvenile male patients,3–5 affecting worldwide populations ranging from 1:5000 to 1:25,0004 and commonly leading to vision loss.4–6 Retinoschisin is a secreted octameric complex consisting of identical subunits linked by disulfide bonds.1,7 Each subunit contains a single discoidin domain that has been implicated in cellular adhesion or cell-cell interactions, which may function to maintain cellular organization and synaptic structure of the retina.1,3,8,9 The Rs1 knockout mouse has a retinal phenotype closely resembling human XLRS with a highly disorganized retina.10 Replacement of the RS1 gene leads to improvement in retinal function and morphology,11,12 indicating a role for Retinoschisin in the development and maintenance of retinal architecture. However, in adult animals, the concentration of Retinoschisin remains high in the photoreceptor layer,13 and recent evidence shows that the pineal glands in rodents and humans also express Retinoschisin.14 Although RS1 mutations cause structural delamination of the neural retinal layers in mice, the pineal gland structures are still intact in these mutant animals.14 Thus, the functions of Retinoschisin in adult animals and humans remain unclear, and the mechanism of Retinoschisin secretion is not completely understood. Photoreceptors are nonspiking neurons, and their synapses mediate the continuous release of neurotransmitters, which is an L-type voltage-gated calcium channel (VGCC)-dependent process.15 In retina photoreceptors, the synthesis and release of melatonin are under circadian control, and they are also L-type VGCC dependent.16,17 Because Retinoschisin is secreted from photoreceptors and the mechanisms underlying its secretion are not known, we tested whether the secretion of Retinoschisin could also be an L-type VGCC-dependent process. Circadian oscillators in the retina provide a mechanism for visual systems to initiate more sustained adaptive changes in ambient illumination throughout the day.18,19 Circadian oscillators in photoreceptors are endogenous and able to function independently in the absence of other retinal inputs.20–22 Importantly, photoreceptors are more sensitive to intense light damage at night than during the day, even in animals that have been maintained in constant darkness for several days after circadian light-dark cycle entrainment.23 We have shown that the L-type VGCCs in chick cone photoreceptors are under circadian control.24 mRNA levels, protein expression, and currents of the L-type VGCCs are greater when measured during the subjective night than during the subjective day.24 Because Retinoschisin is present in retinas and pineal glands,14 there is a possible circadian regulation of Retinoschisin or a role of Retinoschisin in the circadian regulation of retina physiology. Here, we report that mRNA levels, protein expression, and secretion of Retinoschisin—all which are high at night and low during the day—are under circadian control. The Ras, Erk, CaMKII pathway is part of the output pathway that regulates the circadian expression of Retinoschisin. The Retinoschisin rhythm is concurrent with the circadian rhythm of L-type VGCCs,24 and the L-type VGCC inhibitor, nitrendipine, abolishes the rhythms of Retinoschisin. The result indicates a relationship between L-type VGCCs and Retinoschisin, in which the circadian rhythms of Retinoschisin total cellular content levels and secretion are VGCC dependent. In addition, acute changes in illumination affect the protein expression of Retinoschisin in the chick retina, which depends on prior light exposure experience. This result implies that the oscillations seen in Retinoschisin are truly circadian in nature, not simply a response to bright light. To our knowledge, this study is the first to elucidate the circadian regulation of Retinoschisin.
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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.
Bernhard H F Weber - One of the best experts on this subject based on the ideXlab platform.
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Retinoschisin binding in the presence of galactose, glucose, and mannose.
2019Co-Authors: Karolina Plössl, Bernhard H F Weber, Kristina Straub, Verena Schmid, Franziska Strunz, Jens Wild, Rainer Merkl, Ulrike FriedrichAbstract:(A) HEK293 cells transfected with a bicistronic expression construct for ATP1A3 and ATP1B2 for 48 h were subjected to recombinant Retinoschisin for 1 h in the presence of 0, 0.5, or 0.75 M galactose, glucose, or mannose, followed by intensive washing. Retinoschisin binding was investigated by Western blot analyses with antibodies against Retinoschisin. ACTB staining served as loading control. Densitometric quantification of Retinoschisin binding was performed on immunoblots from 4 individual experiments. Signals were normalized against ACTB and calibrated against signals for 0 M sugar. Data represent the mean + SD. Underlined asterisks mark statistically significant (* = P < 0.05) differences. (B) HEK293 cells transfected with a bicistronic expression construct for ATP1A3 and ATP1B2 for 48 h were subjected to recombinant Retinoschisin for 1 h in the presence of 0 M (control) or 0.75 M galactose, glucose, or mannose, followed by intensive washing. Subsequently, Retinoschisin binding was analyzed via immunocytochemistry with antibodies against Retinoschisin and ATP1B2. Scale bars, 40 μm.
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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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Retinoschisin binding in dependence of ATP1B2 glycosylation.
2019Co-Authors: Karolina Plössl, Bernhard H F Weber, Kristina Straub, Verena Schmid, Franziska Strunz, Jens Wild, Rainer Merkl, Ulrike FriedrichAbstract:(A) HEK293 cells were co-transfected with expression constructs for ATP1A3 and expression constructs for ATP1B2 mutants deficient in each one glycosylation site (ATP1B2-N96Q, -N118Q, -N153Q, -N159Q, -N193Q, -N197Q, -N238Q, and -N250Q). 48 h after transfection, cells were incubated with recombinant Retinoschisin for 1 h, followed by intensive washing. Cells transfected with only ATP1A3 expression constructs served as a negative control, cells co-transfected with expression constructs for ATP1A3 and for normal ATP1B2 served as a positive control in the Retinoschisin binding assay [13]. Heterologous protein expression as well as Retinoschisin binding was investigated by Western blot analyses with antibodies against Retinoschisin, ATP1A3, and ATP1B2. The ACTB staining served as loading control. (B) Enriched membrane fractions from HEK293 cells transfected with ATP1A3 and ATP1B2 expression constructs and Y79 cells were subjected to enzymatic deglycosylation using PNGase F. A control sample was subjected to the same treatment, but without PNGase F. Subsequently, membrane fractions were incubated with recombinant Retinoschisin for 1 h, followed by intensive washing. Retinoschisin binding, (heterologous) Na/K-ATPase expression, and ATP1B2 deglycosylation were investigated by Western blot analyses with antibodies against Retinoschisin, ATP1A3, and ATP1B2. Membranes from HEK293 cells transfected with only ATP1A3 expression vectors served as negative control in this Retinoschisin binding assay. (C) HEK293 cells co-transfected with expression constructs for ATP1A3 and for ATP1B2 for 48 h were subjected to enzymatic deglycosylation using PNGase F as described in (B). A control sample was subjected to the same treatment, but without PNGase F. Subsequently, cells were incubated with recombinant Retinoschisin for 1 h, followed by intensive washing. Retinoschisin binding was analyzed via immunocytochemistry with antibodies against Retinoschisin and ATP1B2. Scale bars, 40 μm.
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Pathomechanism of mutated and secreted Retinoschisin in X-linked juvenile retinoschisis.
Experimental eye research, 2018Co-Authors: Karolina Plössl, Bernhard H F Weber, Kristina Straub, Verena Schmid, Rainer Merkl, Carina Schmid, Mirjam Ammon, Ulrike FriedrichAbstract:Mutations in the RS1 gene encoding Retinoschisin cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy in males. While most of the XLRS associated mutations strongly interfere with cellular secretion, this is not true for mutants RS1-F108C, -R141G, -R141H, -R182C, -H207Q and -R209H. Native Retinoschisin builds double-octamers and binds to retinal membranes, interacting with the retinal Na/K-ATPase. Functionally, it regulates MAP kinase signaling and Na/K-ATPase localization, and hampers photoreceptor degeneration. In this study, we investigated the capacity of the Retinoschisin mutants still secreted extracellularly to fulfil these tasks. We addressed secretion and oligomerization of the heterologously expressed mutants as well as their binding to recombinant retinal Na/K-ATPases and murine Retinoschisin-deficient (Rs1h-/Y) retinal and non-retinal explants. This has refined the categorization of secreted Retinoschisin mutants: (i) no octamerization, unspecific membrane binding (RS1-F108C and -R182C), (ii) double-octamerization but no membrane binding (RS1-R141H), and (iii) double-octamerization and unspecific membrane binding (RS1-R141G, -H207Q, and -R209H). Notably, selected mutants of all categories (RS1-F108C, -R141H, and -R209H) failed to regulate retinal MAP kinase signaling and Na/K-ATPase localization in Rs1h-/Y retinal explants, and could not attenuate photoreceptor degeneration. Bioinformatic modeling of the secreted mutants depicted prominent alterations in the spatial and temporal conformation of a substructure called "spike 3" and its vicinity, implying a crucial role of this substructure for binding capacity and specificity. Taken together, our data point to a pathomechanism for secreted Retinoschisin mutants, specifically to disturbances of the Retinoschisin interface accompanied by unphysiological membrane interactions and impaired regulatory functions.
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The X-linked juvenile retinoschisis protein Retinoschisin is a novel regulator of mitogen-activated protein kinase signalling and apoptosis in the retina.
Journal of cellular and molecular medicine, 2016Co-Authors: Karolina Plössl, Bernhard H F Weber, Ulrike FriedrichAbstract:X-linked juvenile retinoschisis (XLRS) is a hereditary retinal dystrophy in young males, caused by mutations in the RS1 gene. The function of the encoded protein, termed Retinoschisin, and the molecular mechanisms underlying XLRS pathogenesis are still unresolved, although a direct interaction partner of the secreted Retinoschisin, the retinal Na/K-ATPase, was recently identified. Earlier gene expression studies in Retinoschisin-deficient (Rs1h−/Y) mice provided a first indication of pathological up-regulation of mitogen-activated protein (MAP) kinase signalling in disease pathogenesis. To further investigate the role for Retinoschisin in MAP kinase regulation, we exposed Y-79 cells and murine Rs1h−/Y retinae to recombinant Retinoschisin and the XLRS-associated mutant RS1-C59S. Although normal Retinoschisin stably bound to retinal cells, RS1-C59S exhibited a strongly reduced binding affinity. Simultaneously, exposure to normal Retinoschisin significantly reduced phosphorylation of C-RAF and MAP kinases ERK1/2 in Y-79 cells and murine Rs1h−/Y retinae. Expression of MAP kinase target genes C-FOS and EGR1 was also down-regulated in both model systems. Finally, Retinoschisin treatment decreased pro-apoptotic BAX-2 transcript levels in Y-79 cells and Rs1h−/Y retinae. Upon Retinoschisin treatment, these cells showed increased resistance against apoptosis, reflected by decreased caspase-3 activity (in Y-79 cells) and increased photoreceptor survival (in Rs1h−/Y retinal explants). RS1-C59S did not influence C-RAF or ERK1/2 activation, C-FOS or EGR1 expression, or apoptosis. Our data imply that Retinoschisin is a novel regulator of MAP kinase signalling and exerts an anti-apoptotic effect on retinal cells. We therefore discuss that disturbances of MAP kinase signalling by Retinoschisin deficiency could be an initial step in XLRS pathogenesis.
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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...
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RS-1 gene delivery to an adult Rs1h knockout mouse model restores ERG b-wave with reversal of the electronegative waveform of X-linked retinoschisis. Invest Ophthalmol Vis Sci
2013Co-Authors: Yong Zeng, Nizar Smaoui, Ronald A. Bush, Yuichiro Takada, Sten Kjellstrom, Kelaginamane Hiriyanna, A Tanikawa, Eric F Wawrousek, Rafael Caruso, Paul A SievingAbstract:PURPOSE. To create and evaluate a mouse model of human X-linked juvenile retinoschisis (XLRS) and then investigate whether supplementing with the Retinoschisin protein by gene delivery can reverse the abnormal “electronegative ” electroretinogram (ERG) retinal response. METHODS. An X-linked retinoschisis mouse (Rs1h-KO) model was created by substituting a neomycin resistance cassette for exon 1 and 1.6 kb of intron 1 of Rs1h, the murine orthologue of the human RS-1 gene. RS protein was evaluated by immunohistochemistry and Western blot analysis with a polyclonal RS N-terminus antibody. Retinal function was evaluated by conventional, full-field flash ERG recordings. RS protein supplementation therapy was evaluated by gene transfer with an AAV(2/2)-CMV-Rs1h vector containing C57BL/6J Rs1h cDNA under the regulation of a CMV promoter, and ERG functiona