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X-linked juvenile retinoschisis (XLRS) is an early onset inherited condition that affects primarily males and is characterized by cystic lesions of the inner retina, decreased visual acuity and contrast sensitivity, and a selective reduction of the electroretinogram (ERG) b-wave. Although XLRS is genetically heterogeneous, all mouse models develope...
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... F0 mice were then crossed with C57Bl6/Ntac females to generate F1 heterozygotes. Subsequent mouse genotyping was done by Taqman assay and PCR using primers shown in Table 1. ...Citations
... The current knowledge about XLRS is partly derived from animal models. Although the phenotype presented in these models resembles that of XLRS patients, rodent and primate retinas vary significantly [26,[40][41][42]. In this study, hiPSCs with the RS1 missense variant of c.214G > A (p.E72K), and a genetic background comparable to that of XLRS patients, were successfully constructed. ...
... A previous study also reported a significant increase in spontaneous firing rate in Rs1 mutant mice compared with WT controls. They considered that elevated activity acts as a barrier to visual signal discrimination [40]. Furthermore, the spontaneous activity observed before the onset of vision may be related to retinal projection to the central visual system and drive retinal developmental processes [59][60][61]. ...
Background
X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS.
Methods
hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy.
Results
ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation.
Conclusions
The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.
... XLRS is characterized by the presence of intraretinal schisis, decreased visual acuity and contrast sensitivity, and a marked reduction in the b-wave of the flash ERG (Peachey et al., 1987;Tanino et al., 1985). Rodent Rs1 mutants recapitulate many features of the human phenotype and provide valuable opportunities to understand basic pathophysiological features of XLRS (Eleftheriou et al., 2022;Heymann et al., 2022;Jablonski et al., 2005;Liu et al., 2019). The ERG is comprised of multiple components, including those generated by photoreceptors, ON-bipolar cells, and Müller glial cells (MGCs) which can be dissected pharmacologically (Bonezzi et al., 2020(Bonezzi et al., , 2023Frishman and Steinberg, 1989). ...
... In addition to examining the adult retina, we take advantage of the high signal-to-noise feature of the ex vivo ERG to address a second question: When is the onset of the XLRS ERG phenotype? Rs1 mutants as young as postnatal day (P)15 have a particularly severe disease phenotype (Eleftheriou et al., 2022;Liu et al., 2019). We examine Rs1 mutants at earlier ages. ...
... Rs1 KO , Rs1 R141C , Rs1 C59S and a wildtype. Wildtype and mutant mice were maintained on the same background strain as described previously (Liu et al., 2019). In brief, Rs1 KO models were generated by a lacZ replacement of Rs1 exons 1-3. ...
... The protein contains an aminoterminal signal peptide, the RS1 domain, and a discoidin domain, a specialized domain found in a family of extracellular surface proteins that plays an important role in retinal cell adhesion and cell-cell interactions [72]. At present, several XLRS mouse models have been constructed to recapitulate the retinoschisis phenotype [73,74]. A recent study has shown that this specific retinopathy may occur in patient iPSC-derived ROs [75]. ...
Inherited retinal diseases (IRDs) can induce severe sight-threatening retinal degeneration and impose a considerable economic burden on patients and society, making efforts to cure blindness imperative. Transgenic animals mimicking human genetic diseases have long been used as a primary research tool to decipher the underlying pathogenesis, but there are still some obvious limitations. As an alternative strategy, patient-derived induced pluripotent stem cells (iPSCs), particularly three-dimensional (3D) organoid technology, are considered a promising platform for modeling different forms of IRDs, including retinitis pigmentosa, Leber congenital amaurosis, X-linked recessive retinoschisis, Batten disease, achromatopsia, and best vitelliform macular dystrophy. Here, this paper focuses on the status of patient-derived iPSCs and organoids in IRDs in recent years concerning disease modeling and therapeutic exploration, along with potential challenges for translating laboratory research to clinical application. Finally, the importance of human iPSCs and organoids in combination with emerging technologies such as multi-omics integration analysis, 3D bioprinting, or microfluidic chip platform are highlighted. Patient-derived retinal organoids may be a preferred choice for more accurately uncovering the mechanisms of human retinal diseases and will contribute to clinical practice.
... Knockout mice, in contrast, do not express any protein. Indeed, newer mouse models with Rs1 mutations similar to those in humans, such as C59S or R141C point mutant substitutions, show less severe ERG phenotypes, especially at young ages (Liu et al., 2019). ...
X-linked juvenile retinoschisis (XLRS), a hereditary retinal disorder primarily affecting males, is characterized by the formation of cystic spaces between the outer plexiform layer and outer nuclear layer of the retina. Mutations in the RS1 gene, which encodes the extracellular binding protein retinoschisin, are responsible for XLRS pathogenesis. While the role of retinoschisin in maintaining retinal integrity is well established, there is growing evidence suggesting compromised photoreceptor function in XLRS. To investigate the molecular pathways affected by RS1 deficiency, particularly in phototransduction, we performed electroretinographic (ERG) and proteomic analyses on retinae from Rs1 knockout mice, a model of human XLRS. The Rs1 knockout mice had reduced ERG a-wave amplitudes. Correspondingly, differential expression analysis revealed downregulation of proteins crucial for phototransduction, with Ingenuity Pathway Analysis (IPA) highlighting "Phototransduction" as the most significantly downregulated biological theme. Compensatory mechanisms were also observed in the IPA, including upregulation of synaptic remodeling, inflammation, cell adhesion, and G-protein signaling. These findings strongly implicate an underrecognized role of photoreceptor dysfunction in XLRS pathology. We speculate that entrapment of mutant retinoschisin protein within photoreceptor inner segments as well as disrupted reciprocal regulation between L-type voltage-gated calcium channels and retinoschisin contribute to the dysfunction in photoreceptors.
... Animal models of human diseases have been widely used to study features of human monogenic diseases and to explore treatment strategies. Several Rs1h −/y knockout (KO) mouse models have been generated which mimic the human pathology in X-linked juvenile retinoschisis (XLRS) patient [1][2][3][4]. All of these genetically modified mice fail to express normal retinoschisin protein, and they display structural and functional retinal phenotypes in the spectrum of XLRS patients. ...
... reporter gene one Rs1h −/y mouse model, and two knock-in mouse models carrying either a C59S or an R141C point mutant substitution [3]. Except for the exon 1 deletion Rs1h −/y mouse we made previously, the phenotype onset and progression of this rat model roughly follows that of the faster of the current mouse models (Table 1) [2,3,37,38]. ...
... reporter gene one Rs1h −/y mouse model, and two knock-in mouse models carrying either a C59S or an R141C point mutant substitution [3]. Except for the exon 1 deletion Rs1h −/y mouse we made previously, the phenotype onset and progression of this rat model roughly follows that of the faster of the current mouse models (Table 1) [2,3,37,38]. What appears different in this rat model is the rapid emergence and collapse of the schisis cavities. ...
Animal models of X-linked juvenile retinoschisis (XLRS) are valuable tools for understanding basic biochemical function of retinoschisin (RS1) protein and to investigate outcomes of preclinical efficacy and toxicity studies. In order to work with an eye larger than mouse, we generated and characterized an Rs1h −/y knockout rat model created by removing exon 3. This rat model expresses no normal RS1 protein. The model shares features of an early onset and more severe phenotype of human XLRS. The morphologic pathology includes schisis cavities at postnatal day 15 (p15), photoreceptors that are misplaced into the subretinal space and OPL, and a reduction of photoreceptor cell numbers by p21. By 6 mo age only 1–3 rows of photoreceptors nuclei remain, and the inner/outer segment layers and the OPL shows major changes. Electroretinogram recordings show functional loss with considerable reduction of both the a-wave and b-wave by p28, indicating early age loss and dysfunction of photoreceptors. The ratio of b-/a-wave amplitudes indicates impaired synaptic transmission to bipolar cells in addition. Supplementing the Rs1h −/y exon3-del retina with normal human RS1 protein using AAV8- RS1 delivery improved the retinal structure. This Rs1h −/y rat model provides a further tool to explore underlying mechanisms of XLRS pathology and to evaluate therapeutic intervention for the XLRS condition.
... However, there still exists several gaps in knowledge, especially at early developmental stages. RS1 is secreted primarily by photoreceptors in the mature mouse retina (Liu et al., 2019). Whereas RS1 is observed in inner retinal cells during development Liu et al., 2019), the functional consequences of this expression is not clear. ...
... RS1 is secreted primarily by photoreceptors in the mature mouse retina (Liu et al., 2019). Whereas RS1 is observed in inner retinal cells during development Liu et al., 2019), the functional consequences of this expression is not clear. The protein consists of disulfide bond-stabilized homodimers, which assemble intracellularly (Molday et al., 2012) in octamer pairs displaying a symmetrical cog wheel-like structure (Bush et al., 2016;Tolun et al., 2016). ...
... RS1 has also been identified as a binding partner for the a3 b 2 isoform of the Na/K ATPase at the surface of photoreceptors and may also regulate intracellular signaling (Molday et al., 2007;Plössl et al., 2017a,b). Our recent work (Liu et al., 2019) identified that in RS1-deficient mice horizontal cells (HCs) and rod bipolar cells abnormally extended their neurites past the outer plexiform layer (OPL) and into the outer nuclear layer (ONL). These anatomic defects indicated a role for RS1 in controlling glutamate release from photoreceptor terminals, a hypothesis that was further supported by functional studies identifying both presynaptic and postsynaptic abnormalities at the glutamatergic photoreceptor-to-ON bipolar cell (BC) synapse of Rs1 À/À (KO) mice (Ou et al., 2015). ...
Genetic disorders which present during development make treatment strategies particularly challenging because there is a need to disentangle primary pathophysiology from downstream dysfunction caused at key developmental stages. To provide a deeper insight into this question, we studied a mouse model of X-linked juvenile retinoschisis (XLRS), an early-onset inherited condition caused by mutations in the Rs1 gene encoding retinoschisin (RS1) and characterized by cystic retinal lesions and early visual deficits. Using an unbiased approach in expressing the fast intracellular calcium indicator GCaMP6f in neuronal, glial, and vascular cells of the retina of RS1-deficient male mice, we found that initial cyst formation is paralleled by the appearance of aberrant spontaneous neuro-glial signals as early as postnatal day 15, when eyes normally open. These presented as glutamate-driven wavelets of neuronal activity and sporadic radial bursts of activity by Müller glia, spanning all retinal layers and disrupting light-induced signaling. This study confers a role to RS1 beyond its function as an adhesion molecule, identifies an early onset for dysfunction in the course of disease, establishing a potential window for disease diagnosis and therapeutic intervention.Significance StatementDevelopmental disorders make it difficult to distinguish pathophysiology due to ongoing disease from pathophysiology due to disrupted development. Here, we investigated a mouse model for X-linked retinoschisis (XLRS), a well-defined monogenic degenerative disease caused by mutations in the Rs1 gene, which codes for the protein retinoschisin. We evaluated the spontaneous activity of explanted retinas lacking retinoschisin at key stages of development using the unbiased approach of ubiquitously expressing GCaMP6f in all retinal neurons, vasculature and glia. In mice lacking RS1, we found an array of novel phenotypes which present around eye-opening, are linked to glutamatergic neurotransmission, and affect visual processing. These data identify novel pathophysiology linked to RS1, and define a window where treatments might be best targeted.
... Moreover, cell migrationrelated Avl9 was specifically upregulated [46,47], which may be the mechanism underlying ONL disorganization and nuclei migration at the onset of XLRS. In contrast, Avl9 was downregulated at P56, and the corresponding nuclei displacement was alleviated, and had disappeared in long-term observations [17]. Considering the shrinking of schisis at P56, we also found that Chl1, another adhesion-related protein, was specifically upregulated at P56 [48]. ...
X-linked retinoschisis (XLRS) is among the most commonly inherited degenerative retinopathies. XLRS is caused by functional impairment of RS1. However, the molecular mechanisms underlying RS1 malfunction remain largely uncharacterized. Here, we performed a data-independent acquisition-mass spectrometry-based proteomic analysis in RS1-null mouse retina with different postal days (Ps), including the onset (P15) and early progression stage (P56). Gene set enrichment analysis showed that type I interferon-mediated signaling was upregulated and photoreceptor proteins responsible for detection of light stimuli were downregulated at P15. Positive regulation of Tor signaling was downregulated and nuclear transcribed mRNA catabolic process nonsense-mediated decay was upregulated at P56. Moreover, the differentially expressed proteins at P15 were enriched in metabolism of RNA and RNA destabilization. A broader subcellular localization distribution and enriched proteins in visual perception and phototransduction were evident at P56. Combined transcriptomic-proteomic analysis revealed that functional impairments, including detection of visible light, visual perception, and visual phototransduction, occurred at P21 and continued until P56. Our work provides insights into the molecular mechanisms underlying the onset and progression of an XLRS mouse model during the early stages, thus enhancing the understanding of the mechanism of XLRS.
... Each model developed intraretinal schisis with OCT and showed early abnormalities in outer retina neurons with immunohistochemical analysis. In consistent with the morphological changes, the decrease of ERG b-wave amplitude was more prominent than the a-wave, but the a-wave was already abnormal at P15 and remained decreased till later stage (28). These findings indicated that although the major defects were in the inner retina, outer retina photoreceptor degeneration could present concurrently. ...
Purpose
To study the long-term photoreceptor changes and to evaluate the effects of topical application of a carbonic anhydrase inhibitor (CAI) in a mouse model of X-linked retinoschisis (XLRS).
Methods
Conventional electroretinograms (ERGs) and dark-adapted 10-Hz flicker ERGs were recorded in control and Rs1 −/ Y mice generated with CRISPR/Cas9. ON-pathway blocker 2-amino-4-phosphobutyric acid (APB) was injected intravitreally. Morphology was evaluated with histology and optical coherence tomography (OCT). Mice were treated with a CAI inhibitor brinzolamide eye drops (10 mg/ml) three times a day for 3 months. OCT and ERG findings at 1, 4, and 10 months were analyzed.
Results
Negative ERGs and retinal cavities were evident in Rs1 −/ Y mice. Both a-wave and b-wave amplitudes decreased with age when compared with age-matched controls. The APB-isolated a-wave (a′) amplitudes of Rs1 −/ Y mice were reduced in all age groups. In dark-adapted 10-Hz flicker ERG, the amplitude-intensity curve of Rs1 −/ Y mice shifted down. The thickness of ONL and IS/OS decreased in Rs1 −/ Y mice. CAI reduced the splitting retinal cavities but didn't affect the ERG.
Conclusions
In addition to post receptoral impairments, photoreceptor cells underwent progressive dysfunction since early age in Rs1 −/ Y mice. Long-term CAI treatment improved the shrinkage of the splitting retinal cavity, while no functional improvement was observed.
... One potential explanation for the reduced CS comes from single cell recordings in Rs1 mutant mice, which have elevated background activity ("noise"). 12 Specifically, single cell recordings from retinal ganglion cells (RGCs) showed elevated spontaneous RGC activity in young Rs1 mice compared to wild type mice. 12 The elevated spontaneous activity resulted in a decreased signal-to-noise ratio, which is anticipated to reduce sensitivity. ...
... 12 Specifically, single cell recordings from retinal ganglion cells (RGCs) showed elevated spontaneous RGC activity in young Rs1 mice compared to wild type mice. 12 The elevated spontaneous activity resulted in a decreased signal-to-noise ratio, which is anticipated to reduce sensitivity. Consistent with this finding, it has been proposed that a number of retinal diseases can be characterized by high levels of noise within the visual pathway, 13 which was supported by measurements in subjects with retinitis pigmentosa 14 and diabetic retinopathy. ...
... Single-cell RGC electrophysiology in young Rs1 mutant mice demonstrated elevated spiking activity in the absence of visual stimulation and a reduced signal-to-noise ratio in response to light stimuli. 12 Superficially, this finding is consistent with the present report, which showed that high levels of internal noise are associated with low CS. However, the extent to which elevated intrinsic noise in subjects with XLRS is a behavioral manifestation of physiologic RGC noise is unclear. ...
Purpose:
To define relationships among contrast sensitivity (CS), equivalent intrinsic noise (Neq; a measure of noise within the visual pathway), and retinal thickness in X-linked retinoschisis (XLRS).
Methods:
Nine XLRS and 10 visually-normal subjects participated. CS was measured in the presence and absence of luminance noise. These data were fit with a standard model to estimate Neq and sampling efficiency (an estimate of the ability to use stimulus information). Optical coherence tomography images were obtained to quantify outer nuclear layer (ONL+) and outer segment (OS+) thickness. A linear structure-function model was used to describe the relationship between CS and the product of ONL+ and OS+ thickness.
Results:
CS in the absence of noise (CS0) for the XLRS subjects ranged from normal to as much as 1.5× below the lower limit of normal. Four of the nine subjects with XLRS had abnormally high Neq, whereas two others had sampling efficiency that was borderline abnormal. Log CS0 for the subjects with XLRS was correlated significantly with log Neq (r = -0.78, P = 0.01), but not with log efficiency (r = 0.19, P = 0.63). CS0 and Neq, but not efficiency, conformed to the linear ONL+ × OS+ structure-function model.
Conclusions:
The XLRS subjects in this study who had elevated internal noise had abnormally low CS; both internal noise and CS fell within the predicted limits of a structure-function model.
Translational relevance:
Internal noise measurements can provide insight into a source of CS loss in some individuals with XLRS.
... The impact of these mutations on RS1 protein structure and function was assessed in mammalian cell lines expressing recombinant wild-type and/or mutant RS1 Wang et al., 2002Wang et al., , 2006. RS1 mutant knock-in mouse and retinal organoids expressing patient specific RS1 mutations were also used as system models to study mutation-phenotype links Liu et al., 2019). A selected list of XLRS mutations and their phenotypic consequences are described in Table 1. ...
... Since functional RS1 exists as an octamer, a misfolded RS1 mutant capable of oligomerizing with wild-type RS1 can dominantly cripple oligomer function (Gleghorn et al., 2009). This likelihood was tested in female mouse models of XLRS with C59S and R141C mutations (Liu et al., 2019). Schisis was observed only in the retinas of homozygous but not heterozygous females. ...
... Three developmental studies in mice without functional Rs1 (Rs1-KO(2) , Rs1-KO(3) and Rs1-1 h-44TNJ) demonstrated normal retinal lamination and correct localization of synaptic proteins P14 and P17. (Jablonski et al., 2005;Johnson et al., 2006;Liu et al., 2019;Takada et al., 2008). The earliest morphological schisis lesion in different XLRS mouse models without functional Rs1 occurred after synaptic formation between P14-18. ...
X-linked Retinoschisis (XLRS) is an early-onset transretinal dystrophy, often with a prominent macular component, that affects males and generally spares heterozygous females because of X-linked recessive inheritance. It results from loss-of-function RS1 gene mutations on the X-chromosome. XLRS causes bilateral reduced acuities from young age, and on clinical exam and by ocular coherence tomography (OCT) the neurosensory retina shows foveo-macular cystic schisis cavities in the outer plexiform (OPL) and inner nuclear layers (INL). XLRS manifests between infancy and school-age with variable phenotypic presentation and without reliable genotype-phenotype correlations. INL disorganization disrupts synaptic signal transmission from photoreceptors to ON-bipolar cells, and this reduces the electroretinogram (ERG) bipolar b-wave disproportionately to photoreceptor a-wave changes. RS1 gene expression is localized mainly to photoreceptors and INL bipolar neurons, and RS1 protein is thought to play a critical cell adhesion role during normal retinal development and later for maintenance of retinal structure. Several independent XLRS mouse models with mutant RS1 were created that recapitulate features of human XLRS disease, with OPL-INL schisis cavities, early onset and variable phenotype across mutant models, and reduced ERG b-wave to a-wave amplitude ratio. The faithful phenotype of the XLRS mouse has assisted in delineating the disease pathophysiology. Delivery to XLRS mouse retina of an AAV8-RS1 construct under control of the RS1 promoter restores the retinal structure and synaptic function (with increase of b-wave amplitude). It also ameliorates the schisis-induced inflammatory microglia phenotype toward a state of immune quiescence. The results imply that XLRS gene therapy could yield therapeutic benefit to preserve morphological and functional retina particularly when intervention is conducted at earlier ages before retinal degeneration becomes irreversible. A phase I/IIa single-center, open-label, three-dose-escalation clinical trial reported a suitable safety and tolerability profile of intravitreally administered AAV8-RS1 gene replacement therapy for XLRS participants. Dose-related ocular inflammation occurred after dosing, but this resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in dose-dependent fashion, but no antibodies were observed against the RS1 protein. Retinal cavities closed transiently in one participant. Technological innovations in methods of gene delivery and strategies to further reduce immune responses are expected to enhance the therapeutic efficacy of the vector and ultimate success of a gene therapy approach.
