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Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in CHM, encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs). Despite ubiquitous expression of CHM, the phenotype is limited to degeneration of the retina, retinal pigment epithelium (RPE), and choroid, with evidence for primary pathology in RPE cells. However, the spectrum of under-prenylated Rabs in RPE cells and how they contribute to RPE dysfunction remain unknown. A CRISPR/Cas-9-edited CHM-/- iPSC-RPE model was generated with isogenic control cells. Unprenylated Rabs were biotinylated in vitro and identified by tandem mass tag (TMT) spectrometry. Rab12 was one of the least prenylated and has an established role in suppressing mTORC1 signaling and promoting autophagy. CHM-/- iPSC-RPE cells demonstrated increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction. Autophagic flux was rescued in CHM-/- cells by transduction with gene replacement (ShH10-CMV-CHM) and was reduced in control cells by siRNA knockdown of Rab12. This study supports Rab12 under-prenylation as an important cause of RPE cell dysfunction in choroideremia and highlights increased mTORC1 and reduced autophagy as potential disease pathways for further investigation.
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Autofagia , Coroideremia , Células Madre Pluripotentes Inducidas , Epitelio Pigmentado de la Retina , Proteínas de Unión al GTP rab , Humanos , Proteínas Adaptadoras Transductoras de Señales , Coroideremia/patología , Coroideremia/genética , Coroideremia/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Modelos Biológicos , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Transducción de SeñalRESUMEN
PURPOSE: To assess the safety and efficacy of AAV5-hRKp.RPGR in participants with retinitis pigmentosa GTPase regulator (RPGR)-associated X-linked retinitis pigmentosa (XLRP). DESIGN: Open-label, phase 1/2 dose escalation/expansion study (ClinicalTrials.gov Identifier: NCT03252847). METHODS: Males (≥5 years old) with XLRP-RPGR were evaluated. In the dose escalation phase, subretinal AAV5-hRKp.RPGR (low: 1.0 × 1011 vg/ml; intermediate: 2.0 × 1011 vg/ml; high: 4.0 × 1011 vg/ml) was administered to the poorer-seeing eye (n = 10). Dose confirmation (intermediate dose) was carried out in 3 pediatric participants. In the dose expansion phase, 36 participants were randomized 1:1:1 to immediate (low or intermediate dose) or deferred (control) treatment. The primary outcome was safety. Secondary efficacy outcomes included static perimetry, microperimetry, vision-guided mobility, best corrected visual acuity, and contrast sensitivity. Safety and efficacy outcomes were assessed for 52 weeks for immediate treatment participants and 26 weeks for control participants. RESULTS: AAV5-hRKp.RPGR was safe and well tolerated, with no reported dose-limiting events. Most adverse events (AEs) were transient and related to the surgical procedure, resolving without intervention. Two serious AEs were reported with immediate treatment (retinal detachment, uveitis). A third serious AE (increased intraocular pressure) was reported outside the reporting period. All ocular inflammation-related AEs responded to corticosteroids. Treatment with AAV5-hRKp.RPGR resulted in improvements in retinal sensitivity and functional vision compared with the deferred group at Week 26; similar trends were observed at Week 52. CONCLUSIONS: AAV5-hRKp.RPGR demonstrated an anticipated and manageable AE profile through 52 weeks. Safety and efficacy findings support investigation in a phase 3 trial.
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The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.
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Sitios de Empalme de ARN , Retinitis Pigmentosa , Empalmosomas , Humanos , Empalmosomas/genética , Empalmosomas/metabolismo , Proteómica , Empalme del ARN/genética , Empalme Alternativo/genética , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , ARN Mensajero/metabolismo , Mutación , ADN Helicasas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Here, we present a protocol for isolating and culturing mouse photoreceptors in a minimal, chemically defined medium free from serum. We describe steps for retina dissection, enzymatic dissociation, photoreceptor enrichment, cell culture, extracellular vesicles (EVs) enrichment, and EV ultrastructural analysis. This protocol, which has been verified for cultured cells derived from multiple murine strains, allows for the study of several aspects of photoreceptor biology, including EV isolation and nanotube formation. For complete details on the use and execution of this protocol, please refer to Kalargyrou et al. (2021).1.
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Vesículas Extracelulares , Retina , Animales , Ratones , Técnicas de Cultivo de Célula , DisecciónRESUMEN
Retinitis pigmentosa (RP) is a disease characterised by photoreceptor cell death. It can be initiated by mutations in a number of different genes, primarily affecting rods, which will die first, resulting in loss of night vision. The secondary death of cones then leads to loss of visual acuity and blindness. We set out to investigate whether increased mitochondrial reactive oxygen species (ROS) formation, plays a role in this sequential photoreceptor degeneration. To do this we measured mitochondrial H2O2 production within mouse eyes in vivo using the mass spectrometric probe MitoB. We found higher levels of mitochondrial ROS that preceded photoreceptor loss in four mouse models of RP: Pde6brd1/rd1; Prhp2rds/rds; RPGR-/-; Cln6nclf. In contrast, there was no increase in mitochondrial ROS in loss of function models of vision loss (GNAT-/-, OGC), or where vision loss was not due to photoreceptor death (Cln3). Upregulation of Nrf2 transcriptional activity with dimethylfumarate (DMF) lowered mitochondrial ROS in RPGR-/- mice. These findings have important implications for the mechanism and treatment of RP.
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[This corrects the article DOI: 10.1371/journal.pone.0015730.].
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CreTrp1 mice are widely used for conditional retinal pigment epithelium (RPE) gene function studies. Like other Cre/LoxP models, phenotypes in CreTrp1 mice can be affected by Cre-mediated cellular toxicity, leading to RPE dysfunction, altered morphology and atrophy, activation of innate immunity, and consequent impairment of photoreceptor function. These effects are common among the age-related alterations of RPE that feature in early/intermediate forms of age-related macular degeneration. This article characterizes Cre-mediated pathology in the CreTrp1 line to elucidate the impact of RPE degeneration on both developmental and pathologic choroidal neovascularization. Nonredundant roles of the two major components of the hypoxia-inducible factor (HIF) family of transcription regulators, HIF1α and HIF2α, were identified. Genetic ablation of Hif1a protected against Cre-induced degeneration of RPE and choroid, whereas ablation of Hif2a exacerbated this degeneration. Furthermore, HIF1α deficiency protected CreTrp1 mice against laser-induced choroidal neovascularization, whereas HIF2α deficiency exacerbated the phenotype. Cre-mediated degeneration of the RPE in CreTrp1 mice offers an opportunity to investigate the impact of hypoxia signaling in the context of RPE degeneration. These findings indicate that HIF1α promotes Cre recombinase-mediated RPE degeneration and laser-induced choroidal neovascularization, whereas HIF2α is protective.
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Retinitis pigmentosa GTPase regulator (RPGR) gene variants are the predominant cause of X-linked retinitis pigmentosa (XLRP) and a common cause of cone-rod dystrophy (CORD). XLRP presents as early as the first decade of life, with impaired night vision and constriction of peripheral visual field and rapid progression, eventually leading to blindness. In this review, we present RPGR gene structure and function, molecular genetics, animal models, RPGR-associated phenotypes and highlight emerging potential treatments such as gene-replacement therapy.
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Proteínas del Ojo , Retinitis Pigmentosa , Animales , Mutación , Proteínas del Ojo/genética , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapia , Biología MolecularRESUMEN
PURPOSE: To assess the safety and efficacy of AAV8-hCARp.hCNGB3 in participants with CNGB3-associated achromatopsia (ACHM). DESIGN: Prospective, phase 1/2 (NCT03001310), open-label, nonrandomized clinical trial. METHODS: The study enrolled 23 adults and children with CNGB3-associated ACHM. In the dose-escalation phase, adult participants were administered 1 of 3 AAV8-hCARp.hCNGB3 dose levels in the worse-seeing eye (up to 0.5 mL). After a maximum tolerated dose was established in adults, an expansion phase was conducted in children ≥3 years old. All participants received topical and oral corticosteroids. Safety and efficacy parameters, including treatment-related adverse events and visual acuity, retinal sensitivity, color vision, and light sensitivity, were assessed for 6 months. RESULTS: AAV8-hCARp.hCNGB3 (11 adults, 12 children) was safe and generally well tolerated. Intraocular inflammation occurred in 9 of 23 participants and was mainly mild or moderate in severity. Severe cases occurred primarily at the highest dose. Two events were considered serious and dose limiting. All intraocular inflammation resolved following topical and systemic steroids. There was no consistent pattern of change from baseline to week 24 for any efficacy assessment. However, favorable changes were observed for individual participants across several assessments, including color vision (nâ¯=â¯6/23), photoaversion (nâ¯=â¯11/20), and vision-related quality-of-life questionnaires (nâ¯=â¯21/23). CONCLUSIONS: AAV8-hCARp.hCNGB3 for CNGB3-associated ACHM demonstrated an acceptable safety and tolerability profile. Improvements in several efficacy parameters indicate that AAV8-hCARp.hCNGB3 gene therapy may provide benefit. These findings, with the development of additional sensitive and quantitative end points, support continued investigation.
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Defectos de la Visión Cromática , Humanos , Adulto , Niño , Preescolar , Defectos de la Visión Cromática/genética , Defectos de la Visión Cromática/terapia , Estudios Prospectivos , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Terapia Genética , InflamaciónRESUMEN
The use of AAV-RPE65 vectors for gene supplementation has achieved spectacular success as a treatment for individuals with autosomal recessive retinal disease caused by biallelic mutations in the visual cycle gene RPE65. However, the efficacy of this approach in treating autosomal dominant retinitis pigmentosa (adRP) associated with a monoallelic mutation encoding a rare D477G RPE65 variant has not been studied. Although lacking a severe phenotype, we now find that knock-in mice heterozygous for D477G RPE65 (D477G KI mice) can be used to evaluate outcomes of AAV-RPE65 gene supplementation. Total RPE65 protein levels, which are decreased in heterozygous D477G KI mice, were doubled following subretinal delivery of rAAV2/5.hRPE65p.hRPE65. In addition, rates of recovery of the chromophore 11-cis retinal after bleaching were significantly increased in eyes that received AAV-RPE65, consistent with increased RPE65 isomerase activity. While dark-adapted chromophore levels and a-wave amplitudes were not affected, b-wave recovery rates were modestly improved. The present findings establish that gene supplementation enhances 11-cis retinal synthesis in heterozygous D477G KI mice and complement previous studies showing that chromophore therapy results in improved vision in individuals with adRP associated with D477G RPE65.
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Retina , Retinitis Pigmentosa , Animales , Ratones , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Mutación , Retina/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapia , Retinitis Pigmentosa/metabolismoRESUMEN
Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKß/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKß/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKß is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKß signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKß is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKß signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.
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Actomiosina , Proteína Quinasa de Distrofia Miotónica , Fagocitosis , Actinas/metabolismo , Actomiosina/metabolismo , Miosina Tipo II/metabolismo , Proteína Quinasa de Distrofia Miotónica/metabolismo , Fagocitosis/fisiología , Proteínas Tirosina Quinasas , Receptores Fc , Tirosina Quinasa c-Mer/metabolismoRESUMEN
The retinal pigment epithelium (RPE) is a polarized monolayer that secretes growth factors and cytokines towards the retina apically and the choroid basolaterally. Numerous RPE secreted proteins have been linked to the pathogenesis of age-related macular degeneration (AMD). The purpose of this study was to determine the differential apical and basolateral secretome of RPE cells, and the effects of oxidative stress on directional secretion of proteins linked to AMD and angiogenesis. Tandem mass tag spectrometry was used to profile proteins in human iPSC-RPE apical and basolateral conditioned media. Changes in secretion after oxidative stress induced by H2O2 or tert-butyl hydroperoxide (tBH) were investigated by ELISA and western analysis. Out of 926 differentially secreted proteins, 890 (96%) were more apical. Oxidative stress altered the secretion of multiple factors implicated in AMD and neovascularization and promoted a pro-angiogenic microenvironment by increasing the secretion of pro-angiogenic molecules (VEGF, PTN, and CRYAB) and decreasing the secretion of anti-angiogenic molecules (PEDF and CFH). Apical secretion was impacted more than basolateral for PEDF, CRYAB and CFH, while basolateral secretion was impacted more for VEGF, which may have implications for choroidal neovascularization. This study lays a foundation for investigations of dysfunctional RPE polarized protein secretion in AMD and other chorioretinal degenerative disorders.
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Células Madre Pluripotentes Inducidas , Degeneración Macular , Inductores de la Angiogénesis/farmacología , Células Cultivadas , Humanos , Peróxido de Hidrógeno/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Macular/patología , Estrés Oxidativo , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Advances in the study of neurological conditions have been possible because of pluripotent stem cell technologies and organoids. Studies have described the generation of neural ectoderm-derived retinal and brain structures from pluripotent stem cells. However, the field is still troubled by technical challenges, including high culture costs and variability. Here, we describe a simple and economical protocol that reproducibly gives rise to the neural retina and cortical brain regions from confluent cultures of stem cells. The spontaneously generated cortical organoids are transcriptionally comparable with organoids generated by other methods. Furthermore, these organoids showed spontaneous functional network activity and proteomic analysis confirmed organoids maturity. The generation of retinal and brain organoids in close proximity enabled their mutual isolation. Suspension culture of this complex organoid system demonstrated the formation of nerve-like structures connecting retinal and brain organoids, which might facilitate the investigation of neurological diseases of the eye and brain.
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Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Encéfalo , Diferenciación Celular , Organoides , Proteómica , RetinaRESUMEN
Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.
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Glaucoma , Células Ganglionares de la Retina , Animales , Glaucoma/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Retina/metabolismo , Células Ganglionares de la Retina/metabolismoRESUMEN
The generation of retinal organoids from human pluripotent stem cells (hPSC) is now a well-established process that in part recapitulates retinal development. However, hPSC-derived photoreceptors that exhibit well-organized outer segment structures have yet to be observed. To facilitate improved inherited retinal disease modeling, we determined conditions that would support outer segment development in maturing hPSC-derived photoreceptors. We established that the use of antioxidants and BSA-bound fatty acids promotes the formation of membranous outer segment-like structures. Using new protocols for hPSC-derived retinal organoid culture, we demonstrated improved outer segment formation for both rod and cone photoreceptors, including organized stacked discs. Using these enhanced conditions to generate iPSC-derived retinal organoids from patients with X-linked retinitis pigmentosa, we established robust cellular phenotypes that could be ameliorated following adeno-associated viral vector-mediated gene augmentation. These findings should aid both disease modeling and the development of therapeutic approaches for the treatment of photoreceptor disorders.
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Organoides , Células Madre Pluripotentes , Antioxidantes/farmacología , Suplementos Dietéticos , Humanos , Lípidos , Retina , Células Fotorreceptoras Retinianas ConosRESUMEN
INTRODUCTION: Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. METHODS: We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. RESULTS: To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle and other RP-linked tri-snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. CONCLUSIONS: Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.
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Autofagia , Proteínas del Ojo , Células Madre Pluripotentes Inducidas , Agregado de Proteínas , Retinitis Pigmentosa , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Ribonucleoproteínas Nucleares PequeñasRESUMEN
Opticin is an extracellular glycoprotein present in the vitreous. Its antiangiogenic properties offer the potential for therapeutic intervention in conditions such as proliferative diabetic retinopathy and retinopathy of prematurity. Here, we investigated the hypothesis that intravitreal administration of recombinant human opticin can safely protect against the development of pathological angiogenesis and promote its regression. We generated and purified recombinant human opticin and investigated its impact on the development and regression of pathological retinal neovascularization following intravitreal administration in murine oxygen-induced retinopathy. We also investigated its effect on normal retinal vascular development and function, following intravitreal injection in neonatal mice, by histological examination and electroretinography. In oxygen-induced retinopathy, intravitreal administration of human recombinant opticin protected against the development of retinal neovascularization to similar extent as aflibercept, which targets VEGF. Opticin also accelerated regression of established retinal neovascularization, though the effect at 18 h was less than that of aflibercept. Intravitreal administration of human recombinant opticin in neonatal mice caused no detectable perturbation of subsequent retinal vascular development or function. In summary we found that intraocular administration of recombinant human opticin protects against the development of pathological angiogenesis in mice and promotes its regression.
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Hiperoxia , Neovascularización Retiniana , Retinopatía de la Prematuridad , Animales , Modelos Animales de Enfermedad , Humanos , Hiperoxia/complicaciones , Recién Nacido , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica , Oxígeno/toxicidad , Neovascularización Retiniana/tratamiento farmacológico , Retinopatía de la Prematuridad/tratamiento farmacológico , Retinopatía de la Prematuridad/prevención & controlRESUMEN
The retina encompasses a network of neurons, glia and epithelial and vascular endothelia cells, all coordinating visual function. Traditionally, molecular information exchange in this tissue was thought to be orchestrated by synapses and gap junctions. Recent findings have revealed that many cell types are able to package and share molecular information via extracellular vesicles (EVs) and the technological advancements in visualisation and tracking of these delicate nanostructures has shown that the role of EVs in cell communication is pleiotropic. EVs are released under physiological conditions by many cells but they are also released during various disease stages, potentially reflecting the health status of the cells in their cargo. Little is known about the physiological role of EV release in the retina. However, administration of exogenous EVs in vivo after injury suggest a neurotrophic role, whilst photoreceptor transplantation in early stages of retina degeneration, EVs may facilitate interactions between photoreceptors and Müller glia cells. In this review, we consider some of the proposed roles for EVs in retinal physiology and discuss current evidence regarding their potential impact on ocular therapies via gene or cell replacement strategies and direct intraocular administration in the diseased eye.
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The developing retina undergoes dynamic organizational changes involving significant intra-retinal motility of the encompassing cells. Here, we present a protocol for tracking retinal cell motility in live explanted mouse retinae. Although originally applied to rod and cone photoreceptors, this strategy is applicable to any fluorescently labeled cell in mouse retinae and other similar experimental retinal models. Careful tissue handling is critical for the successful acquisition of high-quality live imaging data. Further instructions for semi-automated in silico data handling are provided. For complete details on the use and execution of this protocol, please refer to Aghaizu et al. (2021).
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Movimiento Celular/fisiología , Rastreo Celular/métodos , Retina , Células Fotorreceptoras Retinianas Conos , Células Fotorreceptoras Retinianas Bastones , Animales , Femenino , Proteínas Luminiscentes , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Retina/citología , Retina/diagnóstico por imagen , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/fisiología , Imagen de Lapso de TiempoRESUMEN
Neuronal communication is typically mediated via synapses and gap junctions. New forms of intercellular communication, including nanotubes (NTs) and extracellular vesicles (EVs), have been described for non-neuronal cells, but their role in neuronal communication is not known. Recently, transfer of cytoplasmic material between donor and host neurons ("material transfer") was shown to occur after photoreceptor transplantation. The cellular mechanism(s) underlying this surprising finding are unknown. Here, using transplantation, primary neuronal cultures and the generation of chimeric retinae, we show for the first time that mammalian photoreceptor neurons can form open-end NT-like processes. These processes permit the transfer of cytoplasmic and membrane-bound molecules in culture and after transplantation and can mediate gain-of-function in the acceptor cells. Rarely, organelles were also observed to transfer. Strikingly, use of chimeric retinae revealed that material transfer can occur between photoreceptors in the intact adult retina. Conversely, while photoreceptors are capable of releasing EVs, at least in culture, these are taken up by glia and not by retinal neurons. Our findings provide the first evidence of functional NT-like processes forming between sensory neurons in culture and in vivo.