RESUMO
Retinitis pigmentosa (RP), an inherited retinal disease, affects 1,5 million people worldwide. The initial mutation-driven photoreceptor degeneration leads to chronic inflammation, characterized by Müller cell activation and upregulation of CD44. CD44 is a cell surface transmembrane glycoprotein and the primary receptor for hyaluronic acid. It is involved in many pathological processes, but little is known about CD44's retinal functions. CD44 expression is also increased in Müller cells from our Pde6bSTOP/STOP RP mouse model. To gain a more detailed understanding of CD44's role in healthy and diseased retinas, we analyzed Cd44-/- and Cd44-/-Pde6bSTOP/STOP mice, respectively. The loss of CD44 led to enhanced photoreceptor degeneration, reduced retinal function, and increased inflammatory response. To understand the underlying mechanism, we performed proteomic analysis on isolated Müller cells from Cd44-/- and Cd44-/-Pde6bSTOP/STOP retinas and identified a significant downregulation of glutamate transporter 1 (SLC1A2). This downregulation was accompanied by higher glutamate levels, suggesting impaired glutamate homeostasis. These novel findings indicate that CD44 stimulates glutamate uptake via SLC1A2 in Müller cells, which in turn, supports photoreceptor survival and function.
Assuntos
Células Ependimogliais , Receptores de Hialuronatos , Retinose Pigmentar , Transdução de Sinais , Animais , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/genética , Camundongos , Células Ependimogliais/metabolismo , Transdução de Sinais/fisiologia , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Retinose Pigmentar/genética , Camundongos Knockout , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados/metabolismo , Sobrevivência Celular/fisiologia , Camundongos Transgênicos , Retina/metabolismo , Retina/patologiaRESUMO
Photoreceptor degeneration is a major cause of untreatable blindness worldwide and has recently been targeted by emerging technologies, including cell- and gene-based therapies. Cell types of neural lineage have shown promise for replacing either photoreceptors or retinal pigment epithelial cells following delivery to the subretinal space, while cells of bone marrow lineage have been tested for retinal trophic effects following delivery to the vitreous cavity. Here we explore an alternate approach in which cells from the immature neural retinal are delivered to the vitreous cavity with the goal of providing trophic support for degenerating photoreceptors. Rat and human retinal progenitor cells were transplanted to the vitreous of rats with a well-studied photoreceptor dystrophy, resulting in substantial anatomical preservation and functional rescue of vision. This work provides scientific proof-of-principle for a novel therapeutic approach to photoreceptor degeneration that is currently being evaluated in clinical trials.
Assuntos
Retina , Degeneração Retiniana , Transplante de Células-Tronco , Animais , Ratos , Degeneração Retiniana/terapia , Degeneração Retiniana/patologia , Transplante de Células-Tronco/métodos , Humanos , Retina/patologia , Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/transplante , Modelos Animais de DoençasRESUMO
Transient receptor potential canonical (TRPC) channels are calcium channels with diverse expression profiles and physiological implications in the retina. Neurons and glial cells of rat retinas with photoreceptor degeneration caused by retinitis pigmentosa (RP) exhibit basal calcium levels that are above those detected in healthy retinas. Inner retinal cells are the last to degenerate and are responsible for maintaining the activity of the visual cortex, even after complete loss of photoreceptors. We considered the possibility that TRPC1 and TRPC5 channels might be associated with both the high calcium levels and the delay in inner retinal degeneration. TRPC1 is known to mediate protective effects in neurodegenerative processes while TRPC5 promotes cell death. In order to comprehend the implications of these channels in RP, the co-localization and subsequent physical interaction between TRPC1 and TRPC5 in healthy retina (Sprague-Dawley rats) and degenerating (P23H-1, a model of RP) retina were detected by immunofluorescence and proximity ligation assays. There was an overlapping signal in the innermost retina of all animals where TRPC1 and TRPC5 physically interacted. This interaction increased significantly as photoreceptor loss progressed. Both channels function as TRPC1/5 heteromers in the healthy and damaged retina, with a marked function of TRPC1 in response to retinal degenerative mechanisms. Furthermore, our findings support that TRPC5 channels also function in partnership with STIM1 in Müller and retinal ganglion cells. These results suggest that an increase in TRPC1/5 heteromers may contribute to the slowing of the degeneration of the inner retina during the outer retinal degeneration.
Assuntos
Ratos Sprague-Dawley , Degeneração Retiniana , Canais de Cátion TRPC , Animais , Canais de Cátion TRPC/metabolismo , Ratos , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Retinose Pigmentar/genética , Modelos Animais de DoençasRESUMO
Minocycline, a broad-spectrum tetracycline antibiotic, has been shown to possess anti-inflammatory and antioxidative effects in various neurodegenerative diseases. However, its specific effects on retinitis pigmentosa (RP) have not been thoroughly investigated. Therefore, the objective of this study was to explore the potential role of minocycline in treating RP. In this investigation, we used rd1 to explore the antioxidant effect of minocycline in RP. Minocycline therapy effectively restored retinal function and structure in rd1 mice at 14 days postnatal. Additionally, minocycline inhibited the activation of microglia. Moreover, RNA sequencing analysis revealed a significant downregulation in the expression of mitochondrial genes within the retina of rd1 mice. Further KEGG and GO pathway analysis indicated impaired oxidative phosphorylation and electron transport chain processes. TEM confirmed the presence of damaged mitochondria in photoreceptors, while JC-1 staining demonstrated a decrease in mitochondrial membrane potential, accompanied by an increase in mitochondrial reactive oxygen species (ROS) levels. However, treatment with minocycline successfully reversed the abnormal expression of mitochondrial genes and reduced the levels of mitochondrial ROS, thereby providing protection against photoreceptor degeneration. Collectively, minocycline demonstrated the ability to rescue photoreceptor cells in RP by effectively modulating mitochondrial homeostasis and subsequently inflammation. These findings hold significant implications for the development of potential therapeutic strategies for RP.
Assuntos
Homeostase , Minociclina , Mitocôndrias , Espécies Reativas de Oxigênio , Retinose Pigmentar , Minociclina/farmacologia , Minociclina/uso terapêutico , Animais , Retinose Pigmentar/tratamento farmacológico , Retinose Pigmentar/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Homeostase/efeitos dos fármacos , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/patologia , Degeneração Retiniana/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Retina/efeitos dos fármacos , Retina/patologia , Retina/metabolismo , Humanos , Antioxidantes/farmacologia , Antioxidantes/uso terapêuticoRESUMO
Topical treatment of vitreoretinal diseases remains a challenge due to slow corneal uptake and systemic clearance. Exosomes are emerging nanocarriers for drug delivery due to biocompatibility and cellular targeting properties. To apply them for retinal targeting via the topical route, exosomes must traverse various ocular barriers including the cornea, lens, vitreous humor (VH), and the retina itself. Here we engineered high-purity milk-derived exosomes by anchoring arginine-rich cationic motifs via PEG2000 lipid insertion on their surface. Modification enabled exosomes to use weak-reversible electrostatic interactions with anionic glycosaminoglycan (GAG) and water content of the tissue to enhance their transport rate and retention. Addition of cationic motifs neutralized the anionic surface charge of exosomes (-24 to -2 mV) without impacting size or morphology. Cationic-motif-modified exosomes exhibited two-fold faster steady state diffusivity through bovine corneas compared to unmodified exosomes. Fluorescence recovery after photobleaching confirmed that cationic-motif-modified exosomes can diffuse through VH without steric hindrance. In healthy VH, cationic-motif-modified exosomes demonstrated stronger binding resulting in three-fold lower average diffusivity that enhanced by six-fold in 50% GAG-depleted VH recapitulating advanced liquefaction. Cationic-motif-modified exosomes penetrated through the full-thickness of porcine retinal explants resulting in ten-fold higher uptake in photoreceptors and three-fold greater transfection with encapsulated eGFP mRNA compared to unmodified exosomes. Cationic-motif-modified exosomes are safe to use as they did not adversely affect the mechanical swelling properties of the cornea or lens nor impact retinal cell viability. Cationic-motif-modified exosomes, therefore, offer themselves as a cell-free nanocarrier platform for gene delivery to retinal photoreceptors potentially via the topical route.
Assuntos
Exossomos , RNA Mensageiro , Animais , Exossomos/química , Exossomos/metabolismo , Bovinos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cátions/química , Células Fotorreceptoras de Vertebrados/metabolismoRESUMO
PURPOSE: In this study, differences in retinal feature visualization of high-resolution optical coherence tomography (OCT) devices were investigated with different axial resolutions in quantifications of retinal pigment epithelium and photoreceptors (PRs) in intermediate age-related macular degeneration. METHODS: Patients were imaged with standard SPECTRALIS HRA + OCT and the investigational High-Res OCT device (both by Heidelberg Engineering, Heidelberg, Germany). Drusen, retinal pigment epithelium, and PR layers were segmented using validated artificial intelligence-based algorithms followed by manual corrections. Thickness and drusen maps were computed for all patients. Loss and thickness measurements were compared between devices, drusen versus nondrusen areas, and early treatment diabetic retinopathy study subfields using mixed-effects models. RESULTS: Thirty-three eyes from 28 patients with intermediate age-related macular degeneration were included. Normalized PR integrity loss was significantly higher with 4.6% for standard OCT compared with 2.5% for High-Res OCT. The central and parafoveal PR integrity loss was larger than the perifoveal loss (P < 0.05). Photoreceptor thickness was increased on High-Res OCT and in nondrusen regions (P < 0.001). Retinal pigment epithelium appeared thicker on standard OCT and above drusen (P < 0.01). CONCLUSION: Our study shows that High-Res OCT is able to identify the condition of investigated layers in intermediate age-related macular degeneration with higher precision. This improved in vivo imaging technology might promote our understanding of the pathophysiology and progression of age-related macular degeneration.
Assuntos
Epitélio Pigmentado da Retina , Tomografia de Coerência Óptica , Humanos , Tomografia de Coerência Óptica/métodos , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/diagnóstico por imagem , Feminino , Masculino , Idoso , Idoso de 80 Anos ou mais , Células Fotorreceptoras de Vertebrados/patologia , Acuidade Visual/fisiologia , Drusas Retinianas/diagnóstico , Drusas Retinianas/diagnóstico por imagem , Degeneração Macular/diagnóstico , Degeneração Macular/fisiopatologia , Pessoa de Meia-IdadeRESUMO
Photoreceptor oil droplets (ODs) are spherical organelles placed most commonly within the inner segment of the cone photoreceptors. Comprising neutral lipids, ODs can be either non-pigmented or pigmented and have been considered optically functional in various studies. Among living amphibians, ODs were only reported to occur in frogs and toads (Anura), while they are absent in salamanders and caecilians. Nonetheless, the limited understanding of their taxonomic distribution in anurans impedes a comprehensive assessment of their evolution and relationship with visual ecology. We studied the retinae of 134 anuran species, extending the knowledge of the distribution of ODs to 46 of the 58 currently recognized families, and providing a new perspective on this group that complements the available information from other vertebrates. The occurrence of ODs in anurans shows a strong phylogenetic signal, and our findings revealed that ODs evolved at least six times during the evolutionary history of the group, independently from other vertebrates. Although no evident correlation was found between OD occurrence, adult habits and diel activity, it is inferred that each independent origin involves distinct scenarios in the evolution of ODs concerning photic habits. Furthermore, our results revealed significant differences in the size of the ODs between nocturnal and arrhythmic anurans relative to the length of the cones' outer segment.
Assuntos
Anuros , Evolução Biológica , Filogenia , Animais , Anuros/fisiologia , Gotículas Lipídicas , Bufonidae/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologiaRESUMO
Mitochondria-associated ER membranes (MAMs) are contact sites that enable bidirectional communication between the ER (endoplasmic reticulum) and mitochondria, including the transfer of Ca2+ signals. MAMs are essential for mitochondrial function and cellular energy metabolism. However, unrestrained Ca2+ transfer to the mitochondria can lead to mitochondria-dependent apoptosis. IP3R2 (Inositol 1,4,5-trisphosphate receptor 2) is an important intracellular Ca2+ channel. This study investigated the contribution of IP3R2-MAMs to hypoxia-induced apoptosis in photoreceptor cells. A photoreceptor hypoxia model was established by subretinal injection of hyaluronic acid (1%) in C57BL/6 mice and 1% O2 treatment in 661W cells. Transmission electron microscopy (TEM), ER-mitochondria colocalization, and the MAM reporter were utilized to evaluate MAM alterations. Cell apoptosis and mitochondrial homeostasis were evaluated using immunofluorescence (IF), flow cytometry, western blotting (WB), and ATP assays. SiRNA transfection was employed to silence IP3R2 in 661W cells. Upon hypoxia induction, MAMs were significantly increased in photoreceptors both in vivo and in vitro. This was accompanied by the activation of mitochondrial apoptosis and disruption of mitochondrial homeostasis. Elevated MAM-enriched IP3R2 protein levels induced by hypoxic injury led to mitochondrial calcium overload and subsequent photoreceptor apoptosis. Notably, IP3R2 knockdown not only improved mitochondrial morphology but also restored mitochondrial function in photoreceptors by limiting MAM formation and thereby attenuating mitochondrial calcium overload under hypoxia. Our results suggest that IP3R2-MAM-mediated mitochondrial calcium overload plays a critical role in mitochondrial dyshomeostasis, ultimately contributing to photoreceptor cell death. Targeting MAM constitutive proteins might provide an option for a therapeutic approach to mitigate photoreceptor death in retinal detachment.
Assuntos
Apoptose , Cálcio , Retículo Endoplasmático , Receptores de Inositol 1,4,5-Trifosfato , Mitocôndrias , Animais , Camundongos , Western Blotting , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Hipóxia/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologiaRESUMO
This review examines the pivotal role of photoreceptor cells in ocular refraction development, focusing on dopamine (DA) as a key neurotransmitter. Contrary to the earlier view favoring cone cells, recent studies have highlighted the substantial contributions of both rod and cone cells to the visual signaling pathways that influence ocular refractive development. Notably, rod cells appeared to play a central role. Photoreceptor cells interact intricately with circadian rhythms, color vision pathways, and other neurotransmitters, all of which are crucial for the complex mechanisms driving the development of myopia. This review emphasizes that ocular refractive development results from a coordinated interplay between diverse cell types, signaling pathways, and neurotransmitters. This perspective has significant implications for unraveling the complex mechanisms underlying myopia and aiding in the development of more effective prevention and treatment strategies.
Assuntos
Miopia , Refração Ocular , Miopia/fisiopatologia , Miopia/metabolismo , Miopia/etiologia , Humanos , Refração Ocular/fisiologia , Animais , Dopamina/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Ritmo Circadiano/fisiologia , Transdução de Sinais/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Células Fotorreceptoras de Vertebrados/patologiaRESUMO
Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.
Assuntos
Mitocôndrias , Oxirredutases , Proteínas de Plantas , Epitélio Pigmentado da Retina , Animais , Mitocôndrias/metabolismo , Camundongos , Oxirredutases/metabolismo , Oxirredutases/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Ciona intestinalis/metabolismo , Ubiquinona/metabolismo , Ubiquinona/análogos & derivados , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologiaRESUMO
Müller glia and microglia are capable of phagocytosing fragments of retinal cells in response to retinal injury or degeneration. However, the direct evidence for their mutual interactions between Müller glia and microglia in the progression of retinal degeneration (RD) remains largely unclear. This study aims to construct a progressive RD mouse model and investigate the activated pattern of Müller glia and the interplay between Müller glia and microglia in the early stage or progression of RD. A Prohibitin 2 (Phb2) photoreceptor-specific knockout (RKO) mouse model was generated by crossing Phb2flox/flox mice with Rhodopsin-Cre mice. Optical Coherence Tomography (OCT), histological staining, and Electroretinography (ERG) assessed retinal structure and function, and RKO mice exhibited progressive RD from six weeks of age. In detail, six-week-old RKO mice showed no significant retinal impairment, but severe vision dysfunction and retina thinning were shown in ten-week-old RKO mice. Furthermore, RKO mice were sensitive to Light Damage (LD) and showed severe RD at an early age after light exposure. Bulk retina RNA-seq analysis from six-week-old control (Ctrl) and RKO mice showed reactive retinal glia in RKO mice. The activated pattern of Müller glia and the interplay between Müller glia and microglia was visualized by immunohistology and 3D reconstruction. In six-week-old RKO mice or light-exposed Ctrl mice, Müller glia were initially activated at the edge of the retina. Moreover, in ten-week-old RKO mice or light-exposed six-week-old RKO mice with severe photoreceptor degeneration, abundant Müller glia were activated across the whole retinas. With the progression of RD, phagocytosis of microglia debris by activated Müller glia were remarkably increased. Altogether, our study establishes a Phb2 photoreceptor-specific knockout mouse model, which is a novel mouse model of RD and can well demonstrate the phenotype of progressive RD. We also report that Müller glia in the peripheral retina is more sensitive to the early damage of photoreceptors. Our study provides more direct evidence for Müller glia engulfing microglia debris in the progression of RD due to photoreceptor Phb2 deficiency.
Assuntos
Células Ependimogliais , Microglia , Células Fotorreceptoras de Vertebrados , Proibitinas , Degeneração Retiniana , Animais , Camundongos , Modelos Animais de Doenças , Eletrorretinografia , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , Fagocitose/fisiologia , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/deficiência , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Tomografia de Coerência ÓpticaRESUMO
NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.
Assuntos
Apoptose , Autofagia , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Células Fotorreceptoras de Vertebrados , Sulfonamidas , Animais , Camundongos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Óxidos S-Cíclicos/farmacologia , Células Ependimogliais/metabolismo , Células Ependimogliais/efeitos dos fármacos , Furanos/farmacologia , Hipóxia/metabolismo , Indenos/farmacologia , Inflamassomos/metabolismo , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonas/farmacologiaRESUMO
Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments, which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lacked outer segments and underwent apoptosis. Interestingly, zebrafish mak mutants failed to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies were specified. These data suggest that Mak contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of Mak was found to be critical in ciliary axoneme development and photoreceptor survival. Thus, Mak is required for ciliogenesis and outer segment formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.
Assuntos
Axonema , Cílios , Mutação , Proteínas Serina-Treonina Quinases , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/embriologia , Axonema/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Cílios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Mutação/genética , Apoptose , Masculino , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sobrevivência Celular , Corpos Basais/metabolismo , Serina-Treonina Quinase 3RESUMO
Activated microglia play an important role in driving photoreceptor degeneration-associated neuroinflammation in the retina. Controlling pro-inflammatory activation of microglia holds promise for mitigating the progression of photoreceptor degeneration. Our previous study has demonstrated that pre-light damage treatment of hyperoside, a naturally occurring flavonol glycoside with antioxidant and anti-inflammatory activities, prevents photooxidative stress-induced photoreceptor degeneration and neuroinflammatory responses in the retina. However, the direct impact of hyperoside on microglia-mediated neuroinflammation during photoreceptor degeneration remains unknown. Upon verifying the anti-inflammatory effects of hyperoside in LPS-stimulated BV-2 cells, our results here further demonstrated that post-light damage hyperoside treatment mitigated the loss of photoreceptors and attenuated the functional decline of the retina. Meanwhile, post-light damage hyperoside treatment lowered neuroinflammatory responses and dampened microglial activation in the illuminated retinas. With respect to microglial activation, hyperoside mitigated the pro-inflammatory responses in DNA-stimulated BV-2 cells and lowered DNA-stimulated production of 2'3'-cGAMP in BV-2 cells. Moreover, hyperoside was shown to directly interact with cGAS and suppress the enzymatic activity of cGAS in a cell-free system. In conclusion, the current study suggests for the first time that the DNA sensor cGAS is a direct target of hyperoside. Hyperoside is effective at mitigating DNA-stimulated cGAS-mediated pro-inflammatory activation of microglia, which likely contributes to the therapeutic effects of hyperoside at curtailing neuroinflammation and alleviating neuroinflammation-instigated photoreceptor degeneration.
Assuntos
Microglia , Nucleotidiltransferases , Quercetina , Degeneração Retiniana , Animais , Camundongos , Linhagem Celular , DNA/metabolismo , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Nucleotidiltransferases/efeitos dos fármacos , Nucleotidiltransferases/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/metabolismo , Quercetina/farmacologia , Quercetina/análogos & derivados , Degeneração Retiniana/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/prevenção & controleRESUMO
Prominin 1 (PROM1) is a pentaspan transmembrane glycoprotein localized on the nascent photoreceptor discs. Mutations in PROM1 are linked to various retinal diseases. In this study, we assessed the role of PROM1 in photoreceptor biology and physiology using the PROM1 knockout murine model (rd19). Our study found that PROM1 is essential for vision and photoreceptor development. We found an early reduction in photoreceptor response beginning at post-natal day 12 (P12) before eye opening in the absence of PROM1 with no apparent loss in photoreceptor cells. However, at this stage, we observed an increased glial cell activation, indicative of cell damage. Contrary to our expectations, dark rearing did not mitigate photoreceptor degeneration or vision loss in PROM1 knockout mice. In addition to physiological defects seen in PROM1 knockout mice, ultrastructural analysis revealed malformed outer segments characterized by whorl-like continuous membranes instead of stacked disks. In parallel to the reduced rod response at P12, proteomics revealed a significant reduction in the levels of protocadherin, a known interactor of PROM1, and rod photoreceptor outer segment proteins, including rhodopsin. Overall, our results underscore the indispensable role of PROM1 in photoreceptor development and maintenance of healthy vision.
Assuntos
Antígeno AC133 , Animais , Camundongos , Antígeno AC133/metabolismo , Antígeno AC133/genética , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Rodopsina/metabolismo , Rodopsina/genéticaRESUMO
Zeaxanthin dipalmitate (ZD) is a chemical extracted from wolfberry that protects degenerated photoreceptors in mouse retina. However, the pure ZD is expensive and hard to produce. In this study, we developed a method to enrich ZD from wolfberry on a production line and examined whether it may also protect the degenerated mouse retina. The ZD-enriched wolfberry extract (ZDE) was extracted from wolfberry by organic solvent method, and the concentration of ZD was identified by HPLC. The adult C57BL/6 mice were treated with ZDE or solvent by daily gavage for 2 weeks, at the end of the first week the animals were intraperitoneally injected with N-methyl-N-nitrosourea to induce photoreceptor degeneration. Then optomotor, electroretinogram, and immunostaining were used to test the visual behavior, retinal light responses, and structure. The final ZDE product contained ~30mg/g ZD, which was over 9 times higher than that from the dry fruit of wolfberry. Feeding degenerated mice with ZDE significantly improved the survival of photoreceptors, enhanced the retinal light responses and the visual acuity. Therefore, our ZDE product successfully alleviated retinal morphological and functional degeneration in mouse retina, which may provide a basis for further animal studies for possible applying ZDE as a supplement to treat degenerated photoreceptor in the clinic.
Assuntos
Modelos Animais de Doenças , Lycium , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados , Extratos Vegetais , Degeneração Retiniana , Zeaxantinas , Animais , Lycium/química , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/patologia , Camundongos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Zeaxantinas/farmacologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Eletrorretinografia , Retina/efeitos dos fármacos , Retina/patologia , Retina/metabolismo , Visão Ocular/efeitos dos fármacos , Masculino , Xantofilas/farmacologiaRESUMO
The prolonged intravitreal administration of anti-vascular endothelial growth factor (VEGF) drugs is prone to inducing aberrant retinal vascular development and causing damage to retinal neurons. Hence, we have taken an alternative approach by designing and synthesizing a series of cyclic peptides targeting CC motif chemokine receptor 3 (CCR3). Based on the binding mode of the N-terminal region in CCR3 protein to CCL11, we used computer-aided identification of key amino acid sequence, conformational restriction through different cyclization methods, designed and synthesized a series of target cyclic peptides, and screened the preferred compound IB-2 through affinity. IB-2 exhibits excellent anti-angiogenic activity in HRECs. The apoptosis level of 661W cells demonstrated a significant decrease with the escalating concentration of IB-2. This suggests that IB-2 may have a protective effect on photoreceptor cells. In vivo experiments have shown that IB-2 significantly reduces retinal vascular leakage and choroidal neovascularization (CNV) area in a laser-induced mouse model of CNV. These findings indicate the potential of IB-2 as a safe and effective therapeutic agent for AMD, warranting further development.
Assuntos
Degeneração Macular , Peptídeos Cíclicos , Receptores CCR3 , Animais , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/síntese química , Degeneração Macular/tratamento farmacológico , Degeneração Macular/patologia , Camundongos , Receptores CCR3/antagonistas & inibidores , Receptores CCR3/metabolismo , Humanos , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Estrutura Molecular , Relação Estrutura-Atividade , Camundongos Endogâmicos C57BL , Relação Dose-Resposta a Droga , Apoptose/efeitos dos fármacos , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/patologia , Neovascularização de Coroide/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , AngiogêneseRESUMO
We explored the effect of inhibition of thioredoxin interacting protein (Txnip) on neuroprotection in Müller cells under high glucose. Wild-type (WT) and Txnip knockout (Txnip-/-) mice were used to establish a streptozotocin (STZ)-induced diabetes model and a Müller cells high glucose model. We detected BDNF expression and PI3K/AKT/CREB pathway activation levels in the retina and Müller cells of each group in vivo and in vitro experiments. The Txnip-/- STZ group showed higher expression of BDNF and phosphorylation of PI3K/AKT/CREB in retina, and less retinal photoreceptor apoptosis was observed in Txnip-/- diabetic group than in WT. After using an inhibitor of PI3K signaling pathway, BDNF expression was reduced; In vitro co-cultured with Müller cells in different groups, 661 W cells showed different situations, Txnip-/- Müller cells maximum downregulated Cleaved-caspase 3 expression in 661 W, accompanied by an increase in Bcl-2/Bax ratio. These findings indicate that inhibiting endogenous Txnip in mouse Müller cells can promote their expression and secretion of BDNF, thereby reducing HG induced photoreceptor apoptosis and having important neuroprotective effects on DR. The regulation of BDNF expression by Txnip may be achieved by activating the PI3K/AKT/CREB pathway. This study suggests that regulating Txnip may be a potential target for DR treatment.
Assuntos
Apoptose , Proteínas de Transporte , Diabetes Mellitus Experimental , Células Ependimogliais , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Células Ependimogliais/metabolismo , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/patologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos Knockout , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/patologia , Técnicas de Silenciamento de Genes , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Masculino , Retinopatia Diabética/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Modelos Animais de DoençasRESUMO
Human stem cell-derived organoids enable both disease modeling and serve as a source of cells for transplantation. Human retinal organoids are particularly important as a source of human photoreceptors; however, the long differentiation period required and lack of vascularization in the organoid often results in a necrotic core and death of inner retinal cells before photoreceptors are fully mature. Manipulating the in vitro environment of differentiating retinal organoids through the incorporation of extracellular matrix components could influence retinal development. We investigated the addition of hyaluronan (HA), a component of the interphotoreceptor matrix, as an additive to promote long-term organoid survival and enhance retinal maturation. HA treatment had a significant reduction in the proportion of proliferating (Ki67+) cells and increase in the proportion of photoreceptors (CRX+), suggesting that HA accelerated photoreceptor commitment in vitro. HA significantly upregulated genes specific to photoreceptor maturation and outer segment development. Interestingly, prolonged HA-treatment significantly decreased the length of the brush border layer compared to those in control retinal organoids, where the photoreceptor outer segments reside; however, HA-treated organoids also had more mature outer segments with organized discs structures, as revealed by transmission electron microscopy. The brush border layer length was inversely proportional to the molar mass and viscosity of the hyaluronan added. This is the first study to investigate the role of exogenous HA, viscosity, and polymer molar mass on photoreceptor maturation, emphasizing the importance of material properties on organoid culture. STATEMENT OF SIGNIFICANCE: Retinal organoids are a powerful tool to study retinal development in vitro, though like many other organoid systems, can be highly variable. In this work, Shoichet and colleagues investigated the use of hyaluronan (HA), a native component of the interphotoreceptor matrix, to improve photoreceptor maturation in developing human retinal organoids. HA promoted human photoreceptor differentiation leading to mature outer segments with disc formation and more uniform and healthy retinal organoids. These findings highlight the importance of adding components native to the developing retina to generate more physiologically relevant photoreceptors for cell therapy and in vitro models to drive drug discovery and uncover novel disease mechanisms.
Assuntos
Diferenciação Celular , Ácido Hialurônico , Organoides , Retina , Ácido Hialurônico/farmacologia , Ácido Hialurônico/química , Humanos , Organoides/efeitos dos fármacos , Organoides/citologia , Organoides/metabolismo , Diferenciação Celular/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/citologia , Retina/crescimento & desenvolvimento , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismoRESUMO
Flicker electroretinography (ERG) has served as a valuable noninvasive objective tool for investigating retinal physiological function through the measurement of electrical signals originating from retinal neurons in response to temporally modulated light stimulation. Deficits in the response at certain frequencies can be used as effective biomarkers of cone-pathway dysfunction. In this Letter, we present the progress we made on its optical counterpart-photopic flicker optoretinography (f-ORG). Specifically, we focus on the measurement of the response of light-adapted retinal photoreceptors to a flicker stimulus with chirped frequency modulation. In contrast to measurements performed at discrete frequencies, this technique enables a significantly accelerated characterization of photoreceptor outer segment optical path length modulation amplitudes in the nanometer range as a function of stimulus frequency, enabling the acquisition of the characteristic frequency response in less than 2â sec.