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1.
Biochim Biophys Acta Mol Cell Res ; 1870(1): 119384, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36302465

RESUMO

Adverse effects of spaceflight on the human body are attritubuted to microgravity and space radiation. One of the most sensitive organs affected by them is the eye, particularly the retina. The conditions that astronauts suffer, such as visual acuity, is collectively called a spaceflight-associated neuro-ocular syndrome (SANS); however, the underlying molecular mechanism of the microgravity-induced ocular pathogenesis is not clearly understood. The current study explored how microgravity affects the retina function in ARPE19 cells in vitro under time-averaged simulated microgravity (µG) generated by clinostat. We found multicellular spheroid (MCS) formation and a significantly decreased cell migration potency under µG conditions compared to 1G in ARPE19 cells. We also observed that µG increases intracellular reactive oxygen species (ROS) and causes mitochondrial dysfunction in ARPE19 cells. Subsequently, we showed that µG activates autophagic pathways and ciliogenesis. Furthermore, we demonstrated that mitophagy activation is triggered via the mTOR-ULK1-BNIP3 signaling axis. Finally, we validated the effectiveness of TPP-Niacin in mitigating µG-induced oxidative stress and mitochondrial dysfunction in vitro, which provides the first experimental evidence for TPP-Niacin as a potential therapeutic agent to ameliorate the cellular phenotypes caused by µG in ARPE19 cells. Further investigations are, however, required to determine its physiological functions and biological efficacies in primary human retinal cells, in vivo models, and target identification.


Assuntos
Niacina , Ausência de Peso , Humanos , Niacina/metabolismo , Niacina/farmacologia , Estresse Oxidativo , Células Epiteliais/metabolismo , Retina/metabolismo , Mitocôndrias/metabolismo
2.
Methods Mol Biol ; 2582: 323-334, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370360

RESUMO

Vascular stiffness is an independent predictor of human vascular diseases and is linked to ischemia, diabetes, high blood pressure, hyperlipidemia, and/or aging. Blood vessel stiffening increases owing to changes in the microscale architecture and/or content of extracellular, cytoskeletal, and nuclear matrix proteins. These alterations, while best appreciated in large blood vessels, also gradually occur in the microvasculature and play an important role in the initiation and progression of numerous microangiopathies including diabetic retinopathy. Although macroscopic measurements of arterial stiffness by pulse wave velocity are often used for clinical diagnosis, stiffness changes of intact microvessels and their causative factors have not been characterized. Herein, we describe the use of atomic force microscopy (AFM) to determine stiffness of mouse retinal capillaries and assess its regulation by the cellular communication network (CCN) 1, a stiffness-sensitive gene-encoded matricellular protein. AFM yields reproducible measurements of retinal capillary stiffness in lightly fixed freshly isolated retinal flat mounts. AFM measurements also show significant changes in compliance properties of the retinal microvasculature of mice with endothelial-specific deletion of CCN1, indicating that CCN1 expression, or lack thereof, affects the mechanical properties of microvascular cells in vivo. Thus, AFM has the force sensitivity and the spatial resolution necessary to measure the local modulus of retinal capillaries in situ and eventually to investigate microvascular compliance heterogeneities as key components of disease pathogenesis.


Assuntos
Análise de Onda de Pulso , Doenças Vasculares , Camundongos , Humanos , Animais , Microscopia de Força Atômica , Retina/metabolismo , Endotélio , Microvasos , Doenças Vasculares/metabolismo
3.
Nat Commun ; 13(1): 7037, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396639

RESUMO

Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective cytokine in multiple models of retinal degeneration. To understand mechanisms underlying its broad neuroprotective effects, we have investigated the influence of CNTF on metabolism in a mouse model of photoreceptor degeneration. CNTF treatment improves the morphology of photoreceptor mitochondria, but also leads to reduced oxygen consumption and suppressed respiratory chain activities. Molecular analyses show elevated glycolytic pathway gene transcripts and active enzymes. Metabolomics analyses detect significantly higher levels of ATP and the energy currency phosphocreatine, elevated glycolytic pathway metabolites, increased TCA cycle metabolites, lipid biosynthetic pathway intermediates, nucleotides, and amino acids. Moreover, CNTF treatment restores the key antioxidant glutathione to the wild type level. Therefore, CNTF significantly impacts the metabolic status of degenerating retinas by promoting aerobic glycolysis and augmenting anabolic activities. These findings reveal cellular mechanisms underlying enhanced neuronal viability and suggest potential therapies for treating retinal degeneration.


Assuntos
Fator Neurotrófico Ciliar , Degeneração Retiniana , Camundongos , Animais , Fator Neurotrófico Ciliar/genética , Fator Neurotrófico Ciliar/metabolismo , Degeneração Retiniana/terapia , Neuroproteção , Retina/metabolismo , Glicólise
4.
Invest Ophthalmol Vis Sci ; 63(12): 9, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36331259

RESUMO

Purpose: Cone and rod photoreceptors in the retina convert light to electrical signals which are transmitted to the visual cortex of the brain. Abnormal photoreceptor development and degeneration results in blindness. So far, the mechanism that controls photoreceptor specification and its subsequent fate bifurcation remain elusive. Methods: To trace and enrich the human photoreceptor lineage, we first engineered H9 human embryonic stem cell (hESC) reporter line by fusing EGFP to endogenous BLIMP1 using CRISPR/CAS9 gene-editing technology, and then used the cell line to generate 3D retinal organoids. Following EGFP-based cell sorting, single-cell RNA-sequencing was conducted via 10x Genomics Chromium system, and the data were analyzed using Seurat. Immunofluorescence combined with lentivirus-mediated knockdown and overexpression experiments were used as validation approaches. Results: Single-cell transcriptomic profiling revealed that retinal progenitor cells were temporally programmed to differentiate to cone and rod sequentially. We identified PHLDA1 as a novel regulator of photoreceptor specification. PHLDA1 mediated the effects of IGF1 through IGF1R, and inhibited AKT phosphorylation during photoreceptor development. Conclusions: Our data established a transcriptomic cell atlas of the human photoreceptor lineage, and identified IGF1-PHLDA1 axis to regulate human photoreceptor development.


Assuntos
Organoides , Células Fotorreceptoras Retinianas Bastonetes , Humanos , Organoides/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transcriptoma , Diferenciação Celular/fisiologia , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células-Tronco Embrionárias , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo
5.
Genet Res (Camb) ; 2022: 4112307, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36381427

RESUMO

Aim: We purposed to evaluate the KCTD10 effects of angiogenesis in diabetic retinopathy (DR). Methods: We induced a DR cell model using high glucose (HG) treatment of HRECs and ARPE-19 cells. A DR rat was established by injecting streptozotocin. Small interference RNA targeted KCTD10 (si-KCTD10) was used to mediate KCTD10 inhibition in cell and animal models. The roles of KCTD10 on cell viability, apoptosis, angiogenesis, and related proteins (VEGF and HIF-1α) were observed by RT-qPCR, Western blot, CCK-8 assay, TUNEL staining, tube formation assay, ELISA, and immunohistochemistry assay. Results: KCTD10 expression was upregulated in DR cells and retinal tissue of DR rats. Treatment of the cells with si-KCTD10 increased cell viability and decreased apoptosis and angiogenesis in DR cells. Inhibition of KCTD10 could reduce the expression of VEGF and HIF-1α in DR cells. Furthermore, KCTD10 inhibition reduced VEGF levels in the retinal tissue of DR rats. Conclusion: This work showed that inhibition of KCTD10 relieved angiogenesis in DR.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Ratos , Animais , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Retina/metabolismo , Diabetes Mellitus/metabolismo
6.
Cell Rep ; 41(7): 111671, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36384115

RESUMO

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in the elderly population with unclear pathogenic mechanism. Herein, we detect downregulated circSPECC1 expression in retinal pigment epithelium (RPE) of AMD patients. In RPE cells, circSPECC1 insufficiency leads to oxidative stress-induced ferroptosis, depolarization, and irregular lipid metabolism. Consistently, in mice, circSPECC1 deficiency induces visual impairments and RPE anomalies and interrupts retinal homeostasis. Mechanically, nuclear export of circSPECC1 transcript depends on its N6-methyladenosine (m6A) level with YTHDC1 as the reader. CircSPECC1 directly sponges miR-145-5p to block its interaction with CDKN1A. Overexpressing miR-145-5p aggravates RPE dysfunctions, mimicking circSPECC1 silencing effects. Retinal phenotypes induced by circSPECC1 insufficiency are alleviated by miR-145-5p inhibition and are aggravated by miR-145-5p overexpression. Collectively, circSPECC1, mediated by m6A modification and sponging miR-145-5p, resists oxidative stress injuries and maintains lipid metabolism in RPE. Pharmacological supplementation of circSPECC1 is a promising therapeutic option for atrophic retinopathies like AMD.


Assuntos
Degeneração Macular , MicroRNAs , Estresse Oxidativo , RNA Circular , Idoso , Animais , Humanos , Camundongos , Homeostase , Degeneração Macular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Estresse Oxidativo/genética , Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , RNA Circular/genética
7.
Cells ; 11(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36359808

RESUMO

Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.


Assuntos
Organoides , Células-Tronco Pluripotentes , Humanos , Organoides/metabolismo , Cromatina/metabolismo , Retina/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular/genética
8.
Sci Rep ; 12(1): 19584, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36379991

RESUMO

Mammalian Müller glia express transcription factors and cell cycle regulators essential for the function of retinal progenitors, indicating the latent neurogenic capacity; however, the role of these regulators remains unclear. To gain insights into the role of these regulators in Müller glia, we analyzed expression of transcription factors (Pax6, Vsx2 and Nfia) and cell cycle regulators (cyclin D1 and D3) in rodent Müller glia, focusing on their age- and cell cycle-related expression patterns. Expression of Pax6, Vsx2, Nfia and cyclin D3, but not cyclin D1, increased in Müller glia during development. Photoreceptor injury induced cell cycle-associated increase of Vsx2 and cyclin D1, but not Pax6, Nfia, and cyclin D3. In dissociated cultures, cell cycle-associated increase of Pax6 and Vsx2 was observed in Müller glia from P10 mice but not from P21 mice. Nfia levels were highly correlated with EdU incorporation suggesting their activation during S phase progression. Cyclin D1 and D3 were transiently upregulated in G1 phase but downregulated after S phase entry. Our findings revealed previously unknown links between cell cycle progression and regulator protein expression, which likely affect the cell fate decision of proliferating Müller glia.


Assuntos
Neuroglia , Fatores de Transcrição , Camundongos , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ciclina D3/metabolismo , Proliferação de Células , Neuroglia/metabolismo , Ciclo Celular/fisiologia , Retina/metabolismo , Mamíferos/metabolismo , Proteínas de Homeodomínio/metabolismo
9.
BMC Neurosci ; 23(1): 66, 2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36384553

RESUMO

AIMS: Esketamine upregulates Zn2+-dependent matrix metalloproteinase 9 (MMP9) and increases the neuronal apoptosis in retinal ganglion cell layer during the early development. We aimed to test whether albumin can alleviate esketamine-induced apoptosis through downregulating Zn2+-dependent MMP9. METHODS: We investigate the role of Zn2+ in esketamine-induced neuronal apoptosis by immunofluorescence. MMP9 protein expression and enzyme activity were investigated by zymography in situ., western blot and immunofluorescence. Whole-mount retinas from P7 Sprague-Dawley rats were used. RESULTS: We demonstrated that esketamine exposure increased Zn2+ in the retinal GCL during the early development. Zn2+-dependent MMP9 expression and enzyme activity up-regulated, which eventually aggravated apoptosis. Albumin effectively down-regulated MMP9 expression and activity via binding of free zinc, ultimately protected neurons from apoptosis. Meanwhile albumin treatment promoted activated microglia into multi-nucleated macrophagocytes and decreased the inflammation. CONCLUSION: Albumin alleviates esketamine-induced neuronal apoptosis through decreasing Zn2+ accumulation in GCL and downregulating Zn2+-dependent MMP9.


Assuntos
Metaloproteinase 9 da Matriz , Retina , Ratos , Animais , Metaloproteinase 9 da Matriz/metabolismo , Regulação para Baixo , Ratos Sprague-Dawley , Retina/metabolismo , Apoptose , Albuminas/metabolismo , Albuminas/farmacologia , Zinco/farmacologia
10.
Genes (Basel) ; 13(11)2022 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-36360271

RESUMO

BACKGROUND: The presence of hyperreflective foci (HRF) in retinitis pigmentosa (RP) is a potentially new finding. We investigated the presence of HRF in SD-OCT images in eyes with RP and its relation to vascular, morphologic and metabolic findings in RP. METHODS: The study was performed on 42 RP patients and 24 controls. Using SD-OCT, we calculated the amount of HRF within the entire retina (HRF-ER) and the outer nuclear layer (HRF-ONL). Retinal vessel diameters (µm) and oxygen saturation (%) values were measured using Oxymap T1. We evaluated the mean diameter in retinal arterioles (D-A) and venules (D-V), the corresponding oxygen saturation values (A-SO2, V-SO2) and the oxygen saturation difference (A-V SO2). RESULTS: RP differed from controls by HRF-ER, HRF-ON and EZ-length (p < 0.001). D-A and D-V were narrower and A-SO2 and V-SO2 were higher in RP (p ≤ 0.001). Within RP, significant interactions were found between the HRF-ER* group and: BCVA, EZ length, D-A, A-SO2 and A-V SO2 (p ≤ 0.018). The HRF-ONL* group interactions were significant for: BCVA, EZ length, D-A, A-SO2 and A-V SO2 (p ≤ 0.014). CONCLUSION: The present study highlights the presence of HRF to reflect the vascular, morphologic and metabolic alterations in RP. These biomarkers seem to be associated with remodeling and apoptosis that occur with the progression of degeneration.


Assuntos
Oximetria , Retinite Pigmentosa , Humanos , Oximetria/métodos , Oxigênio/metabolismo , Retinite Pigmentosa/metabolismo , Vasos Retinianos/diagnóstico por imagem , Vasos Retinianos/metabolismo , Retina/metabolismo
11.
Genes (Basel) ; 13(11)2022 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-36360284

RESUMO

Chickens are excellent models for the study of retinal development and function. Gene expression at the correct time is crucial to retinal development and function. The present study aimed to investigate retinal gene expression and morphology in locally grown chickens at various developmental stages. RNA was extracted from the retina at the embryonic and post-hatch stages, and the retinal layers were stained with haematoxylin and eosin (H&E). RT-PCR and RT-qPCR were used for gene expression analysis of 14 selected genes. The results showed that all the retinal genes were expressed at different developmental stages. However, there were slight noticeable variations in expression patterns. At the morphological level, all retinal layers were well observed, except for the outer plexiform layer that became visible in the fifteen-day chick embryo. The current study provides a baseline for standard retinal gene expression of 14 genes and retinal histological staining. The selected genes have different roles in retinal development and function, and most of these genes are associated with retinal diseases. The results obtained here can be applied to molecular retinal research and retinal diseases with genetic factors in retina animal models or human diseases.


Assuntos
Galinhas , Doenças Retinianas , Humanos , Embrião de Galinha , Animais , Galinhas/genética , Regulação da Expressão Gênica no Desenvolvimento , Retina/metabolismo , Doenças Retinianas/metabolismo , Expressão Gênica
12.
Invest Ophthalmol Vis Sci ; 63(12): 7, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36326725

RESUMO

Purpose: Progressive retinal ganglion cell (RGC) loss induced by retinal ischemia/reperfusion (RIR) injury leads to irreversible visual impairment. Pregabalin (PGB) is a promising drug for neurodegenerative diseases. However, with regard to RGC survival, its specific role and exact mechanism after RIR injury remain unclear. In this study, we sought to investigate whether PGB could protect RGCs from mitochondria-related apoptosis induced by RIR and explore the possible mechanisms. Methods: C57BL/6J mice and primary RGCs were pretreated with PGB prior to ischemia/reperfusion modeling. The retinal structure and cell morphology were assessed by immunochemical assays and optical coherence tomography. CCK8 was used to assay cell viability, and an electroretinogram was performed to detect RGC function. Mitochondrial damage was assessed by a reactive oxygen species (ROS) assay kit and transmission electron microscopy. Western blot and immunofluorescence assays quantified the expression of proteins associated with the Akt/GSK3ß/ß-catenin pathway. Results: Treatment with PGB increased the viability of RGCs in vitro. Consistently, PGB preserved the normal thickness of the retina, upregulated Bcl-2, reduced the ratio of cleaved caspase-3/caspase-3 and the expression of Bax in vivo. Meanwhile, PGB improved mitochondrial structure and prevented excessive ROS production. Moreover, PGB restored the amplitudes of oscillatory potentials and photopic negative responses following RIR. The mechanisms underlying its neuroprotective effects were attributed to upregulation of the Akt/GSK3ß/ß-catenin pathway. However, PGB-mediated neuroprotection was suppressed when using MK2206 (an Akt inhibitor), whereas it was preserved when treated with TWS119 (a GSK3ß inhibitor). Conclusions: PGB exerts a protective effect against RGC apoptosis induced by RIR injury, mediated by the Akt/GSK3ß/ß-catenin pathway.


Assuntos
Traumatismo por Reperfusão , Células Ganglionares da Retina , Animais , Camundongos , Apoptose , beta Catenina/metabolismo , Caspase 3/metabolismo , Sobrevivência Celular , Glicogênio Sintase Quinase 3 beta/metabolismo , Isquemia/metabolismo , Camundongos Endogâmicos C57BL , Pregabalina/farmacologia , Pregabalina/uso terapêutico , Pregabalina/metabolismo , Prostaglandinas B , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução de Sinais
13.
Invest Ophthalmol Vis Sci ; 63(12): 5, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36326727

RESUMO

Purpose: Uveal coloboma is a congenital eye malformation caused by failure of the optic fissure to close in early human development. Despite significant progress in identifying genes whose regulation is important for executing this closure, mutations are detected in a minority of cases using known gene panels, implying additional genetic complexity. We have previously shown knockdown of znf503 (the ortholog of mouse Zfp503) in zebrafish causes coloboma. Here we characterize Zfp503 knockout (KO) mice and evaluate transcriptomic profiling of mutant versus wild-type (WT) retinal pigment epithelium (RPE)/choroid. Methods: Zfp503 KO mice were generated by gene targeting using homologous recombination. Embryos were characterized grossly and histologically. Patterns and level of developmentally relevant proteins/genes were examined with immunostaining/in situ hybridization. The transcriptomic profile of E11.5 KO RPE/choroid was compared to that of WT. Results: Zfp503 is dynamically expressed in developing mouse eyes, and loss of its expression results in uveal coloboma. KO embryos exhibit altered mRNA levels and expression patterns of several key transcription factors involved in eye development, including Otx2, Mitf, Pax6, Pax2, Vax1, and Vax2, resulting in a failure to maintain the presumptive RPE, as evidenced by reduced melanin pigmentation and its differentiation into a neural retina-like lineage. Comparison of RNA sequencing data from WT and KO E11.5 embryos demonstrated reduced expression of melanin-related genes and significant overlap with genes known to be dynamically regulated at the optic fissure. Conclusions: These results demonstrate a critical role of Zfp503 in maintaining RPE fate and optic fissure closure.


Assuntos
Coloboma , Neuropeptídeos , Humanos , Camundongos , Animais , Epitélio Pigmentado da Retina/metabolismo , Coloboma/genética , Coloboma/metabolismo , Peixe-Zebra/genética , Melaninas/metabolismo , Retina/metabolismo , Camundongos Knockout , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Neuropeptídeos/genética , Proteínas do Tecido Nervoso/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo
14.
ASN Neuro ; 14: 17590914221136365, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36317314

RESUMO

Müller glial cells (MGCs), the main glial component of the retina, play an active role in retinal homeostasis during development and pathological processes. They strongly monitor retinal environment and, in response to retinal imbalance, activate neuroprotective mechanisms mainly characterized by the increase of glial fibrillary acidic protein (GFAP). Under these circumstances, if homeostasis is not reestablished, the retina can be severely injured and GFAP contributes to neuronal degeneration, as they occur in several proliferative retinopathies such as diabetic retinopathy, sickle cell retinopathy and retinopathy of prematurity. In addition, MGCs have an active participation in inflammatory responses releasing proinflammatory mediators and metalloproteinases to the extracellular space and vitreous cavity. MGCs are also involved in the retinal neovascularization and matrix extracellular remodeling during the proliferative stage of retinopathies. Interestingly, low-density lipoprotein receptor-related protein 1 (LRP1) and its ligand α2-macroglobulin (α2M) are highly expressed in MGCs and they have been established to participate in multiple cellular and molecular activities with relevance in retinopathies. However, the exact mechanism of regulation of retinal LRP1 in MGCs is still unclear. Thus, the active participation of MGCs and LRP1 in these diseases, strongly supports the potential interest of them for the design of novel therapeutic approaches. In this review, we discuss the role of LRP1 in the multiple MGCs activities involved in the development and progression of proliferative retinopathies, identifying opportunities in the field that beg further research in this topic area.Summary StatementMGCs and LRP1 are active players in injured retinas, participating in key features such as gliosis and neurotoxicity, neovascularization, inflammation, and glucose control homeostasis during the progression of ischemic diseases, such as proliferative retinopathies.


Assuntos
Células Ependimogliais , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Neovascularização Retiniana , Humanos , Células Ependimogliais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Retina/metabolismo , Retina/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia
15.
Sci Rep ; 12(1): 20195, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36424523

RESUMO

Understanding the physiology of the retina, and especially of the highly polarized photoreceptors, is essential not only to broaden our knowledge of the processes required for normal vision, but also to develop effective therapies to prevent or slow retinal degenerative diseases. However, the molecular analysis of photoreceptors is a challenge due to the heterogeneity of the retinal tissue and the lack of easy and reliable methods for cell separation. Here we present the ReLayS method-a simple technique for the separation of photoreceptor segments (PS) containing both inner and outer segments, outer nuclear layer (ONL), and inner retina (InR) that contains the remaining retinal layers. The layer-specific material isolated from a mouse half-retina with the ReLayS method was sufficient for protein isolation and Western blotting or RNA isolation and real-time PCR studies. The separation of PS, ONL, and InR was successfully validated by Western blotting and real-time PCR using proteins and genes with known expression profiles within the retina. Furthermore, the separation of the PS from the ONL enabled the detection of light-driven translocation of transducin from the PS to the soma. ReLayS is a simple and useful method to address protein and possibly metabolites distribution in photoreceptor compartments in various situations including development, ageing, and degenerative diseases.


Assuntos
Corpo Celular , Retina , Camundongos , Animais , Corpo Celular/metabolismo , Retina/metabolismo , Células Fotorreceptoras/metabolismo , Transducina/metabolismo
16.
Nat Commun ; 13(1): 5827, 2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207300

RESUMO

Chromatin organization and enhancer-promoter contacts establish unique spatiotemporal gene expression patterns in distinct cell types. Non-coding genetic variants can influence cellular phenotypes by modifying higher-order transcriptional hubs and consequently gene expression. To elucidate genomic regulation in human retina, we mapped chromatin contacts at high resolution and integrated with super-enhancers (SEs), histone marks, binding of CTCF and select transcription factors. We show that topologically associated domains (TADs) with central SEs exhibit stronger insulation and augmented contact with retinal genes relative to TADs with edge SEs. Merging genome-wide expression quantitative trait loci (eQTLs) with topology map reveals physical links between 100 eQTLs and corresponding eGenes associated with retinal neurodegeneration. Additionally, we uncover candidate genes for susceptibility variants linked to age-related macular degeneration and glaucoma. Our study of high-resolution genomic architecture of human retina provides insights into genetic control of tissue-specific functions, suggests paradigms for missing heritability, and enables the dissection of common blinding disease phenotypes.


Assuntos
Cromatina , Locos de Características Quantitativas , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Humanos , Regiões Promotoras Genéticas , Locos de Características Quantitativas/genética , Retina/metabolismo , Fatores de Transcrição/metabolismo
17.
Acta Neuropathol Commun ; 10(1): 145, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36199154

RESUMO

Amyloid beta (Aß) deposits in the retina of the Alzheimer's disease (AD) eye may provide a useful diagnostic biomarker for AD. This study focused on the relationship of Aß with macroglia and microglia, as these glial cells are hypothesized to play important roles in homeostasis and clearance of Aß in the AD retina. Significantly higher Aß load was found in AD compared to controls, and specifically in the mid-peripheral region. AD retina showed significantly less immunoreactivity against glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) compared to control eyes. Immunoreactivity against ionized calcium binding adapter molecule-1 (IBA-1), a microglial marker, demonstrated a higher level of microgliosis in AD compared to control retina. Within AD retina, more IBA-1 immunoreactivity was present in the mid-peripheral retina, which contained more Aß than the central AD retina. GFAP co-localized rarely with Aß, while IBA-1 co-localized with Aß in more layers of control than AD donor retina. These results suggest that dysfunction of the Müller and microglial cells may be key features of the AD retina.


Assuntos
Doença de Alzheimer , Microglia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Células Ependimogliais , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Amônia Ligase/metabolismo , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Retina/metabolismo
18.
Cells ; 11(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36231018

RESUMO

The avian magnetic compass allows orientation during migration and is shown to function properly under short-wavelength but not long-wavelength visible light. Therefore, the magnetoreceptive system is assumed to be light- and wavelength-dependent and localized in the retina of the eye. Putative candidates for the role of primary magnetosensory molecules are the cryptochromes that are known to be expressed in the avian retina and must be able to interact with phototransduction proteins. Previously, we reported that in migratory birds change in magnetic field direction induces significant effects on electroretinogram amplitude in response to blue flashes, and such an effect was observed only in the nasal quadrant of the retina. Here, we report new electroretinographic, microscopic and microspectrophotometric data on European robins, confirming the magnetosensitivity of the retinal nasal quadrant after applying the background illumination. We hypothesized that magnetoreceptive distinction of this region may be related to its morphology and analyzed the retinal distribution and optical properties of oil droplets, the filtering structures within cones. We found that the nasal quadrant contains double cones with the most intensely colorized oil droplets compared to the rest of the retina, which may be related to its magnetosensory function.


Assuntos
Migração Animal , Criptocromos , Migração Animal/fisiologia , Animais , Aves , Criptocromos/metabolismo , Campos Magnéticos , Retina/metabolismo
19.
Int J Mol Sci ; 23(19)2022 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-36232896

RESUMO

Mutations in the Ceramide Kinase-like (CERKL) gene cause retinal dystrophies, characterized by progressive degeneration of retinal neurons, which eventually lead to vision loss. Among other functions, CERKL is involved in the regulation of autophagy, mitochondrial dynamics, and metabolism in the retina. However, CERKL is nearly ubiquitously expressed, and it has been recently described to play a protective role against brain injury. Here we show that Cerkl is expressed in the hippocampus, and we use mouse hippocampal neurons to explore the impact of either overexpression or depletion of CERKL on mitochondrial trafficking and dynamics along axons. We describe that a pool of CERKL localizes at mitochondria in hippocampal axons. Importantly, the depletion of CERKL in the CerklKD/KO mouse model is associated with changes in the expression of fusion/fission molecular regulators, induces mitochondrial fragmentation, and impairs axonal mitochondrial trafficking. Our findings highlight the role of CERKL, a retinal dystrophy gene, in the regulation of mitochondrial health and homeostasis in central nervous system anatomic structures other than the retina.


Assuntos
Neurônios , Fosfotransferases (Aceptor do Grupo Álcool) , Retina , Distrofias Retinianas , Animais , Hipocampo/citologia , Camundongos , Dinâmica Mitocondrial , Neurônios/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Retina/metabolismo
20.
Int J Mol Sci ; 23(19)2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36233006

RESUMO

Age-related macular degeneration (AMD) is an irreversible chronic degenerative pathology that affects the retina. Despite therapeutic advances thanks to the use of anti-vascular endothelial growth factor (VEGF) agents, resistance mechanisms have been found to accentuate the visual deficit. In the present study, we explored whether a nutraceutical formulation composed of omega-3 fatty acids and resveratrol, called Resvega®, was able to disrupt VEGF-A secretion in human ARPE-19 retina cells. We found that Resvega® inhibits VEGF-A secretion through decreases in both the PI3K-AKT-mTOR and NFκB signaling pathways. In NFκB signaling pathways, Resvega® inhibits the phosphorylation of the inhibitor of NFκB, IκB, which can bind NFκB dimers and sequester them in the cytoplasm. Thus, the NFκB subunits cannot migrate to the nucleus where they normally bind and stimulate the transcription of target genes such as VEGF-A. The IκB kinase complex (IKK) is also affected by Resvega® since the nutraceutical formulation decreases both IKKα and IKKß subunits and the IKKγ subunit which is required for the stimulation of IKK. Very interestingly, we highlight that Resvega® could prolong the anti-angiogenic effect of Avastin®, which is an anti-VEGF agent typically used in clinical practice. Our results suggest that Resvega® may have potential interest as nutritional supplementation against AMD.


Assuntos
Ácidos Graxos Ômega-3 , Degeneração Macular , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Suplementos Nutricionais , Fatores de Crescimento Endotelial , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-3/uso terapêutico , Humanos , Quinase I-kappa B , Degeneração Macular/tratamento farmacológico , NF-kappa B , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Resveratrol/uso terapêutico , Retina/metabolismo , Serina-Treonina Quinases TOR , Fator A de Crescimento do Endotélio Vascular/metabolismo
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