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1.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34556573

RESUMO

Many sensory systems utilize parallel ON and OFF pathways that signal stimulus increments and decrements, respectively. These pathways consist of ensembles or grids of ON and OFF detectors spanning sensory space. Yet, encoding by opponent pathways raises a question: How should grids of ON and OFF detectors be arranged to optimally encode natural stimuli? We investigated this question using a model of the retina guided by efficient coding theory. Specifically, we optimized spatial receptive fields and contrast response functions to encode natural images given noise and constrained firing rates. We find that the optimal arrangement of ON and OFF receptive fields exhibits a transition between aligned and antialigned grids. The preferred phase depends on detector noise and the statistical structure of the natural stimuli. These results reveal that noise and stimulus statistics produce qualitative shifts in neural coding strategies and provide theoretical predictions for the configuration of opponent pathways in the nervous system.


Assuntos
Modelos Neurológicos , Ruído , Retina/fisiologia , Campos Visuais/fisiologia , Vias Visuais/fisiologia , Animais , Humanos , Estimulação Luminosa , Retina/citologia , Razão Sinal-Ruído , Percepção Visual
2.
Nat Commun ; 12(1): 5261, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489404

RESUMO

The advent of single-cell RNA sequencing (scRNA-seq) technologies has revolutionized transcriptomic studies. However, large-scale integrative analysis of scRNA-seq data remains a challenge largely due to unwanted batch effects and the limited transferabilty, interpretability, and scalability of the existing computational methods. We present single-cell Embedded Topic Model (scETM). Our key contribution is the utilization of a transferable neural-network-based encoder while having an interpretable linear decoder via a matrix tri-factorization. In particular, scETM simultaneously learns an encoder network to infer cell type mixture and a set of highly interpretable gene embeddings, topic embeddings, and batch-effect linear intercepts from multiple scRNA-seq datasets. scETM is scalable to over 106 cells and confers remarkable cross-tissue and cross-species zero-shot transfer-learning performance. Using gene set enrichment analysis, we find that scETM-learned topics are enriched in biologically meaningful and disease-related pathways. Lastly, scETM enables the incorporation of known gene sets into the gene embeddings, thereby directly learning the associations between pathways and topics via the topic embeddings.


Assuntos
Bases de Dados Genéticas , Modelos Genéticos , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/métodos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/patologia , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/estatística & dados numéricos , Genes Mitocondriais , Humanos , Camundongos , Redes Neurais de Computação , RNA Citoplasmático Pequeno , Retina/citologia , Retina/fisiologia , Análise de Sequência de RNA/métodos
3.
Int J Mol Sci ; 22(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34445064

RESUMO

Photoreceptors are critical components of the retina and play a role in the first step of the conversion of light to electric signals. With the discovery of the intrinsically photosensitive retinal ganglion cells, which regulate non-image-forming visual processes, our knowledge of the photosensitive cell family in the retina has deepened. Photoreceptor development is regulated by specific genes and proteins and involves a series of molecular processes including DNA transcription, post-transcriptional modification, protein translation, and post-translational modification. Single-cell sequencing is a promising technology for the study of photoreceptor development. This review presents an overview of the types of human photoreceptors, summarizes recent discoveries in the regulatory mechanisms underlying their development at single-cell resolution, and outlines the prospects in this field.


Assuntos
Células Fotorreceptoras de Vertebrados/citologia , Retina/crescimento & desenvolvimento , Análise de Célula Única/métodos , Animais , Humanos , Organoides/citologia , Organoides/embriologia , Organoides/crescimento & desenvolvimento , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/citologia , Retina/embriologia
4.
FASEB J ; 35(9): e21842, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34418159

RESUMO

Retinopathy of prematurity (ROP) remains one of the major causes of blindness in children worldwide. While current ROP treatments are mostly disruptive to reduce proliferative neovascularization by targeting the hypoxic phase, protection against early hyperoxia-induced retinal vascular loss represents an effective therapeutic window, but no such therapeutic strategy is available. Built upon our recent demonstration that the protection against oxygen-induced retinopathy by adenosine A2A receptor (A2A R) antagonists is most effective when administered at the hyperoxia (not hypoxic) phase, we here uncovered the cellular mechanism underlying the A2A R-mediated protection against early hyperoxia-induced retinal vascular loss by reversing the inhibition of cellular proliferation via possibly multiple signaling pathways. Specifically, we revealed two distinct stages of the hyperoxia phase with greater cellular proliferation and apoptosis activities and upregulation of adenosine signaling at postnatal 9 day (P9) but reduced cellular activities and adenosine-A2A R signaling at P12. Importantly, the A2A R-mediated protection at P9 was associated with the reversal of hyperoxia-induced inhibition of progenitor cells at the peripheral retina at P9 and of retinal endothelial proliferation at P9 and P12. The critical role of cellular proliferation in the hyperoxia-induced retinal vascular loss was validated by the increased avascular areas by siRNA knockdown of the multiple signaling molecules involved in modulation of cellular proliferation, including activin receptor-like kinase 1, DNA-binding protein inhibitor 1, and vascular endothelial growth factor-A.


Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Proliferação de Células/efeitos dos fármacos , Hiperóxia/metabolismo , Substâncias Protetoras/farmacologia , Receptor A2A de Adenosina/metabolismo , Neovascularização Retiniana , Vasos Retinianos/efeitos dos fármacos , Receptores de Activinas Tipo II/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína 1 Inibidora de Diferenciação/metabolismo , Camundongos , Neovascularização Patológica , Oxigênio/efeitos adversos , Retina/citologia , Retina/efeitos dos fármacos , Retina/patologia , Vasos Retinianos/citologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Biol Res ; 54(1): 25, 2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362460

RESUMO

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARα) is associated with diabetic retinopathy (DR), and the underlying mechanism is still unclear. Aim of this work was to investigate the mechanism of PPARα in DR. METHODS: Human retinal capillary pericytes (HRCPs) were treated with high glucose (HG) to induce DR cell model. DR mouse model was established by streptozotocin injection, and then received 5-Aza-2-deoxycytidine (DAC; DNA methyltransferase inhibitor) treatment. Hematoxylin-eosin staining was performed to assess retinal tissue damage. PPARα methylation was examined by Methylation-Specific PCR. Flow cytometry and DCFH-DA fluorescent probe was used to estimate apoptosis and reactive oxygen species (ROS). The interaction between DNA methyltransferase-1 (DNMT1) and PPARα promoter was examined by Chromatin Immunoprecipitation. Quantitative real-time PCR and western blot were performed to assess gene and protein expression. RESULTS: HG treatment enhanced the methylation levels of PPARα, and repressed PPARα expression in HRCPs. The levels of apoptotic cells and ROS were significantly increased in HRCPs in the presence of HG. Moreover, DNMT1 was highly expressed in HG-treated HRCPs, and DNMT1 interacted with PPARα promoter. PPARα overexpression suppressed apoptosis and ROS levels of HRCPs, which was rescued by DNMT1 up-regulation. In DR mice, DAC treatment inhibited PPARα methylation and reduced damage of retinal tissues. CONCLUSION: DNMT1-mediated PPARα methylation promotes apoptosis and ROS levels of HRCPs and aggravates damage of retinal tissues in DR mice. Thus, this study may highlight novel insights into DR pathogenesis.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Retinopatia Diabética , PPAR alfa/genética , Retina/patologia , Animais , Apoptose , Células Cultivadas , Metilação de DNA , Diabetes Mellitus , Modelos Animais de Doenças , Humanos , Metilação , Camundongos , Regiões Promotoras Genéticas , Retina/citologia
6.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445152

RESUMO

The cytoprotective versus cytotoxic role of macroautophagy in ocular ischemia/reperfusion injuries remains controversial and its effects under hyperglycemia are unclear. We investigated the involvement of autophagy in in vitro and in vivo normoglycemic and hyperglycemic models of retinal ischemia/reperfusion injury. Retinal ischemia (2 h) and reperfusion (2 or 22 h) was induced in wild-type and type I diabetic Ins2Akita/+ mice using a middle cerebral artery occlusion model. R28 retinal precursor cells were subjected to CoCl2-induced hypoxia with or without autophagic inhibitor NH4Cl. Autophagic regulation during ischemia/reperfusion was assessed through immunohistochemical detection and Western blotting of microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal associated membrane protein 1 (LAMP1). Effect of autophagic inhibition on cell viability and morphology under hypoxic conditions was also evaluated. Upregulation of autophagic markers in the inner retinae was seen after two hours reperfusion, with tapering of the response following 22 h of reperfusion in vivo. LC3-II turnover assays confirmed an increase in autophagic flux in our hypoxic in vitro model. Pharmacological autophagic inhibition under hypoxic conditions decreased cell survival and induced structural changes not demonstrated with autophagic inhibition alone. Yet no statistically significant different autophagic responses in ischemia/reperfusion injuries were seen between the two glycemic states.


Assuntos
Autofagia , Traumatismo por Reperfusão/patologia , Retina/patologia , Células-Tronco/patologia , Animais , Sobrevivência Celular , Feminino , Masculino , Camundongos Endogâmicos C57BL , Retina/citologia , Células-Tronco/citologia
7.
Artigo em Inglês | MEDLINE | ID: mdl-34348917

RESUMO

INTRODUCTION: This study aims to determine whether high glucose condition and dynamic O-linked N-acetylglucosamine (O-GlcNAc) modification can promote the proliferation and migration of human retinal microvascular endothelial cells (HRMECs) and whether Runt-related transcription factor 1 (RUNX1) could mediate the glucose and O-GlcNAc-driven proliferation and migration of HRMECs. RESEARCH DESIGN AND METHODS: Western blot analysis was used to detect the O-GlcNAc modification level and RUNX1 level in cells and retina tissues, cell growth was studied by cell counting kit-8 assay, cell proliferation was detected by immunofluorescence staining. Then, cell migration and tube formation were investigated by scratch-wound assay, Transwell assay, and tube-forming assay. The changes of retinal structure were detected by H&E staining. The O-GlcNAc modification of RUNX1 was detected by immunoprecipitation. RESULTS: High glucose increases pan-cellular O-GlcNAc modification and the proliferation and migration of HRMECs. Hence, O-GlcNAc modification is critical for the proliferation and migration of HRMECs. RUNX1 mediates the glucose and O-GlcNAc-driven proliferation and migration in HRMECs. RUNX1 can be modified by O-GlcNAc, and that the modification is enhanced in a high glucose environment. CONCLUSIONS: The present study reveals that high glucose condition directly affects retinal endothelial cells (EC) function, and O-GlcNAc modification is critical for the proliferation and migration of HRMECs, RUNX1 may take part in this mechanism, and maybe the function of RUNX1 is related to its O-GlcNAc modification level, which provides a new perspective for studying the mechanism of RUNX1 in diabetic retinopathy.


Assuntos
Acetilglucosamina , Subunidade alfa 2 de Fator de Ligação ao Core , Células Endoteliais/citologia , Movimento Celular , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Glucose , Humanos , Retina/citologia
8.
Biomed Res Int ; 2021: 4982227, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34285913

RESUMO

PDGFRα signaling is critically important in ocular development. Previous data on PDGFRα lacks an expression map with high spatial and temporal resolution and lineage information. In this study, we aim to present a detailed PDGFRα expression and lineage map from early embryogenesis to adulthood. PDGFRα-CreER; mT/mG reporter mice were analyzed. mEGFP-positive cells contributed to multiple ocular lineages in a spatiotemporally regulated manner. A dynamic PDGFRα expression was identified in corneal stromal cells, lens epithelial cells, lens fiber cells, and retinal astrocytes during the entire period of eye development, while PDGFRα expression in retinal astrocytes from E17.5 onwards and in Müller glial cells was identified within two weeks after birth. By revealing detailed characterization of gene expression and function, we present a comprehensive map of PDGFRα-expressing cells in the eye for a better understanding of PDGFRα signaling's role during eye development.


Assuntos
Linhagem da Célula , Olho/citologia , Olho/embriologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Animais Recém-Nascidos , Linhagem da Célula/genética , Córnea/citologia , Córnea/embriologia , Embrião de Mamíferos/citologia , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Retina/citologia
9.
Lab Invest ; 101(9): 1289-1303, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34253851

RESUMO

Retinal ganglion cell (RGC) death occurs after optic nerve injury due to acute trauma or chronic degenerative conditions such as optic neuropathies (e.g., glaucoma). Currently, there are no effective therapies to prevent permanent vision loss resulting from RGC death, underlining the need for research on the pathogenesis of RGC disorders. Modeling human RGC/optic nerve diseases in non-human primates is ideal because of their similarity to humans, but has practical limitations including high cost and ethical considerations. In addition, many retinal degenerative disorders are age-related making the study in primate models prohibitively slow. For these reasons, mice and rats are commonly used to model RGC injuries. However, as nocturnal mammals, these rodents have retinal structures that differ from primates - possessing less than one-tenth of the RGCs found in the primate retina. Here we report the diurnal thirteen-lined ground squirrel (TLGS) as an alternative model. Compared to other rodent models, the number and distribution of RGCs in the TLGS retina are closer to primates. The TLGS retina possesses ~600,000 RGCs with the highest density along the equatorial retina matching the location of the highest cone density (visual streak). TLGS and primate retinas also share a similar interlocking pattern between RGC axons and astrocyte processes in the retina nerve fiber layer (RNFL). In addition, using TLGS we establish a new partial optic nerve injury model that precisely controls the extent of injury while sparing a portion of the retina as an ideal internal control for investigating the pathophysiology of axon degeneration and RGC death. Moreover, in vivo optical coherence tomography (OCT) imaging and ex vivo microscopic examinations of the retina in optic nerve injured TLGS confirm RGC loss precedes proximal axon degeneration, recapitulating human pathology. Thus, the TLGS retina is an excellent model, for translational research in neurodegeneration and therapeutic neuroprotection.


Assuntos
Modelos Animais de Doenças , Doenças do Nervo Óptico , Doenças Retinianas , Células Ganglionares da Retina , Sciuridae/fisiologia , Animais , Feminino , Macaca mulatta , Camundongos , Ratos , Retina/citologia , Retina/patologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/fisiologia
10.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207050

RESUMO

The vertebrate retina develops from a specified group of precursor cells that adopt distinct identities and generate lineages of either the neural retina, retinal pigmented epithelium, or ciliary body. In some species, including teleost fish and amphibians, proliferative cells with stem-cell-like properties capable of continuously supplying new retinal cells post-embryonically have been characterized and extensively studied. This region, termed the ciliary or circumferential marginal zone (CMZ), possibly represents a conserved retinal stem cell niche. In this review, we highlight the research characterizing similar CMZ-like regions, or stem-like cells located at the peripheral margin, across multiple different species. We discuss the proliferative parameters, multipotency and growth mechanisms of these cells to understand how they behave in vivo and how different molecular factors and signalling networks converge at the CMZ niche to regulate their activity. The evidence suggests that the mature retina may have a conserved propensity for homeostatic growth and plasticity and that dysfunction in the regulation of CMZ activity may partially account for dystrophic eye growth diseases such as myopia and hyperopia. A better understanding of the properties of CMZ cells will enable important insight into how an endogenous generative tissue compartment can adapt to altered retinal physiology and potentially even restore vision loss caused by retinal degenerative conditions.


Assuntos
Retina/citologia , Retina/fisiologia , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Suscetibilidade a Doenças , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Neurogênese , Organogênese , Neurônios Retinianos/citologia , Neurônios Retinianos/metabolismo , Epitélio Pigmentado da Retina , Vertebrados
11.
Commun Biol ; 4(1): 721, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117344

RESUMO

Hyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. We developed ultrafast phasor-based hyperspectral snapshot microscopy based on sine/cosine interference filters for biomedical imaging not feasible with conventional hyperspectral detection methods. Current approaches rely on slow spatial or spectral scanning limiting their application in living biological tissues, while faster snapshot methods such as image mapping spectrometry and multispectral interferometry are limited in spatial and/or spectral resolution, are computationally demanding, and imaging devices are very expensive to manufacture. Leveraging light sheet microscopy, phasor-based hyperspectral snapshot microscopy improved imaging speed 10-100 fold which, combined with minimal light exposure and high detection efficiency, enabled hyperspectral metabolic imaging of live, three-dimensional mouse tissues not feasible with other methods. As a fit-free method that does not require any a priori information often unavailable in complex and evolving biological systems, the rule of linear combinations of the phasor could spectrally resolve subtle differences between cell types in the developing zebrafish retina and spectrally separate and track multiple organelles in 3D cultured cells over time. The sine/cosine snapshot method is adaptable to any microscope or imaging device thus making hyperspectral imaging and fit-free analysis based on linear combinations broadly available to researchers and the public.


Assuntos
Imageamento Hiperespectral/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Animais , Colo/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3/ultraestrutura , Organelas/ultraestrutura , Retina/citologia , Retina/ultraestrutura , Peixe-Zebra/embriologia
12.
Bioconjug Chem ; 32(6): 1078-1093, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34081855

RESUMO

The prevalence of retinal disorders associated with visual impairment and blindness is increasing worldwide, while most of them remain without effective treatment. Pharmacological and molecular therapy development is hampered by the lack of effective drug delivery into the posterior segment of the eye. Among molecular approaches, RNA-interference (RNAi) features strong advantages, yet delivering it to the inner layer of the retina appears extremely challenging. To address this, we developed an original magnetic nanoparticles (MNPs)-based transfection method that allows the efficient delivery of siRNA in all retinal layers of rat adult retinas through magnetic targeting. To establish delivery of RNAi throughout the retina, we have chosen organotypic retinal explants as an ex vivo model and for future high content screening of molecular drugs. Conversely to classic Magnetofection, and similar to conditions in the posterior chamber of the eye, our methods allows attraction of siRNA complexed to MNPs from the culture media into the explant. Our method termed "Reverse Magnetofection" provides a novel and nontoxic strategy for RNAi-based molecular as well as gene therapy in the retina that can be transferred to a wide variety of organ explants.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Fenômenos Magnéticos , RNA Interferente Pequeno/metabolismo , Retina/citologia , Animais , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Transfecção
13.
FASEB J ; 35(7): e21689, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34085737

RESUMO

Knockout of the chloride channel protein 2 (CLC-2; CLCN2) results in fast progressing blindness in mice. Retinal Pigment Epithelium (RPE) and photoreceptors undergo, in parallel, rapid, and profound morphological changes and degeneration. Immunohistochemistry and electron microscopy of the outer retina and electroretinography of the CLC-2 KO mouse demonstrated normal morphology at postnatal day 2, followed by drastic changes in RPE and photoreceptor morphology and loss of vision during the first postnatal month. To investigate whether the RPE or the photoreceptors are the primary cause of the degeneration, we injected lentiviruses carrying HA-tagged CLC-2 with an RPE-specific promotor in the subretinal space of CLC-2-KO mice at the time of eye opening. As expected, CLC-2-HA was expressed exclusively in RPE; strikingly, this procedure rescued the degeneration of both RPE and photoreceptors. Light response in transduced eyes was also recovered. Only a fraction of RPE was transduced with the lentivirus; however, the entire RPE monolayer appears healthy, even the RPE cells not expressing the CLC-2-HA. Surprisingly, in contrast with previous physiological observations that postulate that CLC-2 has a basolateral localization in RPE, our immunofluorescence experiments demonstrated CLC-2 has an apical distribution, facing the subretinal space and the photoreceptor outer segments. Our findings suggest that CLC-2 does not play the postulated role in fluid transport at the basolateral membrane. Rather, they suggest that CLC-2 performs a critical homeostatic role in the subretinal compartment involving a chloride regulatory mechanism that is critical for the survival of both RPE and photoreceptors.


Assuntos
Canais de Cloreto/fisiologia , Células Fotorreceptoras/citologia , Retina/citologia , Degeneração Retiniana , Epitélio Pigmentado da Retina/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras/metabolismo , Retina/metabolismo
14.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063002

RESUMO

Cone Dystrophy with Supernormal Rod Response (CDSRR) is a rare autosomal recessive disorder leading to severe visual impairment in humans, but little is known about its unique pathophysiology. We have previously shown that CDSRR is caused by mutations in the KCNV2 (Potassium Voltage-Gated Channel Modifier Subfamily V Member 2) gene encoding the Kv8.2 subunit, a modulatory subunit of voltage-gated potassium (Kv) channels. In a recent study, we validated a novel mouse model of Kv8.2 deficiency at a late stage of the disease and showed that it replicates the human electroretinogram (ERG) phenotype. In this current study, we focused our investigation on young adult retinas to look for early markers of disease and evaluate their effect on retinal morphology, electrophysiology and immune response in both the Kv8.2 knockout (KO) mouse and in the Kv2.1 KO mouse, the obligate partner of Kv8.2 in functional retinal Kv channels. By evaluating the severity of retinal dystrophy in these KO models, we demonstrated that retinas of Kv KO mice have significantly higher apoptotic cells, a thinner outer nuclear cell layer and increased activated microglia cells in the subretinal space. Our results indicate that in the murine retina, the loss of Kv8.2 subunits contributes to early cellular and physiological changes leading to retinal dysfunction. These results could have potential implications in the early management of CDSRR despite its relatively nonprogressive nature in humans.


Assuntos
Envelhecimento/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Subunidades Proteicas/metabolismo , Retina/citologia , Retina/metabolismo , Canais de Potássio Shab/metabolismo , Animais , Morte Celular , Eletrorretinografia , Gliose/patologia , Imunidade , Camundongos Knockout , Microglia/patologia , Visão Noturna , Retina/fisiologia
15.
Nature ; 594(7862): 277-282, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34040258

RESUMO

Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours1. Although the role of neurons in tumour progression has previously been demonstrated2, the importance of neuronal activity to tumour initiation is less clear-particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with the neurofibromatosis 1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood3,4, raising  the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis 1 tumour suppressor gene (Nf1)5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin 3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome.


Assuntos
Transformação Celular Neoplásica/genética , Genes da Neurofibromatose 1 , Mutação , Neurofibromina 1/genética , Neurônios/metabolismo , Glioma do Nervo Óptico/genética , Glioma do Nervo Óptico/patologia , Animais , Astrocitoma/genética , Astrocitoma/patologia , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Transformação Celular Neoplásica/efeitos da radiação , Feminino , Mutação em Linhagem Germinativa , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos da radiação , Nervo Óptico/citologia , Nervo Óptico/efeitos da radiação , Estimulação Luminosa , Retina/citologia , Retina/efeitos da radiação
16.
Biochem Biophys Res Commun ; 559: 113-120, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33940381

RESUMO

Retinal damage caused by blue light has become an important public health concern. Mitochondria have been found to play a key role in light-induced retinal cell death. In this study, we aimed to clarify the molecular mechanism involved in mitochondrion-related retinal cell damage caused by blue light, the major component of light-emitting diodes (LEDs). Our results show that blue light (450 nm, 300lux)-induced R28 cell death is caspase independent and can be attenuated by necrostatin-1. Apoptosis-inducing factor (AIF) cleavage and translocation to the nucleus are involved in the cell death progress. Blue light exposure causes mitochondrial fragmentation, which is mediated by phosphorylation at dynamin-related protein 1 (Drp1) Ser616 site, but it does not alter the protein levels of fission or fusion machinery. Knocking down Drp1 or treatment with Drp1 inhibitor Mdivi-1 protects R28 cells from blue light. Overproduction of reactive oxygen species (ROS) is induced by blue light. The ROS scavenger Trolox decreases Drp1 Ser616 phosphorylation level and mitochondrial fragmentation upon blue light exposure. Moreover, Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 blocks Drp1 phosphorylation and rescues mitochondrial fragmentation and AIF-mediated cell death caused by blue light. In conclusion, our data suggest that the CaMKII-Drp1 pathway plays a major role in blue light-induced AIF-mediated retinal cell damage.


Assuntos
Fator de Indução de Apoptose/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dinaminas/metabolismo , Necroptose , Retina/citologia , Animais , Linhagem Celular , Fosforilação , Ratos , Retina/metabolismo
17.
Exp Eye Res ; 207: 108566, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33838142

RESUMO

Emerging treatment strategies for retinal degeneration involve replacing lost photoreceptors using supportive scaffolds to ensure cells survive the implantation process. While many design aspects of these scaffolds, including material chemistry and microstructural cues, have been studied in depth, a full set of design constraints has yet to be established. For example, while known to be important in other tissues and systems, the influence of mechanical properties on surgical handling has not been quantified. In this study, photocrosslinked poly(ethylene glycol) dimethacrylate (PEGDMA) was used as a model polymer to study the effects of scaffold modulus (stiffness) on surgical handling, independent of material chemistry. This was achieved by modulating the molecular weight and concentrations of the PEGDMA in various prepolymer solutions. Scaffold modulus of each formulation was measured using photo-rheology, which enabled the collection of real-time polymerization data. In addition to measuring scaffold mechanical properties, this approach gave insight on polymerization kinetics, which were used to determine the polymerization time required for each sample. Scaffold handling characteristics were qualitatively evaluated using both in vitro and ex vivo trials that mimicked the surgical procedure. In these trials, scaffolds with shear moduli above 35 kPa performed satisfactorily, while those below this limit performed poorly. In other words, scaffolds below this modulus were too fragile for reliable transplantation. To better compare these results with literature values, the compressive modulus was measured for select samples, with the lower shear modulus limit corresponding to roughly 115 kPa compressive modulus. While an upper mechanical property limit was not readily apparent from these results, there was increased variability in surgical handling performance in samples with shear moduli above 800 kPa. Overall, the knowledge presented here provides important groundwork for future studies designed to examine additional retinal scaffold considerations, including the effect of scaffold mechanical properties on retinal progenitor cell fate.


Assuntos
Metacrilatos/química , Polietilenoglicóis/química , Retina/citologia , Degeneração Retiniana/cirurgia , Transplante de Células-Tronco , Células-Tronco/citologia , Tecidos Suporte/química , Animais , Reagentes para Ligações Cruzadas , Módulo de Elasticidade/fisiologia , Degeneração Retiniana/fisiopatologia , Suínos
18.
Exp Eye Res ; 207: 108584, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33910034

RESUMO

Microglia are the resident immune cells in the retina. To investigate their properties and behaviour, a reliable and yielding procedure to culture them is necessary. We here describe a way of isolation of microglial cells from the porcine retina, as pig eyes are similar to human eyes in size, structure and vasculature, including similarities in proteins and pathways. Retina was isolated from fresh pig eyes, dissociated by a mixture of collagenase, hyaluronidase and DNAse, and passed through a cell strainer. After triple centrifugation with decreasing velocity and re-suspension, cells were seeded into poly-d-lysine coated culture flasks and cultured using DMEM and macrophage-colony stimulating factor (M-CSF). Number of cells increased gradually during the first 10-14 days, till they could be split and used for experiments. Identity of isolated cells as microglia was assessed by immunostaining against the microglia/macrophage markers Iba1, CD11b, CD68, CD45 and TMEM119. Phagocytic function of microglia could be demonstrated by phagocytosis of fluorescence beads and their response to lipopolysaccharide (LPS). As a conclusion, we developed a protocol for isolation and cultivation of pig retinal microglial cells that are suitable for research in the laboratory.


Assuntos
Separação Celular/métodos , Microglia/citologia , Retina/citologia , Animais , Biomarcadores/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Imuno-Histoquímica , Microglia/metabolismo , Fagocitose/fisiologia , Retina/metabolismo , Sus scrofa
19.
J Vis Exp ; (170)2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33900292

RESUMO

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Retina/citologia , Células Fotorreceptoras Retinianas Cones/citologia , Degeneração Retiniana , Humanos , Reprodutibilidade dos Testes , Células Fotorreceptoras Retinianas Cones/fisiologia
20.
Genes Dev ; 35(9-10): 677-691, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33888564

RESUMO

During the development of the vertebrate nervous systems, genetic programs assemble an immature circuit that is subsequently refined by neuronal activity evoked by external stimuli. However, prior to sensory experience, the intrinsic property of the developing nervous system also triggers correlated network-level neuronal activity, with retinal waves in the developing vertebrate retina being the best documented example. Spontaneous activity has also been found in the visual system of Drosophila Here, we compare the spontaneous activity of the developing visual system between mammalian and Drosophila and suggest that Drosophila is an emerging model for mechanistic and functional studies of correlated spontaneous activity.


Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Retina/citologia , Retina/embriologia , Células Receptoras Sensoriais/fisiologia , Animais , Drosophila melanogaster/fisiologia , Olho/citologia , Olho/crescimento & desenvolvimento , Humanos , Modelos Animais , Retina/fisiologia , Células Receptoras Sensoriais/citologia
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