RESUMO
Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.
Assuntos
Envelhecimento/genética , Reprogramação Celular/genética , Metilação de DNA , Epigênese Genética , Olho , Regeneração Nervosa/genética , Visão Ocular/genética , Visão Ocular/fisiologia , Envelhecimento/fisiologia , Animais , Axônios/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Dioxigenases , Modelos Animais de Doenças , Olho/citologia , Olho/inervação , Olho/patologia , Feminino , Vetores Genéticos/genética , Glaucoma/genética , Glaucoma/patologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Fator 3 de Transcrição de Octâmero/genética , Traumatismos do Nervo Óptico/genética , Proteínas Proto-Oncogênicas/genética , Células Ganglionares da Retina/citologia , Fatores de Transcrição SOXB1/genética , Transcriptoma/genéticaRESUMO
The shortfin mako, Isurus oxyrinchus is an oceanic pelagic shark found worldwide in tropical and subtropical waters. However, the understanding of its biology at molecular level is still incipient. We sequenced the messenger RNA isolated from eye and liver tissues. De novo transcriptome yielded a total of 705,940 transcripts. A total of 3774 genes were differentially expressed (DEGs), with 1612 in the eye and 2162 in the liver. Most DEGs in the eye were related to structural and signaling functions, including nonocular and ocular opsin genes, whereas nine out of ten most overexpressed genes in the liver were related to tumor suppression, wound healing, and human diseases. Furthermore, DEGs findings provide insights on the monochromatic shark vision and a repertory of cancer-related genes, which may be insightful to elucidate shark resistance to cancer. Therefore, our results provide valuable sequence resources for future functional and population studies.
Assuntos
Resistência à Doença/genética , Proteínas do Olho/genética , Fígado , Neoplasias/genética , Tubarões/genética , Animais , Olho , Expressão Gênica , Anotação de Sequência Molecular , Opsinas/genética , RNA Mensageiro/genética , Transcriptoma , Visão Ocular/genéticaRESUMO
Retinitis pigmentosa (RP) is a generic term for a group of genetic diseases characterized by loss of rod and cone photoreceptor cells. Although the genetic causes of RP frequently only affect the rod photoreceptor cells, cone photoreceptors become stressed in the absence of rods and undergo a secondary degeneration. Changes in the gene expression profile of cone photoreceptor cells are likely to occur prior to observable physiological changes. To this end, we sought to achieve greater understanding of the changes in cone photoreceptor cells early in the degeneration process of the Rho-/- mouse model. To account for gene expression changes attributed to loss of cone photoreceptor cells, we normalized PCR in the remaining number of cones to a cone cell reporter (OPN1-GFP). Gene expression profiles of key components involved in the cone phototransduction cascade were correlated with tests of retinal cone function prior to cell loss. A significant downregulation of the photoreceptor transcription factor Crx was observed, which preceded a significant downregulation in cone opsin transcripts that coincided with declining cone function. Our data add to the growing understanding of molecular changes that occur prior to cone dysfunction in a model of rod-cone dystrophy. It is of interest that gene supplementation of CRX by adeno-associated viral vector delivery prior to cone cell loss did not prevent cone photoreceptor degeneration in this mouse model.
Assuntos
Distrofias de Cones e Bastonetes/genética , Distrofias de Cones e Bastonetes/fisiopatologia , Animais , Distrofias de Cones e Bastonetes/terapia , Modelos Animais de Doenças , Eletrorretinografia , Regulação da Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Proteínas de Fluorescência Verde/genética , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/farmacologia , Humanos , Camundongos Transgênicos , Oftalmoscopia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Rodopsina/genética , Opsinas de Bastonetes/genética , Tomografia de Coerência Óptica , Transativadores/genética , Transativadores/farmacologia , Visão Ocular/genéticaRESUMO
BACKGROUND: p19arf, primarily known as a tumor suppressor, has also been reported to play an essential role in normal development of mouse eyes. Consistently, lack of p19arf has been associated with ocular defects, but the mixed background of the knockout (KO) mouse strain used raised a concern on the accuracy of the phenotypes observed in association with the targeted gene due to genetic heterogeneity. OBJECT: We carried out a study to investigate into the effect of genetic background on the manifestation of p19arf KO associated phenotypes. METHODS: We characterized the phenotypes of novel p19arf KO mouse lines generated in FVB/N and C57BL/6J using a transcription activator-like effector nuclease (TALEN) system in comparison to the reported phenotypes of three other p19arf-deficient mouse lines generated using homologous recombination. RESULTS: Ninety-five percent of FVB/N-p19arf KO mice showed ocular opacity from week 4 after birth which worsened rapidly until week 6, while such abnormality was absent in C57BL/6J-p19arf KO mice up to the age of 26 weeks. Histopathological analysis revealed retrolental masses and dysplasia in the retinal layer in FVB/N-p19arf KO mice from week 4. Besides these, both strains developed normally from birth to week 26 without increased tumorigenesis except for a subcutaneous tumor found in a C57BL/6J-p19arf KO mouse. CONCLUSION: Our findings demonstrated surprisingly variable manifestation of p19arf-linked phenotypes between FVB/N and C57BL/6J mice, and furthermore between our mouse lines and the established lines, indicating a critical impact of genetic background on functional study of genes using gene targeting strategies in mice.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Camundongos Endogâmicos/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Animais , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Olho/embriologia , Olho/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenômenos Fisiológicos Oculares/genética , Fenótipo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/fisiologia , Efetores Semelhantes a Ativadores de Transcrição/genética , Visão Ocular/genética , Visão Ocular/fisiologiaRESUMO
The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.
Assuntos
Especiação Genética , Genoma de Inseto , Interações Hospedeiro-Parasita/genética , Himenópteros/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Elementos de DNA Transponíveis , Feminino , Dosagem de Genes , Glicoproteínas/genética , Herbivoria/genética , Imunidade/genética , Proteínas de Insetos/genética , Masculino , Família Multigênica , Receptores Odorantes/genética , Comportamento Social , Visão Ocular/genéticaRESUMO
BACKGROUND: This study aimed to identify and evaluate potential molecular targets associated with the development of proliferative diabetic retinopathy (DR). METHODS: The microarray dataset "GSE60436" generated from fibrovascular membranes (FVMs) associated with proliferative DR was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) from the active FVMs and control or inactive FVMs and control were evaluated and co-DEGs were identified using VEEN analysis. Functional enrichment analysis, and protein-protein interactions (PPI) network and module analyses were performed on the upregulated and downregulated coDEGs. Finally, several predictions regarding microRNAs (miRNAs) and transcription factors (TFs) were made to construct a putative TF-miRNA-target network. RESULTS: A total of 1475 co-DEGs were screened in active/inactive FVM samples, including 461 upregulated and 1014 downregulated genes, which were enriched for angiogenesis [Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A) and Placental Growth Factor (PGF)] and visual perception, respectively. In the case of the upregulated co-DEGs, Kinesin Family Member 11 (KIF11), and BUB1 Mitotic Checkpoint Serine/Threonine Kinase (BUB1) exhibited the highest values in both the PPI network and module analyses, as well as the genes related to mitosis. In the case of downregulated co-DEGs, several G protein subunits, including G Protein Subunit Beta 3 (GNB3), exhibited the highest values in both the PPI network and module analyses. The genes identified in the module analysis were found to be from the signal transduction-related pathways. In addition, we were able to identify four miRNAs and five TFs, including miR-136 and miR-374. CONCLUSIONS: In brief, HIF1A, PGF, KIF11, G protein subunits, and miR-136, miR-374 may all be involved in angiogenesis, retinal endothelial cell proliferation, and visual signal transduction in proliferative DR. This study provides a number of novel insights that may aid the development of future studies dedicated to discovering novel therapeutic targets in proliferative DR.
Assuntos
Retinopatia Diabética/genética , Células Endoteliais/patologia , Perfilação da Expressão Gênica , Neovascularização Retiniana/genética , Proliferação de Células , Conjuntos de Dados como Assunto , Proteínas de Ligação ao GTP/genética , Redes Reguladoras de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Cinesinas/genética , MicroRNAs/genética , Análise em Microsséries , Terapia de Alvo Molecular , Fator de Crescimento Placentário/genética , Domínios e Motivos de Interação entre Proteínas/genética , Fatores de Transcrição/genética , Visão Ocular/genéticaRESUMO
Von Hippel-Lindau (VHL) syndrome is a rare, autosomal dominant disorder, characterised by hypervascularised tumour formation in multiple organ systems. Vision loss associated with retinal capillary hemangioblastomas remains one of the earliest complications of VHL disease. The mortality of Vhl-/- mice in utero restricted modelling of VHL disease in this mammalian model. Zebrafish harbouring a recessive germline mutation in the vhl gene represent a viable, alternative vertebrate model to investigate associated ocular loss-of-function phenotypes. Previous studies reported neovascularisation of the brain, eye and trunk together with oedema in the vhl-/- zebrafish eye. In this study, we demonstrate vhl-/- zebrafish almost entirely lack visual function. Furthermore, hyaloid vasculature networks in the vhl-/- eye are improperly formed and this phenotype is concomitant with development of an ectopic intraretinal vasculature. Sunitinib malate, a multi tyrosine kinase inhibitor, market authorised for cancer, reversed the ocular behavioural and morphological phenotypes observed in vhl-/- zebrafish. We conclude that the zebrafish vhl gene contributes to an endogenous molecular barrier that prevents development of intraretinal vasculature, and that pharmacological intervention with sunitinib can improve visual function and hyaloid vessel patterning while reducing abnormally formed ectopic intraretinal vessels in vhl-/- zebrafish.
Assuntos
Olho/irrigação sanguínea , Retina/embriologia , Proteínas Supressoras de Tumor/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Doença de von Hippel-Lindau/genética , Animais , Antineoplásicos/farmacologia , Cegueira/genética , Modelos Animais de Doenças , Olho/embriologia , Hemangioblastoma/genética , Sunitinibe/farmacologia , Visão Ocular/genética , Doença de von Hippel-Lindau/patologia , Doença de von Hippel-Lindau/prevenção & controleRESUMO
BACKGROUND: The phyla Cnidaria, Placozoa, Ctenophora, and Porifera emerged before the split of proto- and deuterostome animals, about 600 million years ago. These early metazoans are interesting, because they can give us important information on the evolution of various tissues and organs, such as eyes and the nervous system. Generally, cnidarians have simple nervous systems, which use neuropeptides for their neurotransmission, but some cnidarian medusae belonging to the class Cubozoa (box jellyfishes) have advanced image-forming eyes, probably associated with a complex innervation. Here, we describe a new transcriptome database from the cubomedusa Tripedalia cystophora. RESULTS: Based on the combined use of the Illumina and PacBio sequencing technologies, we produced a highly contiguous transcriptome database from T. cystophora. We then developed a software program to discover neuropeptide preprohormones in this database. This script enabled us to annotate seven novel T. cystophora neuropeptide preprohormone cDNAs: One coding for 19 copies of a peptide with the structure pQWLRGRFamide; one coding for six copies of a different RFamide peptide; one coding for six copies of pQPPGVWamide; one coding for eight different neuropeptide copies with the C-terminal LWamide sequence; one coding for thirteen copies of a peptide with the RPRAamide C-terminus; one coding for four copies of a peptide with the C-terminal GRYamide sequence; and one coding for seven copies of a cyclic peptide, of which the most frequent one has the sequence CTGQMCWFRamide. We could also identify orthologs of these seven preprohormones in the cubozoans Alatina alata, Carybdea xaymacana, Chironex fleckeri, and Chiropsalmus quadrumanus. Furthermore, using TBLASTN screening, we could annotate four bursicon-like glycoprotein hormone subunits, five opsins, and 52 other family-A G protein-coupled receptors (GPCRs), which also included two leucine-rich repeats containing G protein-coupled receptors (LGRs) in T. cystophora. The two LGRs are potential receptors for the glycoprotein hormones, while the other GPCRs are candidate receptors for the above-mentioned neuropeptides. CONCLUSIONS: By combining Illumina and PacBio sequencing technologies, we have produced a new high-quality de novo transcriptome assembly from T. cystophora that should be a valuable resource for identifying the neuronal components that are involved in vision and other behaviors in cubomedusae.
Assuntos
Cubomedusas/genética , Peptídeos/genética , Transmissão Sináptica/genética , Transcriptoma/genética , Animais , Cubomedusas/fisiologia , Humanos , Neurônios/metabolismo , Neuropeptídeos , Opsinas/genética , Receptores Acoplados a Proteínas G/genética , Visão Ocular/genética , Visão Ocular/fisiologiaRESUMO
Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1ß were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Transdução Genética/métodos , Animais , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Vetores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras/metabolismo , Regiões Promotoras Genéticas/genética , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transgenes , Visão Ocular/genética , Visão Ocular/fisiologiaRESUMO
To study whether ectopic human melanopsin (hMel) in retinal ganglion cells (RGCs) could restore the visual function in end-stage retinal degeneration, AAV2/8-CMV-hMel/FYP was injected into the intravitreal space of Royal College of Surgeons (RCS) rats. It was observed that ectopic hMel/yellow fluorescent protein (YFP) was dominantly expressed in the RGCs of the RCS rat retinae. At 30-45 days after administration of AAV2/8-CMV-hMel/FYP in RCS rats, the flash visual evoked potentials and behavioral results demonstrated that visual function was significantly improved compared to that in the control group, while no improvement in flash electroretinography was observed at this time point. To translate this potential therapeutic approach to the clinic, the safety of viral vectors in the retinae of normal macaques was then studied, and the expression profile of exogenous hMel with/without internal limiting membrane peeling was compared before viral vector administration. The data revealed that there was no significant difference in the number of RGCs containing exogenous hMel/YFP between the two groups. Whole-cell patch-clamp recordings demonstrated that the hMel/YFP-positive RGCs of the macaque retinae reacted to the intense light stimulation, generating inward currents and action potentials. This result confirms that the ectopic hMel expressed in RGCs is functional. Moreover, the introduction of AAV2/8-CMV-hMel/FYP does not cause detectable pathological effects. Thus, this study suggests that AAV2/8-CMV-hMel/FYP administration without internal limiting membrane peeling is safe and feasible for efficient transduction and provides therapeutic benefits to restore the visual function of patients suffering photoreceptor loss.
Assuntos
Expressão Ectópica do Gene , Células Ganglionares da Retina/metabolismo , Opsinas de Bastonetes/genética , Visão Ocular/genética , Animais , Biomarcadores , Dependovirus/genética , Feminino , Genes Reporter , Vetores Genéticos/genética , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Macaca , Masculino , Imagem Molecular , Técnicas de Patch-Clamp , Ratos , Reprodutibilidade dos Testes , Retina/metabolismo , Retina/fisiopatologiaRESUMO
Retinal gene therapy is increasingly recognized as a novel molecular intervention that has huge potential in treating common causes of blindness, the majority of which have a genetic aetiology1-5. Choroideremia is a chronic X-linked retinal degeneration that was first described in 18726. It leads to progressive blindness due to deficiency of Rab-escort protein 1 (REP1). We designed an adeno-associated viral vector to express REP1 and assessed it in a gene therapy clinical trial by subretinal injection in 14 patients with choroideremia. The primary endpoint was vision change in treated eyes 2 years after surgery compared to unoperated fellow eyes. Despite complications in two patients, visual acuity improved in the 14 treated eyes over controls (median 4.5 letter gain, versus 1.5 letter loss, P = 0.04), with 6 treated eyes gaining more than one line of vision (>5 letters). The results suggest that retinal gene therapy can sustain and improve visual acuity in a cohort of predominantly late-stage choroideremia patients in whom rapid visual acuity loss would ordinarily be predicted.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Coroideremia/terapia , Terapia Genética , Degeneração Retiniana/fisiopatologia , Acuidade Visual/genética , Proteínas Adaptadoras de Transdução de Sinal/uso terapêutico , Adulto , Idoso , Coroideremia/genética , Coroideremia/fisiopatologia , Coroideremia/cirurgia , Dependovirus/genética , Vetores Genéticos/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Retina/fisiopatologia , Degeneração Retiniana/genética , Degeneração Retiniana/cirurgia , Visão Ocular/genética , Visão Ocular/fisiologiaRESUMO
Stress-associated premature senescence plays a major role in retinal diseases. In this study, we investigated the relationship between endothelial dysfunction, endoplasmic reticulum (ER) stress, and cellular senescence in the development of retinal dysfunction. We tested the hypothesis that constant endothelial activation by transmembrane tumor necrosis factor-α (tmTNF-α) exacerbates age-induced visual deficits via senescence-mediated ER stress in this model. To address this, we employed a mouse model of chronic vascular activation using endothelial-specific TNF-α-expressing (tie2-TNF) mice at 5 and 10 months of age. Visual deficits were exhibited by tie2-TNF mice at both 5 months and 10 months of age, with the older mice showing statistically significant loss of visual acuity compared with tie2-TNF mice at age 5 months. The neural defects, as measured by electroretinogram (ERG), also followed a similar trend in an age-dependent fashion, with 10-month-old tie2-TNF mice showing the greatest decrease in "b" wave amplitude at 25 cd.s.m2 compared with age-matched wildtype (WT) mice and five-month-old tie2-TNF mice. While gene and protein expression from the whole retinal extracts demonstrated increased inflammatory (Icam1, Ccl2), stress-associated premature senescence (p16, p21, p53), and ER stress (Grp78, p-Ire1α, Chop) markers in five-month-old tie2-TNF mice compared with five-month-old WT mice, a further increase was seen in 10-month-old tie2-TNF mice. Our data demonstrate that tie2-TNF mice exhibit age-associated increases in visual deficits, and these data suggest that inflammatory endothelial activation is at least partly at play. Given the correlation of increased premature senescence and ER stress in an age-dependent fashion, with the loss of visual functions and increased endothelial activation, our data suggest a possible self-enhanced loop of unfolded protein response pathways and senescence in propagating neurovascular defects in this model. Impact statement Vision loss in most retinal diseases affects the quality of life of working age adults. Using a novel animal model that displays constant endothelial activation by tmTNF-α, our results demonstrate exacerbated age-induced visual deficits via premature senescence-mediated ER stress. We have compared mice of 5 and 10 months of age, with highly relevant human equivalencies of approximately 35- and 50-year-old patients, representing mature adult and middle-aged subjects, respectively. Our studies suggest a possible role for a self-enhanced loop of ER stress pathways and senescence in the propagation of retinal neurovascular defects, under conditions of constant endothelial activation induced by tmTNF-α signaling.
Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Receptor TIE-2/genética , Fator de Necrose Tumoral alfa/metabolismo , Transtornos da Visão/genética , Visão Ocular/genética , Animais , Células Cultivadas , Senescência Celular , Eletrorretinografia , Chaperona BiP do Retículo Endoplasmático , Células Endoteliais/citologia , Feminino , Inflamação , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reflexo , Transdução de SinaisRESUMO
In many human disorders mitochondrial dysfunction is central to degeneration of retinal ganglion cells. As these cells do not regenerate, vision is irreversibly lost. Here we show reversal of visual dysfunction by a mitochondrially targeted adeno associated virus in transgenic mice harboring a G11778A mutation in the ND4 subunit of complex I persists longterm and it is associated with reduced loss of RGCs and their axons, improved oxidative phosphorylation, persistence of transferred ND4 DNA and transcription of ND4 mRNA.
Assuntos
Técnicas de Transferência de Genes , Genes Mitocondriais/genética , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/fisiopatologia , Visão Ocular/genética , Animais , Contagem de Células , Modelos Animais de Doenças , Camundongos , Mutação , Neurônios/patologia , Atrofia Óptica Hereditária de Leber/patologia , Nervo Óptico/patologia , Células Ganglionares da Retina/patologiaRESUMO
UNLABELLED: Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs, with five subtypes named M1-M5) are a unique subclass of RGCs with axons that project directly to many brain nuclei involved in non-image-forming functions such as circadian photoentrainment and the pupillary light reflex. Recent evidence suggests that melanopsin-based signals also influence image-forming visual function, including light adaptation, but the mechanisms involved are unclear. Intriguingly, a small population of M1 ipRGCs have intraretinal axon collaterals that project toward the outer retina. Using genetic mouse models, we provide three lines of evidence showing that these axon collaterals make connections with upstream dopaminergic amacrine cells (DACs): (1) ipRGC signaling to DACs is blocked by tetrodotoxin both in vitro and in vivo, indicating that ipRGC-to-DAC transmission requires voltage-gated Na(+) channels; (2) this transmission is partly dependent on N-type Ca(2+) channels, which are possibly expressed in the axon collateral terminals of ipRGCs; and (3) fluorescence microscopy reveals that ipRGC axon collaterals make putative presynaptic contact with DACs. We further demonstrate that elimination of M1 ipRGCs attenuates light adaptation, as evidenced by an impaired electroretinogram b-wave from cones, whereas a dopamine receptor agonist can potentiate the cone-driven b-wave of retinas lacking M1 ipRGCs. Together, the results strongly suggest that ipRGCs transmit luminance signals retrogradely to the outer retina through the dopaminergic system and in turn influence retinal light adaptation. SIGNIFICANCE STATEMENT: Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) comprise a third class of retinal photoreceptors that are known to mediate physiological responses such as circadian photoentrainment. However, investigation into whether and how ipRGCs contribute to vision has just begun. Here, we provide convergent anatomical and physiological evidence that axon collaterals of ipRGCs constitute a centrifugal pathway to DACs, conveying melanopsin-based signals from the innermost retina to the outer retina. We further demonstrate that retrograde signals likely influence visual processing because elimination of axon collateral-bearing ipRGCs impairs light adaptation by limiting dopamine-dependent facilitation of the cone pathway. Our findings strongly support the hypothesis that retrograde melanopsin-based signaling influences visual function locally within the retina, a notion that refutes the dogma that RGCs only provide physiological signals to the brain.
Assuntos
Potenciais da Membrana/fisiologia , Retina/citologia , Células Ganglionares da Retina/fisiologia , Visão Ocular/fisiologia , Vias Visuais/fisiologia , Animais , Animais Recém-Nascidos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Luz , Masculino , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células Ganglionares da Retina/classificação , Células Ganglionares da Retina/efeitos dos fármacos , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Transducina/genética , Transducina/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Visão Ocular/genética , beta-Galactosidase/metabolismoRESUMO
The neural cell adhesion molecule (NCAM) is involved in developmental processes and age-associated cognitive decline; however, little is known concerning the effects of NCAM in the visual system during aging. Using anatomical, electrophysiological, and behavioral assays, we analyzed age-related changes in visual function of NCAM deficient (-/-) and wild-type mice. Anatomical analyses indicated that aging NCAM -/- mice had fewer retinal ganglion cells, thinner retinas, and fewer photoreceptor cell layers than age-matched controls. Electroretinogram testing of retinal function in young adult NCAM -/- mice showed a 2-fold increase in a- and b-wave amplitude compared with wild-type mice, but the retinal activity dropped dramatically to control levels when the animals reached 10 months. In behavioral tasks, NCAM -/- mice had no visual pattern discrimination ability and showed premature loss of vision as they aged. Together, these findings demonstrate that NCAM plays significant roles in the adult visual system in establishing normal retinal anatomy, physiology and function, and in maintaining vision during aging.
Assuntos
Envelhecimento/genética , Envelhecimento/fisiologia , Antígeno CD56/metabolismo , Transtornos da Visão/etiologia , Transtornos da Visão/genética , Visão Ocular/genética , Visão Ocular/fisiologia , Envelhecimento/patologia , Animais , Antígeno CD56/genética , Eletrorretinografia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Fotorreceptoras/patologia , Retina/citologia , Retina/metabolismo , Retina/patologia , Retina/fisiologia , Transtornos da Visão/patologiaRESUMO
Retinal disease is one of the most active areas of gene therapy, with clinical trials ongoing in the United States for five diseases. There are currently no treatments for patients with late-stage disease in which photoreceptors have been lost. Optogenetic gene therapies are in development, but, to date, have suffered from the low light sensitivity of microbial opsins, such as channelrhodopsin and halorhodopsin, and azobenzene-based photoswitches. Several groups have shown that photoreceptive G-protein-coupled receptors (GPCRs) can be expressed heterologously, and photoactivate endogenous Gi/o signaling. We hypothesized such a GPCR could increase sensitivity due to endogenous signal amplification. We targeted vertebrate rhodopsin to retinal ON-bipolar cells of blind rd1 mice and observed restoration of: (i) light responses in retinal explants, (ii) visually-evoked potentials in visual cortex in vivo, and (iii) two forms of visually-guided behavior: innate light avoidance and discrimination of temporal light patterns in the context of fear conditioning. Importantly, both the light responses of the retinal explants and the visually-guided behavior occurred reliably at light levels that were two to three orders of magnitude dimmer than required for channelrhodopsin. Thus, gene therapy with native light-gated GPCRs presents a novel approach to impart light sensitivity for visual restoration in a useful range of illumination.
Assuntos
Optogenética/métodos , Rodopsina/genética , Visão Ocular/genética , Animais , Dependovirus/genética , Expressão Ectópica do Gene , Potenciais Evocados Visuais/genética , Potenciais Evocados Visuais/efeitos da radiação , Terapia Genética , Vetores Genéticos/genética , Luz , Camundongos , Estimulação Luminosa , Retina/citologia , Retina/metabolismo , Células Bipolares da Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução Genética , Percepção VisualRESUMO
Achromatopsia is a hereditary form of day blindness caused by cone photoreceptor dysfunction. Affected patients suffer from congenital color blindness, photosensitivity, and low visual acuity. Mutations in the CNGA3 gene are a major cause of achromatopsia, and a sheep model of this disease was recently characterized by our group. Here, we report that unilateral subretinal delivery of an adeno-associated virus serotype 5 (AAV5) vector carrying either the mouse or the human intact CNGA3 gene under the control of the red/green opsin promoter results in long-term recovery of visual function in CNGA3-mutant sheep. Treated animals demonstrated shorter maze passage times and a reduced number of collisions with obstacles compared with their pretreatment status, with values close to those of unaffected sheep. This effect was abolished when the treated eye was patched. Electroretinography (ERG) showed marked improvement in cone function. Retinal expression of the transfected human and mouse CNGA3 genes at the mRNA level was shown by polymerase chain reaction (PCR), and cone-specific expression of CNGA3 protein was demonstrated by immunohistochemisrty. The rescue effect has so far been maintained for over 3 years in the first-treated animals, with no obvious ocular or systemic side effects. The results support future application of subretinal AAV5-mediated gene-augmentation therapy in CNGA3 achromatopsia patients.
Assuntos
Defeitos da Visão Cromática/genética , Defeitos da Visão Cromática/terapia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Terapia Genética , Retina/metabolismo , Visão Ocular/genética , Animais , Defeitos da Visão Cromática/fisiopatologia , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Feminino , Expressão Gênica , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Homozigoto , Humanos , Injeções Intraoculares , Masculino , Aprendizagem em Labirinto , Camundongos , Mutação , RNA Mensageiro/genética , OvinosRESUMO
Peptide's bioregulators promotes restoration of the physiological activity of the retina in retinitis pigmentosa in older adults and in animal models. The molecular mechanism of physiological activity of peptides is connected with its ability to regulate synthesis of protein markers of differentiation of neurons and retinal pigment epithelium epigenetically.
Assuntos
Peptídeos/genética , Retina/patologia , Retinose Pigmentar/genética , Visão Ocular/genética , Adulto , Animais , Humanos , Peptídeos/metabolismo , Retina/metabolismo , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Visão Ocular/fisiologia , Acuidade Visual/fisiologiaRESUMO
The activity of Na(+)/K(+)-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na(+)/K(+)-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na(+)/K(+)-ATPase deficiency to in vivo neurodegeneration has been lacking. In this study, we use Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na(+)/K(+) ATPase. Loss of ATPα, an α subunit of Na(+)/K(+)-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K(+). Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na(+)/K(+)-ATPase ß subunit, partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na(+)/K(+)-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na(+)/K(+) ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na(+)/K(+) ATPase deficiency-mediated neurodegeneration.
Assuntos
Envelhecimento , Cegueira/patologia , Células Fotorreceptoras de Invertebrados/metabolismo , Degeneração Retiniana/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Animais Geneticamente Modificados , Cegueira/genética , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Eletrorretinografia , Líquido Extracelular/metabolismo , Luz/efeitos adversos , Microscopia Eletrônica de Transmissão , Nervo Óptico/patologia , Células Fotorreceptoras de Invertebrados/ultraestrutura , Potássio/metabolismo , Interferência de RNA/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , ATPase Trocadora de Sódio-Potássio/genética , Temperatura , Visão Ocular/genéticaRESUMO
PURPOSE: Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE. METHODS: iPS-RPE was derived from human iPS cells. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids. RESULTS: Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol, iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media. CONCLUSIONS: iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE.