RESUMEN
Inherited retinopathies (IR) are common untreatable blinding conditions. Most of them are inherited as monogenic disorders, due to mutations in genes expressed in retinal photoreceptors (PR) and in retinal pigment epithelium (RPE). The retina's compatibility with gene transfer has made transduction of different retinal cell layers in small and large animal models via viral and non-viral vectors possible. The ongoing identification of novel viruses as well as modifications of existing ones based either on rational design or directed evolution have generated vector variants with improved transduction properties. Dozens of promising proofs of concept have been obtained in IR animal models with both viral and non-viral vectors, and some of them have been relayed to clinical trials. To date, recombinant vectors based on the adeno-associated virus (AAV) represent the most promising tool for retinal gene therapy, given their ability to efficiently deliver therapeutic genes to both PR and RPE and their excellent safety and efficacy profiles in humans. However, AAVs' limited cargo capacity has prevented application of the viral vector to treatments requiring transfer of genes with a coding sequence larger than 5 kb. Vectors with larger capacity, i.e. nanoparticles, adenoviral and lentiviral vectors are being exploited for gene transfer to the retina in animal models and, more recently, in humans. This review focuses on the available platforms for retinal gene therapy to fight inherited blindness, highlights their main strengths and examines the efforts to overcome some of their limitations.
Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Enfermedades de la Retina/terapia , Adenoviridae/genética , Virus ADN/genética , Humanos , Lentivirus/genética , Enfermedades de la Retina/congénito , Enfermedades de la Retina/genéticaRESUMEN
Sorting of specific mRNAs to particular cellular locations and regulation of their translation is an essential mechanism underlying cell polarization. The transport of RNAs by kinesins and dyneins has been clearly established in several cell models, including neurons in culture. A similar role appears to exist in higher eukaryotes for the myosins. Myosin Va (Myo5a) has been described as a component of ribonucleoprotein particles (RNPs) in the adult rat nervous system and associated to ZBP1 and ribosomes in ribosomal periaxoplasmic plaques (PARPs), making it a likely candidate for mediating some aspects of RNA transport in neurons. To test this hypothesis, we have characterized RNPs containing Myo5a in adult brains of rats and mice. Microarray analysis of RNAs co-immunoprecipitated with Myo5a indicates that this motor may associate with a specific subpopulation of neuronal mRNAs. We found mRNAs encoding α-synuclein and several proteins with functions in translation in these RNPs. Immunofluorescence analyses of RNPs showed apparent co-localization of Myo5a with ribosomes, mRNA and RNA-binding proteins in discrete structures present both in axons of neurons in culture and in myelinated fibers of medullary roots. Our data suggest that PARPs include RNPs bearing the mRNA coding for Myo5a and are equipped with kinesin and Myo5a molecular motors. In conclusion, we suggest that Myo5a is involved in mRNA trafficking both in the central and peripheral nervous systems.
Asunto(s)
Axones/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Actinas/metabolismo , Animales , Encéfalo/metabolismo , Células Cultivadas , Ganglios Espinales/metabolismo , Bulbo Raquídeo , Ratones , Ratones Endogámicos C57BL , Fibras Nerviosas Mielínicas/metabolismo , Ratas , Ratas Sprague-Dawley , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
Gene transfer to both cone and rod photoreceptors (PRs) is essential for gene therapy of inherited retinal degenerations that are caused by mutations in genes expressed in both PR types. Vectors based on the adeno-associated virus (AAV) efficiently transduce PRs of different species. However, these are predominantly rods and little is known about the ability of the AAV to transduce cones in combination with rods. Here we show that AAV2/8 transduces pig cones to levels that are similar to AAV2/9, and the outer nuclear layer (mainly rods) to levels that are on average higher, although not statistically significant, than both AAV2/5 and AAV2/9. We additionally found that the ubiquitous cytomegalovirus (CMV), but not the PR-specific GRK1 promoter, transduced pig cones efficiently, presumably because GRK1 is not expressed in pig cones as observed in mice and humans. Indeed, the GRK1 and CMV promoters transduce a similar percentage of murine cones with the CMV reaching the highest expression levels. Consistent with this, the AAV2/8 vectors with either the CMV or the GRK1 promoter restore cone function in a mouse model of Leber congenital amaurosis type 1 (LCA1), supporting the use of AAV2/8 for gene therapy of LCA1 as well as of other retinal diseases requiring gene transfer to both PR types.
Asunto(s)
Terapia Genética , Amaurosis Congénita de Leber/terapia , Degeneración Retiniana/terapia , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Amaurosis Congénita de Leber/genética , Ratones , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/genética , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Transducción GenéticaRESUMEN
Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.
Asunto(s)
Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Retina , Transducción Genética/métodos , Animales , Línea Celular , Ratones , PorcinosRESUMEN
BACKGROUND: Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. METHODOLOGY/PRINCIPAL FINDINGS: To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3'UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy. CONCLUSIONS: We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters.
Asunto(s)
Regulación de la Expresión Génica , MicroARNs/metabolismo , Retina/metabolismo , Transgenes/genética , Animales , Secuencia de Bases , Dependovirus/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Sus scrofa , Transducción GenéticaRESUMEN
BACKGROUND: Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca(2+). Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP(3)) and also regulates actin-binding proteins, PIP2 might be involved in these two processes. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca(2+) and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca(2+) wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca(2+) signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis.
Asunto(s)
Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estrellas de Mar/metabolismo , Cigoto/metabolismo , Actinas/metabolismo , Animales , Sitios de Unión/genética , Calcio/metabolismo , Señalización del Calcio , Femenino , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Microscopía Confocal , Microscopía Electrónica de Transmisión , Oocitos/metabolismo , Fosfolipasa C delta/genética , Polimerizacion , Unión Proteica , Transducción de Señal , Interacciones Espermatozoide-Óvulo , Estrellas de Mar/genética , Cigoto/ultraestructuraRESUMEN
BACKGROUND: Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca(2+) and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca(2+) signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation. METHODOLOGY/PRINCIPAL FINDINGS: By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPgammaS) or inactive (GDPbetaS) forms of G-proteins, we have demonstrated that: i) GTPgammaS induces Ca(2+) release that mimics the effect of 1-MA; ii) GDPbetaS completely blocks 1-MA-induced Ca(2+); iii) GDPbetaS has little effect on the amplitude of the Ca(2+) peak, but significantly expedites the initial Ca(2+) waves induced by InsP(3) photoactivation, iv) GDPbetaS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca(2+) release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPbetaS, or actinase E, all led to significant changes of 1-MA-induced Ca(2+) signaling. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca(2+) release.
Asunto(s)
Actinas/metabolismo , Señalización del Calcio/fisiología , Citoesqueleto/fisiología , Nucleótidos de Guanina/fisiología , Meiosis/fisiología , Oocitos/citología , Animales , Microinyecciones , Microscopía Confocal , Estrellas de MarRESUMEN
Ca2+ is the most universal second messenger in cells from the very first moment of fertilization. In all animal species, fertilized eggs exhibit massive mobilization of intracellular Ca2+ to orchestrate the initial events of development. Echinoderm eggs have been an excellent model system for studying fertilization and the cell cycle due to their large size and abundance. In preparation for fertilization, the cell cycle-arrested oocytes must undergo meiotic maturation. Studies of starfish oocytes have shown that Ca2+ signaling is intimately involved in this process. Our knowledge of the molecular mechanism of meiotic maturation and fertilization has expanded greatly in the past two decades due to the discovery of cell cycle-related kinases and Ca2+-mobilizing second messengers. However, the molecular details of their actions await elucidation of other cellular elements that assist in the creation and transduction of Ca2+ signals. In this regard, the actin cytoskeleton, the receptors for second messengers and the Ca2+-binding proteins also require more attention. This article reviews the physiological significance and the mechanism of intracellular Ca2+ mobilization in starfish oocytes during maturation and fertilization.
Asunto(s)
Actinas/fisiología , Citoesqueleto/fisiología , Fertilización , Oocitos/fisiología , Animales , Calcio/metabolismo , Señalización del Calcio , ADP-Ribosa Cíclica/metabolismo , Exocitosis , Fertilización In Vitro , Meiosis , Modelos Biológicos , Contracción Muscular , Oocitos/metabolismo , Transducción de Señal , Estrellas de MarRESUMEN
Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP(3)-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP(3), despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis.
Asunto(s)
Señalización del Calcio , Citoesqueleto/metabolismo , Oocitos/citología , Oocitos/metabolismo , Actinas , Animales , Exocitosis , Meiosis , Estrellas de MarRESUMEN
Periaxoplasmic ribosomal plaques (PARPs) are periodic structural formations containing ribosomes, which are likely cortical sites of translation along myelinated fibers. beta-actin mRNA, and its trans-acting binding factor, zipcode-binding protein-1, were co-distributed within PARP domains of axoplasmic whole-mounts isolated from goldfish Mauthner, rabbit and rat nerve fibers. The distribution of co-localization signals of fluorophore pixels, however, was asymmetric in PARP domains, possibly indicative of endpoint trafficking of RNPs. beta-actin mRNA in RNA extracted from axoplasm of single Mauthner fibers was confirmed by RT-PCR. A metabolically active isolated Mauthner fiber system, which required cAMP to activate translation, was developed in order to probe cycloheximide-sensitivity, and the importance of the actin cytoskeleton. cAMP greatly stimulated protein synthesis in axoplasm after a period of pre-incubation, while being inhibited strongly by cycloheximide, or by cytochalasin D. Cytochalasin D reduced incorporation only modestly in the associated myelin sheath. We conclude that mechanisms for targeting and localizing beta-actin mRNA to discrete PARP domains are probably similar to those described for dendritic synaptic domains. Moreover, optimal translation in axoplasm depends on the integrity of the actin cytoskeleton, and can be modulated by cAMP as well.