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Purpose: To determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5. Methods: Five patients (ages 6-31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease. Results: Patients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue. Conclusions: LCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.
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Dependovirus/genética , Proteínas del Ojo/genética , Terapia Genética , Amaurosis Congénita de Leber/terapia , Proteínas Asociadas a Microtúbulos/genética , Retina/fisiopatología , Adulto , Animales , Niño , Modelos Animales de Enfermedad , Electrorretinografía , Femenino , Terapia Genética/métodos , Vectores Genéticos , Humanos , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Imagen Óptica , Fenotipo , Pupila/fisiología , Tomografía de Coherencia Óptica , Agudeza Visual/fisiología , Pruebas del Campo Visual , Campos Visuales/fisiología , Adulto JovenRESUMEN
Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.
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Mutations of the SPAST gene, which encodes the microtubule-severing protein spastin, are the most common cause of hereditary spastic paraplegia (HSP). Haploinsufficiency is the prevalent opinion as to the mechanism of the disease, but gain-of-function toxicity of the mutant proteins is another possibility. Here, we report a new transgenic mouse (termed SPASTC448Y mouse) that is not haploinsufficient but expresses human spastin bearing the HSP pathogenic C448Y mutation. Expression of the mutant spastin was documented from fetus to adult, but gait defects reminiscent of HSP (not observed in spastin knockout mice) were adult onset, as is typical of human patients. Results of histological and tracer studies on the mouse are consistent with progressive dying back of corticospinal axons, which is characteristic of the disease. The C448Y-mutated spastin alters microtubule stability in a manner that is opposite to the expectations of haploinsufficiency. Neurons cultured from the mouse display deficits in organelle transport typical of axonal degenerative diseases, and these deficits were worsened by depletion of endogenous mouse spastin. These results on the SPASTC448Y mouse are consistent with a gain-of-function mechanism underlying HSP, with spastin haploinsufficiency exacerbating the toxicity of the mutant spastin proteins. These findings reveal the need for a different therapeutic approach than indicated by haploinsufficiency alone.
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Paraplejía Espástica Hereditaria/genética , Espastina/genética , Animales , Transporte Axonal/fisiología , Axones/metabolismo , Modelos Animales de Enfermedad , Mutación con Ganancia de Función/genética , Haploinsuficiencia , Haplotipos , Ratones , Ratones Transgénicos , Microtúbulos/metabolismo , Proteínas Mutantes/genética , Mutación , Neuronas/metabolismo , Paraplejía Espástica Hereditaria/fisiopatología , Espastina/fisiologíaRESUMEN
Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an â¼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.
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Ceguera/metabolismo , Ceguera/terapia , Dependovirus/genética , Terapia Genética/métodos , Animales , Electrorretinografía , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Femenino , Humanos , Amaurosis Congénita de Leber/metabolismo , Amaurosis Congénita de Leber/terapia , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Choroideremia (CHM) is a rare monogenic, X-linked recessive inherited retinal degeneration resulting from mutations in the Rab Escort Protein-1 (REP1) encoding CHM gene. The primary retinal cell type leading to CHM is unknown. In this study, we explored the utility of induced pluripotent stem cell-derived models of retinal pigmented epithelium (iPSC-RPE) to study disease pathogenesis and a potential gene-based intervention in four different genetically distinct forms of CHM. A number of abnormal cell biologic, biochemical, and physiologic functions were identified in the CHM mutant cells. We then identified a recombinant adeno-associated virus (AAV) serotype, AAV7m8, that is optimal for both delivering transgenes to iPSC-RPEs as well as to appropriate target cells (RPE cells and rod photoreceptors) in the primate retina. To establish the proof of concept of AAV7m8 mediated CHM gene therapy, we developed AAV7m8.hCHM, which delivers the human CHM cDNA under control of CMV-enhanced chicken ß-actin promoter (CßA). Delivery of AAV7m8.hCHM to CHM iPSC-RPEs restored protein prenylation, trafficking and phagocytosis. The results confirm that AAV-mediated delivery of the REP1-encoding gene can rescue defects in CHM iPSC-RPE regardless of the type of disease-causing mutation. The results also extend our understanding of mechanisms involved in the pathophysiology of choroideremia.
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Coroideremia/metabolismo , Coroideremia/patología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Epitelio Pigmentado de la Retina/citología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Dependovirus/genética , Técnica del Anticuerpo Fluorescente , Humanos , Fagocitosis/fisiología , PrimatesRESUMEN
Cornelia de Lange syndrome (CdLS) is a complex disorder with multiple structural and developmental defects caused by mutations in structural and regulatory proteins involved in the cohesin complex. NIPBL, a cohesin regulatory protein, has been identified as a critical protein responsible for the orchestration of transcriptomic regulatory networks necessary for embryonic development. Mutations in NIPBL are responsible for the majority of cases of CdLS. Through RNA-sequencing of human induced pluripotent stem cells and in vitro-derived cardiomyocytes, we identified hundreds of mRNAs, pseudogenes, and non-coding RNAs with altered expression in NIPBL+/- patient-derived cells. We demonstrate that NIPBL haploinsufficiency leads to upregulation of gene sets identified in functions related to nucleosome, chromatin assembly, RNA modification and downregulation of Wnt signaling, cholesterol biosynthesis and vesicular transport in iPSC and cardiomyocytes. Mutations in NIPBL result in the dysregulation of many genes responsible for normal heart development likely resulting in the variety of structural cardiac defects observed in the CdLS population.
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Diferenciación Celular/genética , Regulación de la Expresión Génica , Haploinsuficiencia , Mioblastos Cardíacos/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas/genética , Transcriptoma , Biomarcadores , Proteínas de Ciclo Celular , Biología Computacional/métodos , Síndrome de Cornelia de Lange/genética , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Genotipo , Cardiopatías Congénitas/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mioblastos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citologíaRESUMEN
Mutations of various genes cause hereditary spastic paraplegia (HSP), a neurological disease involving dying-back degeneration of upper motor neurons. From these, mutations in the SPAST gene encoding the microtubule-severing protein spastin account for most HSP cases. Cumulative genetic and experimental evidence suggests that alterations in various intracellular trafficking events, including fast axonal transport (FAT), may contribute to HSP pathogenesis. However, the mechanisms linking SPAST mutations to such deficits remain largely unknown. Experiments presented here using isolated squid axoplasm reveal inhibition of FAT as a common toxic effect elicited by spastin proteins with different HSP mutations, independent of microtubule-binding or severing activity. Mutant spastin proteins produce this toxic effect only when presented as the tissue-specific M1 isoform, not when presented as the ubiquitously-expressed shorter M87 isoform. Biochemical and pharmacological experiments further indicate that the toxic effects of mutant M1 spastins on FAT involve casein kinase 2 (CK2) activation. In mammalian cells, expression of mutant M1 spastins, but not their mutant M87 counterparts, promotes abnormalities in the distribution of intracellular organelles that are correctable by pharmacological CK2 inhibition. Collectively, these results demonstrate isoform-specific toxic effects of mutant M1 spastin on FAT, and identify CK2 as a critical mediator of these effects.
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Adenosina Trifosfatasas/genética , Transporte Axonal/genética , Adenosina Trifosfatasas/metabolismo , Animales , Transporte Axonal/fisiología , Quinasa de la Caseína II/metabolismo , Células Cultivadas , Decapodiformes , Modelos Animales de Enfermedad , Fibroblastos , Humanos , Microtúbulos/metabolismo , Neuronas Motoras/metabolismo , Proteínas Mutantes/metabolismo , Mutación , Isoformas de Proteínas/genética , Transporte de Proteínas/fisiología , Ratas , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismo , EspastinaRESUMEN
Neurons are terminally differentiated cells that use their microtubule arrays not for cell division but rather as architectural elements required for the elaboration of elongated axons and dendrites. In addition to acting as compression-bearing struts that provide for the shape of the neuron, microtubules also act as directional railways for organelle transport. The stability properties of neuronal microtubules are commonly discussed in the biomedical literature as crucial to the development and maintenance of the nervous system, and have recently gained attention as central to the etiology of neurodegenerative diseases. Drugs that affect microtubule stability are currently under investigation as potential therapies for disease and injury of the nervous system. There is often a lack of consistency, however, in how the issue of microtubule stability is discussed in the literature, and this can affect the design and interpretation of experiments as well as potential therapeutic regimens. Neuronal microtubules are considered to be more stable than microtubules in dividing cells. On average, this is true, but in addition to an abundant stable microtubule fraction in neurons, there is also an abundant labile microtubule fraction. Both are functionally important. Individual microtubules consist of domains that differ in their stability properties, and these domains can also differ markedly in their composition as well as how they interact with various microtubule-related proteins in the neuron. Myriad proteins and pathways have been discussed as potential contributors to microtubule stability in neurons. © 2016 Wiley Periodicals, Inc.
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División Celular/fisiología , Microtúbulos/metabolismo , Neuronas/metabolismo , Animales , Humanos , Neuronas/citologíaRESUMEN
Testing functional hypotheses on the roles played by individual microtubule-related proteins in developing neurons benefits from having an effective means for depleting an individual protein from a population of cultured vertebrate neurons over an appropriate window. Small interfering RNA (siRNA) has the advantage of high transfection efficiency, but has certain limitations that require strategic experimental design. Here we describe the insights that we have gained over the years from using this approach.
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Axones/metabolismo , Corteza Cerebral/citología , Dendritas/metabolismo , Hipocampo/citología , Proteínas Asociadas a Microtúbulos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Ratas , TransfecciónRESUMEN
Individual microtubules (MTs) in the axon consist of a stable domain that is highly acetylated and a labile domain that is not. Traditional MT-severing proteins preferentially cut the MT in the stable domain. In Drosophila, fidgetin behaves in this fashion, with targeted knockdown resulting in neurons with a higher fraction of acetylated (stable) MT mass in their axons. Conversely, in a fidgetin knockout mouse, the fraction of MT mass that is acetylated is lower than in the control animal. When fidgetin is depleted from cultured rodent neurons, there is a 62% increase in axonal MT mass, all of which is labile. Concomitantly, there are more minor processes and a longer axon. Together with experimental data showing that vertebrate fidgetin targets unacetylated tubulin, these results indicate that vertebrate fidgetin (unlike its fly ortholog) regulates neuronal development by tamping back the expansion of the labile domains of MTs.
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Axones/fisiología , Proteínas de Drosophila/metabolismo , Microtúbulos/fisiología , Proteínas Nucleares/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Drosophila , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos , Microtúbulos/metabolismo , Datos de Secuencia Molecular , RatasRESUMEN
Müller cell activation is an early finding in diabetic retinopathy (DR), but its physiopathologic role in the disease is still unclear, especially in the early phases. We investigated on Müller glial activation in primary rat retinal cultures, exposed to High Glucose (HG), and in retinas from streptozotocin (stz)-induced diabetic rats. First of all, we checked if the presence of Müller glia influenced HG neurotoxicity. In mixed glial/neuronal retinal cultures, a single HG administration (sHG) for 48 h induced activation of Müller glia, in absence of neuronal damage. In contrast, in pure neuronal cultures, a marked neurotoxic effect was detected, suggesting that in this cell model Müller glia protect neurons from HG neurotoxicity. To better mimic the diabetic milieu, where retinal cells are constantly bathed in hyperglycemic fluid, and to further characterize astrocytic neuroprotective ability, mixed retinal cultures were exposed to repeated daily replacement of HG (rHG). In this paradigm, starting from 48 h, increased apoptosis and synaptic loss were observed, even in the presence of Müller cells. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whose activation triggers a prosurvival pathway, was increased by sHG, while it was down-regulated by rHG, suggesting that ERK1/2 activation is involved in neuroprotection. Consistently, in presence of ERK1/2 inhibitor PD98059, sHG exerted a proapoptotic effect also in glial/neuronal retinal cultures. In line with the in vitro data, early changes in diabetic retinas from stz-injected rats included Müller cell activation and increased pERK1/2 levels, but no signs of neuronal damage. These results suggest that, in the early phases of DR, Müller glial activation does not contribute to neurodegeneration, but may indeed have a neuroprotective activity against HG-induced neurotoxicity through a mechanism involving pERK1/2.
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Diabetes Mellitus Experimental , Retinopatía Diabética/fisiopatología , Células Ependimogliales/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Retina/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Retinopatía Diabética/inducido químicamente , Células Ependimogliales/efectos de los fármacos , Glucosa/toxicidad , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
PURPOSE: Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDA-induced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). METHODS: NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca(2+)](i). RESULTS: Curcumin dose- and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDA-induced [Ca(2+)](i) rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca(2+)](i). The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. CONCLUSIONS: These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs.
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Curcumina/farmacología , Hipocampo/efectos de los fármacos , N-Metilaspartato/toxicidad , Fármacos Neuroprotectores/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Calcio/metabolismo , Células Cultivadas , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Agonistas de Aminoácidos Excitadores/toxicidad , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Hipocampo/embriología , Hipocampo/metabolismo , Hipocampo/patología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ácido Kaínico/toxicidad , Técnicas de Placa-Clamp , Embarazo , Ratas , Ratas Wistar , Retina/embriología , Retina/metabolismo , Retina/patología , Factores de Tiempo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/toxicidadRESUMEN
The early effects of the diabetic milieu on retinal tissue and their relation to the Renin-Angiotensin system (RAS) activation are poorly known. Here we investigated RAS signaling in retinas explanted from adult rats exposed for 48 h to high glucose (HG), with or without the Angiotensin Converting Enzyme inhibitor enalaprilat, which blocks RAS. HG was observed to i) initiate a phosphotyrosine-dependent signaling cascade; ii) up-regulate Angiotensin(1) Receptor (AT(1)R); iii) activate src tyrosine kinase and increase phosphorylation of Pyk2, PLCgamma1 and ERK1/2; and iv) activate Akt and the transcription factor CREB. In the presence of enalaprilat, tyrosine phosphorylation signal and AT(1)R upregulation decreased and activation of PLCgamma1 and CREB reverted, showing their relation to RAS signaling. In line with Akt activation, no apoptosis or synapse degeneration was found. Müller glia was activated, but in a RAS-independent manner. Our results suggest that, in early phases of HG exposure, a pro-survival cell program may be induced in the retina.