RESUMEN
The isomerization of all-trans retinol (vitamin A) to 11-cis retinol in the retinal pigment epithelium (RPE) is a key step in the visual process for the regeneration of the visual pigment chromophore, 11-cis retinal. LRAT and RPE65 are recognized as the minimal isomerase catalytic components. However, regulators of this rate-limiting step are not fully identified and could account for the phenotypic variability associated with inherited retinal degeneration (RD) caused by mutations in the RPE65 gene. To identify new RPE65 partners, we screened a porcine RPE mRNA library using a yeast two-hybrid assay with full-length human RPE65. One identified clone (here named FATP1c), containing the cytosolic C-terminal sequence from the fatty acid transport protein 1 (FATP1 or SLC27A1, solute carrier family 27 member 1), was demonstrated to interact dose-dependently with the native RPE65 and with LRAT. Furthermore, these interacting proteins colocalize in the RPE. Cellular reconstitution of human interacting proteins shows that FATP1 markedly inhibits 11-cis retinol production by acting on the production of all-trans retinyl esters and the isomerase activity of RPE65. The identification of this new visual cycle inhibitory component in RPE may contribute to further understanding of retinal pathogenesis.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Ojo/metabolismo , Proteínas de Transporte de Ácidos Grasos/metabolismo , Vitamina A/antagonistas & inhibidores , Animales , Glutatión Transferasa/metabolismo , Humanos , Insectos , Ratones , Fenotipo , Retina/metabolismo , Fracciones Subcelulares/metabolismo , Porcinos , Factores de Tiempo , Técnicas del Sistema de Dos Híbridos , Vitamina A/química , cis-trans-IsomerasasRESUMEN
Systemic anticancer chemotherapy is immunosuppressive and mostly induces nonimmunogenic tumor cell death. Here, we show that even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit an effective antitumor immune response that suppresses the growth of inoculated tumors or leads to the regression of established neoplasia. Although both antracyclins and mitomycin C induced apoptosis with caspase activation, only anthracyclin-induced immunogenic cell death was immunogenic. Caspase inhibition by Z-VAD-fmk or transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin (DX)-induced cell death, yet suppressed the immunogenicity of dying tumor cells in several rodent models of neoplasia. Depletion of dendritic cells (DCs) or CD8+T cells abolished the immune response against DX-treated apoptotic tumor cells in vivo. Caspase inhibition suppressed the capacity of DX-killed cells to be phagocytosed by DCs, yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors became immunogenic upon DX treatment in vitro, and intratumoral inoculation of DX could trigger the regression of established tumors in immunocompetent mice. These results delineate a procedure for the generation of cancer vaccines and the stimulation of anti-neoplastic immune responses in vivo.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Doxorrubicina/farmacología , Mitomicina/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Antibióticos Antineoplásicos/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Inhibidores de Caspasas , Línea Celular Tumoral , Células Dendríticas/inmunología , Doxorrubicina/uso terapéutico , Immunoblotting , Etiquetado Corte-Fin in Situ , Ratones , Mitomicina/uso terapéutico , Neoplasias/prevención & control , Ratas , Vacunación/métodos , Proteínas Virales/genética , Proteínas Virales/farmacologíaRESUMEN
PURPOSE: Electroretinography (ERG) is a widely used technique to test retinal function in humans and animals. Recordings are particularly dependent on the type of electrodes used, with the best electrodes often being expensive and not always easy to use. The need of a simple and effective electrode type has led us to search the efficacy of different types of electrodes used in practice and compare them with the modified cotton wick electrode. MATERIAL AND METHODS: A modified type of electrode made of a cotton wick and impregnated with NaCl is described, and the ERG results were compared with other types of electrodes. RESULTS: Compared with standard metal wire loop electrodes, the cotton wick electrode results in obtaining higher amplitudes, a better inter-eye correlation in the same animal and a better reproducibility of the recordings over time. CONCLUSION: This cotton electrode is simple to make and easy to place. It provides reliable recordings during the entire life span of the animal and reliable comparisons between contralateral eyes, thus providing a powerful tool for ERG studies.
Asunto(s)
Fibra de Algodón , Electrodos , Electrorretinografía/instrumentación , Retina/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Ratas , Reproducibilidad de los ResultadosRESUMEN
We investigated the molecular determinants of Ca(2+)-activated chloride current (CaCC) expressed in adult sensory neurons after a nerve injury. Dorsal root ganglia express the transcripts of three gene families known to induce CaCCs in heterologous systems: bestrophin, tweety, and TMEM16. We found with quantitative transcriptional analysis and in situ hybridization that nerve injury induced upregulation of solely bestrophin-1 transcripts in sensory neurons. Gene screening with RNA interference in single neurons demonstrated that mouse Best1 is required for the expression of CaCC in injured sensory neurons. Transfecting injured sensory neurons with bestrophin-1 mutants inhibited endogenous CaCC. Exogenous expression of the fusion protein green fluorescent protein-Bestrophin-1 in naive neurons demonstrated a plasma membrane localization of the protein that generates a CaCC with biophysical and pharmacological properties similar to endogenous CaCC. Our data suggest that Best1 belongs to a group of genes upregulated by nerve injury and supports functional CaCC expression in injured sensory neurons.
Asunto(s)
Calcio/metabolismo , Cloruros/metabolismo , Proteínas del Ojo/metabolismo , Ganglios Espinales/fisiología , Nervio Ciático/lesiones , Células Receptoras Sensoriales/fisiología , Animales , Bestrofinas , Membrana Celular/metabolismo , Proteínas del Ojo/genética , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Canales Iónicos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Técnicas de Placa-Clamp , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven-transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debré-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-betagal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns(-/-) mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns(-/-) mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns(-/-) mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns(-/-) mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis.
Asunto(s)
Cistina/metabolismo , Cistinosis/genética , Glicoproteínas , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Alelos , Sistemas de Transporte de Aminoácidos Neutros , Animales , Huesos/diagnóstico por imagen , Huesos/patología , Cistinosis/diagnóstico por imagen , Cistinosis/metabolismo , Citosina/metabolismo , Perros , Electrorretinografía , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Genéticos , Regiones Promotoras Genéticas , Radiografía , Proteínas Recombinantes/metabolismo , Retina/anomalías , Retina/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Distribución TisularRESUMEN
The susceptibility of cells to apoptosis induction is deeply influenced by their position in the cell cycle. Unfortunately, however, current methods for the enrichment of cells in defined phases of the cell cycle are mostly based on the synchronization of cells by agents or conditions that are intrinsically toxic and induce apoptosis on their own. We developed a novel procedure for the purification of cells in distinct phases of the cell cycle. This method is based on the stable transfection of cells with a chimeric protein made up by histone H2B and green fluorescent protein (GFP). Cytofluorometric purification of cells defined by their size and their H2B-GFP-dependent fluorescence (which reflects chromatin and hence DNA content) allowed for the efficient separation of diploid and tetraploid cells in the fluorescence-activated cell sorter (FACS). Moreover, when applied to diploid cells, this method allowed for the enrichment of live, functional cells in the G1, S and G2 phases of the cell cycle. FACS-purified cells were viable and readily resumed the cell cycle upon reculture. While staurosporine was equally toxic for cells in any phase of the cell cycle, camptothecin was particularly toxic for cells in the S phase. Moreover, BAY11-7082, a specific inhibitor of the IKK complex required for NF-kappaB activation, exhibited a particular cell cycle-specific profile of toxicity (G2>S>G1). These results delineate a novel procedure for studying the intersection between cell cycle regulation and cell death mechanisms.
Asunto(s)
Apoptosis , Neoplasias del Colon/patología , Citometría de Flujo/métodos , Interfase , Apoptosis/efectos de los fármacos , Bromodesoxiuridina/metabolismo , Camptotecina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Interfase/efectos de los fármacos , Nitrilos/farmacología , Ploidias , Estaurosporina/farmacología , Sulfonas/farmacologíaRESUMEN
The class-B type-I scavenger receptor (SR-BI) plays a key role in cholesterol homeostasis; it mediates the selective uptake of lipoprotein cholesterol to steroidogenic tissues. We show by RT-PCR, western blot, in situ hybridization and immunohistochemistry analysis that SR-BI is highly expressed in different neuro-retinal and non-neuronal cells types on rat eye. Immunohistochemistry of the steroidogenic acute regulatory protein (StAR) involved in neurosteroid production showed the same expression pattern than SR-BI in rat eye. Our results may suggest a key role of these genes in the ocular cholesterol metabolism for membranes biosynthesis and neurosteroidogenesis.
Asunto(s)
Fenómenos Fisiológicos Oculares , Fosfoproteínas/genética , Receptores Inmunológicos/genética , Animales , Encéfalo/fisiología , Antígenos CD36 , Regulación de la Expresión Génica , Hibridación in Situ , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Ratas Mutantes , Receptores Depuradores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores Depuradores de Clase B , Transcripción GenéticaRESUMEN
Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM (-/y) patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC-derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model.
RESUMEN
FATP1 is involved in lipid transport into cells and in intracellular lipid metabolism. We showed previously that this protein interacts with and inhibits the limiting-step isomerase of the visual cycle RPE65. Here, we aimed to analyze the effect of Fatp1-deficiency in vivo on the visual cycle, structure and function, and on retinal aging. Among the Fatp family members, we observed that only Fatp1 and 4 are expressed in the control retina, in both the neuroretina and the retinal pigment epithelium. In the neuroretina, Fatp1 is mostly expressed in photoreceptors. In young adult Fatp1(-/-) mice, Fatp4 expression was unchanged in retinal pigment epithelium and reduced two-fold in the neuroretina as compared to Fatp1(+/+) mice. The Fatp1(-/-) mice had a preserved retinal structure but a decreased electroretinogram response to light. These mice also displayed a delayed recovery of the b-wave amplitude after bleaching, however, visual cycle speed was unchanged, and both retinal pigment epithelium and photoreceptors presented the same fatty acid pattern compared to controls. In 2 year-old Fatp1(-/-) mice, transmission electron microscopy studies showed specific abnormalities in the retinas comprising choroid vascularization anomalies and thickening of the Bruch membrane with material deposits, and sometimes local disorganization of the photoreceptor outer segments. These anomalies lead us to speculate that the absence of FATP1 accelerates the aging process.
Asunto(s)
Envejecimiento/genética , Adaptación a la Oscuridad/fisiología , Proteínas de Transporte de Ácidos Grasos/metabolismo , Luz , Retina/efectos de la radiación , Envejecimiento/fisiología , Animales , Cartilla de ADN/genética , Adaptación a la Oscuridad/genética , Electrorretinografía , Proteínas de Transporte de Ácidos Grasos/deficiencia , Ácidos Grasos/metabolismo , Fluorescencia , Técnicas Histológicas , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa , Retina/metabolismo , Retina/ultraestructura , Epitelio Pigmentado de la Retina/metabolismo , Rodopsina/metabolismo , Estadísticas no Paramétricas , cis-trans-Isomerasas/metabolismoRESUMEN
Tetraploidy can result in cancer-associated aneuploidy. As shown here, freshly generated tetraploid cells arising due to mitotic slippage or failed cytokinesis are prone to undergo Bax-dependent mitochondrial membrane permeabilization and subsequent apoptosis. Knockout of Bax or overexpression of Bcl-2 facilitated the survival of tetraploid cells at least as efficiently as the p53 or p21 knockout. When tetraploid cells were derived from diploid p53 and Bax-proficient precursors, such cells exhibited an enhanced transcription of p53 target genes. Tetraploid cells exhibited an enhanced rate of spontaneous apoptosis that could be suppressed by inhibition of p53 or by knockdown of proapoptotic p53 target genes such as BBC3/Puma, GADD45A and ferredoxin reductase. Unexpectedly, tetraploid cells were more resistant to DNA damaging agents (cisplatin, oxaliplatin and camptothecin) than their diploid counterparts, and this difference disappeared upon inhibition of p53 or knockdown of p53-inducible ribonucleotide reductase. Tetraploid cells were also more resistant against UVC and gamma-irradiation. These data indicate the existence of p53-dependent alterations in apoptosis regulation in tetraploid cells.
Asunto(s)
Apoptosis/fisiología , Neoplasias/genética , Poliploidía , Animales , Antineoplásicos/metabolismo , Línea Celular Tumoral , Cisplatino/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Daño del ADN , Femenino , Eliminación de Gen , Humanos , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Nocodazol/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.