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PURPOSE: To determine the differential profiles of sphingomyelin, sphingoid base, sphingoid base-1-phosphate and ceramide lipid species and their quantitative differences between control and glaucomatous aqueous humor (AQH) derived from human donors. METHODS: AQH from control and primary open-angle glaucoma donors was collected and subjected to lipid extraction using suitable modifications of the Bligh and Dyer method. Proteins were estimated using Bradford's method. Lipids were identified and ratiometrically quantified in a two-step process using precursor ion scan or neutral loss scan (NLS) with appropriate class-specific lipid standards on a TSQ Quantum Access Max mass spectrometer following established procedures. Primary human trabecular meshwork cells and video microscopic imaging were used to assess changes in cell shape and motility upon exposure to 20 pmol of Cer(d18:0/18:1(9Z)) in 10% dimethyl sulfoxide (vehicle). RESULTS: We identified several species of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramides that were common between control and glaucomatous AQH. Some unique lipid species in these classes were also identified in controls but not in glaucoma and vice versa. We found exposure to 20 pmol of Cer(d18:0/18:1(9Z)) resulted in changes in the trabecular meshwork cell shape and observed motility changes compared to vehicle-only control. CONCLUSIONS: Most lipids belonging to the sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramide species were common between control and primary open-angle glaucoma donors. However, some sphingolipids and ceramides were found to be uniquely present in control but absent in the glaucomatous AQH and vice versa. Identification of unique lipid species present or absent in the pathophysiological context may contribute further insight into glaucoma pathology.
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Humor Acuoso/metabolismo , Ceramidas/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Movimiento Celular , Forma de la Célula , Ceramidas/química , Femenino , Glaucoma/metabolismo , Glaucoma/patología , Humanos , Masculino , Persona de Mediana Edad , Espectrometría de Masa por Ionización de Electrospray , Esfingomielinas/química , Esfingomielinas/metabolismo , Espectrometría de Masas en Tándem , Donantes de Tejidos , Malla Trabecular/metabolismo , Malla Trabecular/patologíaRESUMEN
Background: Glaucoma is one of the main causes of irreversible visual field loss and blindness worldwide. Vision loss in this multifactorial neurodegenerative disease results from progressive degeneration of retinal ganglion cells (RGCs) and their axons. Identifying molecular markers that can be measured objectively and quantitatively may provide essential insights into glaucoma diagnosis and enhance pathophysiology understanding. Methods: The chronic, progressive DBA/2J glaucomatous mouse model of glaucoma and C57BL6/J optic nerve crush (ONC) mouse model were used in this study. Changes in PVALB expression with RGC and optic nerve degeneration were assessed via gene expression microarray analysis, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot and immunohistochemistry. Results: Microarray analysis of the retinal gene expression in the DBA/2J mice at different ages showed that the expression of PVALB was downregulated as the mice aged and developed glaucoma with retinal ganglion cell loss. Analysis of qRT-PCR results demonstrated PVALB at the mRNA level was reduced in the retinas and optic nerves of old DBA/2J mice and in those after ONC compared to baseline young DBA2/J mice. PVALB protein expression measured by Western blot was also significantly reduced signal in the retinas and optic nerves of old DBA/2J mice and those eyes with crushed nerves. Immunohistochemical staining results demonstrated that there were fewer PVALB-positive cells in the ganglion cell layer (GCL) of the retina and staining pattern changed in the optic nerve from old DBA/2J mice as well as in mice eyes following ONC. Conclusion: PVALB is abundantly expressed both by RGCs' soma in the retinas and RGCs' axons in the optic nerves of C57BL/6J. Furthermore, the expression level of PVALB decreases with RGC degeneration in the glaucomatous DBA/2J mice and after ONC injury of C57BL6/6J, indicating that PVALB is a reliable RGC molecular marker that can be used to study retinal and optic nerve degeneration.
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Pinin (Pnn), a nuclear speckle-associated protein, has been shown to function in maintenance of epithelial integrity through altering expression of several key adhesion molecules. Here we demonstrate that Pnn plays a crucial role in small intestinal development by influencing expression of an intestinal homeobox gene, Cdx2. Conditional inactivation of Pnn within intestinal epithelia resulted in significant downregulation of a caudal type homeobox gene, Cdx2, leading to obvious villus dysmorphogenesis and severely disrupted epithelial differentiation. Additionally, in Pnn-deficient small intestine, we observed upregulated Tcf/Lef reporter activity, as well as misregulated expression/distribution of beta-catenin and Tcf4. Since regulation of Cdx gene expression has been closely linked to Wnt/beta-catenin signaling activity, we explored the possibility of Pnn's interaction with beta-catenin, a major effector of the canonical Wnt signaling pathway. Co-immunoprecipitation assays revealed that Pnn, together with its interaction partner CtBP2, a transcriptional co-repressor, was in a complex with beta-catenin. Moreover, both of these proteins were found to be recruited to the proximal promoter area of Cdx2. Taken together, our results suggest that Pnn is essential for tight regulation of Wnt signaling and Cdx2 expression during small intestinal development.
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Moléculas de Adhesión Celular/metabolismo , Genes Homeobox , Proteínas de Homeodominio/genética , Intestino Delgado/crecimiento & desarrollo , Morfogénesis/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Animales , Factor de Transcripción CDX2 , Moléculas de Adhesión Celular/genética , Proteínas de Unión al ADN , Embrión de Mamíferos/metabolismo , Proteínas de Homeodominio/metabolismo , Ratones , Proteínas Nucleares/genética , Transducción de Señal , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
PURPOSE: To determine retinal vesicular glutamate transporter 3 (VGLUT3) expression alterations in a mouse model of progressive optic neuropathy (glaucoma). METHODS: Tissue specimens were obtained from age-matched DBA/2J and control C57BL/6J mice for western blot analysis. Enucleated globes from DBA/2J, C57BL/6J, and BALB/cJ mice were fixed in formalin, paraffin-embedded, and sectioned for VGLUT3 protein localization. RESULTS: western blot analysis of the control retinas revealed the expression of a ~55 kDa immunoreactive VGLUT3 protein that is to be expected in tissues such as retina, brain, liver, heart, and kidney tissue, but not in intestinal or lung tissue. Furthermore, a strong ~130 kDa immunoreactive VGLUT3 isoform that is restricted to the central nervous system (the brain and retinas) was also identified in the controls, but was not detected in the DBA/2J retinas. Immunofluorescence microscopy showed a lack of VGLUT3 expression in the synapses between amacrine and retinal ganglion cells in DBA/2J retinas, in contrast to its strong expression in the C57BL/6J and BALB/cJ controls. CONCLUSIONS: Our results implicate the dysregulated expression of a central nervous system-specific VGLUT3 isoform as a predisposing factor in the development of optic neuropathy in DBA/2J mice, a spontaneous mouse model of glaucoma. In striking parallel to the visual system defects of glaucomatous DBA/2J mice, the inner ear of VGLUT3 knockout mice displays a progressive loss of inner hair cell to spiral-ganglion neuron synapses. A significant reduction in the number of spiral-ganglion neurons leads to age-associated deafness. Thus, we propose that the absence of this biochemically uncharacterized 130 kDa VGLUT3 isoform in the DBA/2J retina is a predisposing factor in synaptic instability, and a contributing factor in the age-dependent and progressive loss of ganglion cells projecting to the brain.
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Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Regulación de la Expresión Génica , Enfermedades del Nervio Óptico/metabolismo , Animales , Encéfalo/metabolismo , Sistema Nervioso Central/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microscopía Fluorescente/métodos , Isoformas de Proteínas , Retina/metabolismo , Especificidad de la EspecieRESUMEN
PURPOSE: Pinin (Pnn/DRS/memA) plays an important role in regulating cell-cell adhesion of corneal epithelial cells. In the nucleus, Pnn interacts with both transcriptional repressor and pre-mRNA processing machinery. Here we investigated the consequences of "knocking down" Pnn expression with short hairpin RNAi (shRNAi) on the corneal epithelial cell phenotype. METHODS: Cultured human corneal epithelial (HCE-T) cells were cotransfected with a shRNAi-expressing construct containing an inverted repeat of a Pnn specific 21 nucleotide sequence (Pnn shRNAi) and a GFP vector as a marker of transfected cells. After 24-48 h, cells were fixed and immunostained with antibodies against Pnn, keratin, desmoplakin, desmoglein, E-cadherin, ZO-1, SR-proteins, and SRm300. To demonstrate specificity of the Pnn knock down, a rescue vector was designed by incorporating three conservative nucleotide substitutions within the Pnn-shRNAi targeting sequences of the full length Pnn-GFP construct, thus generating a Pnn construct to produce mRNA that Pnn shRNAi could not target (Pnn-CS3-GFP). RESULTS: HCE-T cells were cotransfected with Pnn shRNAi and GFP vectors and after 24 and 48 h exhibited significantly reduced immunostaining for Pnn. Western blot analyses of Pnn and E-cadherin protein expression in cells transfected with Pnn-shRNAi and GFP vectors revealed marked reduction in levels of both proteins compared to those observed in cells transfected with GFP alone. The cells receiving Pnn-shRNAi appeared to be less adherent to neighboring nontransfected cells, often exhibited altered cell shape, downregulated cell adhesion and cell junction molecules, and escaped from the epithelium. The Pnn shRNAi transfected cells exhibited fewer keratin filaments anchored to desmosomes and a concurrent increase in the perinuclear bundling of filaments. SR proteins and SRm300 showed an altered distribution in the Pnn knock down cells. Cotransfection of Pnn-CS3-GFP with Pnn shRNAi demonstrated that the conservatively mutated Pnn could maintain cell-cell adhesion. CONCLUSIONS: Our results indicate that knocking down Pnn expression leads to a loss of epithelial cell-cell adhesion, changes in cell shape, and movement of Pnn shRNAi transfected cells out of the epithelium. We suggest that Pnn plays an integral role in the establishment and maintenance of epithelial cell-cell adhesion via its activity within nuclear multi-protein complexes.
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Moléculas de Adhesión Celular/fisiología , Adhesión Celular/fisiología , Epitelio Corneal/fisiología , Proteínas Nucleares/fisiología , ARN sin Sentido/genética , ARN Interferente Pequeño/genética , Western Blotting , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Movimiento Celular/fisiología , Forma de la Célula/fisiología , Proteínas del Citoesqueleto/metabolismo , Desmosomas/metabolismo , Epitelio Corneal/citología , Expresión Génica , Silenciador del Gen/fisiología , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Uniones Estrechas/metabolismo , Transfección , Proteína de la Zonula Occludens-1RESUMEN
PURPOSE: We investigated the progressive nature of neurodegenerative structural changes following injury to retinal ganglion cell (RGC) axons using quantifiable and noninvasive in vivo imaging techniques. METHODS: To track degenerative RGC progression in retinas following optic nerve crush (ONC) injury, spectral-domain optical coherence tomography (SD-OCT) was used to quantitate the RGC nerve fiber layer (NFL) density. The RGC soma cell density (RCD) was measured by confocal scanning laser ophthalmoscopy (CSLO). The RCD counts were performed using blood vessels as landmarks to anatomically track defined progressive changes in enhanced yellow fluorescent fusion protein (EYFP)-labeled RGCs. RESULTS: Following ONC injury, 68% of the observed decrease in RCD measured by CSLO and 54% of the NFL thickness obtained by SD-OCT imaging (N=4 retinas) occurred within the first week. Between days 7 and 14, an additional 22% decrease in RCD was concurrent with a 31% decrease in overall NFL thickness. Finally, between days 14 and 21, an additional 10% decrease in RCD measured in vivo by CSLO and 15% decrease in NFL thickness by SD-OCT was observed. CONCLUSIONS: Our data suggest that in vivo CSLO imaging of EYFP-RGC expression and SD-OCT measured NFL thickness are fast and reliable methods that longitudinally track neurodegenerative progression following ONC injury. Neurodegenerative changes in NFL thickness measured by SD-OCT imaging have the same overall trajectory as those observed by CSLO for RCD; however, changes in NFL thickness initially lag behind in vivo RGC soma counts with a slower decline in overall measurable change.
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Axones/patología , Disco Óptico/patología , Traumatismos del Nervio Óptico/patología , Células Ganglionares de la Retina/patología , Animales , Recuento de Células , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Compresión Nerviosa/efectos adversos , Tomografía de Coherencia ÓpticaRESUMEN
PURPOSE: Recent advances in technology now provide tools capable of tracking genome-wide expression changes occurring in progressive pathological processes. The present experiments were carried out to determine if acetylcholine receptor α 6 subunit (Chrna6) is a reliable retinal ganglion cell (RGC) marker in adult mouse eyes and if Chrna6 expression can be used to track progressive loss of RGCs, such as is observed in the DBA/2J glaucoma model. METHODS: Data sets derived from the BXD strains were used to extract gene expression signatures for RGCs. Pooled retinas from DBA/2J or C57BL/6J cases at 1-3 months, 12 months, and 16-17 months were prepared for gene-array and RT-PCR analysis. Globes were fixed in paraformaldehyde and sectioned for immunofluorescence with antibodies against Chrna6. RESULTS: Chrna6 has a cellular expression signature for RGCs with high correlation to Thy1 (r = 0.65), a recognized RGC marker. Immunofluorescence experiments confirm that in the young and adult mouse retina, Chrna6 is preferentially expressed by RGCs. We further show that C3H/HeJ retinas, which lack photoreceptors, also express Chrna6 in the RGC layer. Gene expression array analyses, confirmed by RT-PCR, show progressive loss of Chrna6 expression in retinas of the DBA/2J glaucomatous mouse retinas. CONCLUSIONS: Quantitative trait locus analysis provides support for Chrna6 as a RGC marker. Chrna6 expression decreases with death of RGCs in glaucomatous DBA/2J mice and after optic nerve crush injury, further supporting Chrna6 as a reliable RGC marker. High expression of RGC Chrna6 in the absence of photoreceptors is suggestive that Chrna6 expression by RGCs is independent of photoreceptor-derived stimuli.
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Regulación de la Expresión Génica , Glaucoma/genética , ARN/genética , Receptores Nicotínicos/genética , Retina/metabolismo , Animales , Modelos Animales de Enfermedad , Glaucoma/metabolismo , Glaucoma/patología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microscopía Electrónica , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Nicotínicos/biosíntesis , Retina/ultraestructuraRESUMEN
PURPOSE: Mice with a targeted disruption of Brn3b (knockout Brn3b(-/-)) undergo the loss of a majority of retinal ganglion cells (RGCs) before birth. Spectral domain optical coherence tomography (SD-OCT) allows for the noninvasive examination of Brn3b(-/-) cellular loss in vivo. METHODS: The central retinas of Brn3b(-/-) and phenotypically wild-type (Brn3b(+/+) and Brn3b(±)) mice were imaged by SD-OCT. The combined nerve fiber layer (NFL) and inner plexiform layer (IPL) were manually segmented and thickness maps were generated. The results were confirmed by histologic and immunofluorescence cell counts of the RGC layer (RGCL) of the same retinas. RESULTS: The combined NFL and IPL of the Brn3b(-/-) retinas were significantly thinner, and the histologic cell counts significantly lower, than those of the phenotypically wild-type retinas (paired t-test; P < 0.01 and P < 0.01, respectively). The combined NFL and IPL thickness and the histologic cell count correlated highly (R(2) = 0.9612). Immunofluorescence staining revealed significant RGC-specific loss in Brn3b(-/-) retinas (paired t-test; P < 0.01). The distribution of combined central NFL and IPL loss was not localized or sectorial. CONCLUSIONS: The strong correlation between the combined layer thickness and histologic cell counts validates manual OCT segmentation as a method of monitoring cell loss in the RGCL. A retinal thickness map assessed if combined NFL and IPL thickness loss in Brn3b(-/-) eyes was topographically specific. Generalized RGC and combined NFL and IPL loss was observed in the Brn3b(-/-) retinas, in contrast to topographically specific RGC loss observed in glaucomatous DBA2/J eyes.
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Anomalías del Ojo/genética , Anomalías del Ojo/patología , Glaucoma/genética , Glaucoma/patología , Proteínas de Homeodominio/genética , Células Ganglionares de la Retina/patología , Factor de Transcripción Brn-3B/genética , Animales , Recuento de Células , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Fibras Nerviosas/patología , Fenotipo , Tomografía de Coherencia Óptica/métodosRESUMEN
CtBP is a transcriptional corepressor with tumorigenic potential that targets the promoter of the tumor suppressor gene E-cadherin. Pnn/DRS (Pnn) is a "nuclear speckle"-associated protein involved in mRNA processing as well as transcriptional regulation of E-cadherin via its binding to CtBP. Here, we show that CtBP can recruit Pnn to CtBP-associated complexes, resulting in Pnn-dependent chromatin remodeling at the E-cadherin promoter. In addition, CtBP and Pnn can differentially modulate E-cadherin mRNA splicing, with polymerase II serving as an interface in this event. Therefore, the Pnn/CtBP functional interplay represents a novel mechanism linking the corepressor CtBP and Pnn to the transcription-coupled mRNA splicing of a major tumor suppressor gene. Our findings implicate the existence of the molecular switches involved in tumorigenesis, which coordinate promoter-specific events and mRNA processing, by serving as bridging elements between the regulatory complexes both at gene promoters and within the mRNA splicing machineries.
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Oxidorreductasas de Alcohol/metabolismo , Cadherinas/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Empalme del ARN , Transcripción Genética , Oxidorreductasas de Alcohol/genética , Moléculas de Adhesión Celular/genética , Línea Celular , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , ADN Complementario , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Hemaglutininas/metabolismo , Humanos , Riñón/citología , Luciferasas/metabolismo , Modelos Biológicos , Proteínas Nucleares/genética , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , TransfecciónRESUMEN
Previous in vitro studies have indicated multiple and varied roles of Pinin (PNN); however, its in vivo role has remained unclear. Here, we report generation of null, hypomorphic, and conditional Pnn alleles in mice. We found that insertion of neomycin-resistance cassette into intron 8 of Pnn resulted in knockdown of Pnn, which allowed Pnn hypomorphic embryos to pass peri-implantation lethality. These mice are lethal at perinatal stages and exhibit defects in the cardiac outflow tract, palate, dorsal dermis, and axial skeleton. Since Wnt/beta-catenin signaling has been shown to play pivotal roles in development of all tissues affected by Pnn hypomorphism, we speculated that Pnn may affect Wnt/beta-catenin signaling. Supporting this view, we demonstrate abnormal activities of Tcf/Lef transcription factors, and alterations in beta-catenin level in multiple Pnn hypomorphic tissues. Taken together, the data suggest that Pnn plays important roles during mouse development through its involvement in regulation of Tcf/Lef activity.