RESUMEN
Purpose: To determine major differences in lipid profile between human control and glaucomatous optic nerve. To assess major enzymes in lipid pathway if aberration is revealed for a lipid class by profiling. Methods: Optic nerve (ON) samples were obtained from human cadaveric donors [control (n = 11) and primary open-angle glaucoma (POAG; n = 12)]; the lipids were extracted using Bligh and Dyer methods. Control and glaucoma donors were all Caucasians age 72.3 ± 5.9 and 70.3 ± 10.5 (inclusive of both sexes), respectively. Lipids were extracted after weighing the tissue; the protein amounts in the corresponding aqueous phase of organic solvent extraction were recorded. High-resolution mass spectrometry was performed using a Q-exactive mass spectrometer coupled with an EASY-nLC 1000 liquid chromatograph instrument. Bioinformatics and statistical analysis were performed using LipidSearch v.4.1 and MetaboAnalyst 4.0/STATA 14.2. Protein amounts were determined using Bradford's method. Western blot, ELISA, and immunohistochemistry utilized established protocols and were performed for protein quantification and localization, respectively. Additional donor tissues were utilized for Western blot, ELISA, and immunohistochemistry. Results: Principal component analysis (PCA) placed control and glaucomatous ONs in two distinct groups based on analysis of lipid profiles. Total lipid, total phospholipids, total ceramide, and total sphingolipids were similar (without significant difference) between control and glaucoma. However, we found a significant increase in glucosylsphingosine in glaucoma compared to control samples. We found similar levels of glucocerebrosidase (GBA), ceramide glucosyltransferase (UGCG), decreased nonlysosomal glucocerebrosidase (GBA2), and increased lysosomal and nonlysosomal acylsphingosine amidohydrolase (ASAH1 and ASAH2) levels in glaucomatous ON compared to control. Conclusions: We found significant differences in glucosylsphingosine lipids, consistent with decreased GBA and GBA2 and increased ASAH1 and ASAH2 immunoreactivity in glaucoma, suggesting the potential impairment of sphingolipid enzymatic pathways in lysosomal and nonlysosomal cellular compartments.
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Glaucoma de Ángulo Abierto/metabolismo , Metabolismo de los Lípidos , Lipidosis/metabolismo , Nervio Óptico/metabolismo , Psicosina/análogos & derivados , Ceramidasa Ácida/metabolismo , Anciano , Western Blotting , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Femenino , Glucosilceramidasa/metabolismo , Glucosiltransferasas/metabolismo , Humanos , Inmunohistoquímica , Masculino , Espectrometría de Masas , Ceramidasa Neutra/metabolismo , Psicosina/metabolismo , beta-Glucosidasa/metabolismoRESUMEN
Neurons from the adult central nervous system (CNS) demonstrate limited mRNA transport and localized protein synthesis versus developing neurons, correlating with lower regenerative capacity. We found that deimination (posttranslational conversion of protein-bound arginine into citrulline) undergoes upregulation during early neuronal development while declining to a low basal level in adults. This modification is associated with neuronal arborization from amphibians to mammals. The mRNA-binding proteins (ANP32a, REF), deiminated in neurons, have been implicated in local protein synthesis. Overexpression of the deiminating cytosolic enzyme peptidyl arginine deiminase 2 in nervous systems results in increased neuronal transport and neurite outgrowth. We further demonstrate that enriching deiminated proteins rescues transport deficiencies both in primary neurons and mouse optic nerve even in the presence of pharmacological transport blockers. We conclude that deimination promotes neuronal outgrowth via enhanced transport and local protein synthesis and represents a new avenue for neuronal regeneration in the adult CNS.
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Reprogramación Celular , Iminas/metabolismo , Regeneración Nerviosa , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Polaridad Celular , Proliferación Celular , Modelos Animales de Enfermedad , Ratones , Células PC12 , Desiminasas de la Arginina Proteica/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
PURPOSE: To determine whether there are identifiable, reproducible findings in the trabecular meshwork (TM) of patients with primary open-angle glaucoma (POAG) who underwent Kahook Dual Blade (KDB) goniotomy. DESIGN: Noncomparative retrospective case series. METHODS: Tertiary academic referral center, Veterans Affairs Medical Center. Thirteen patients (14 eyes) with POAG (100%) were treated with KDB goniotomy from May to December 2017. Isolated TM tissue was collected from 9 patients (10 eyes) and submitted for histologic analysis. Hematoxylin-eosin, periodic acid-Schiff, and elastin Van Gieson stains were completed, in addition to immunohistochemistry for collagen IV. RESULTS: Mean age of patients was 74.2 ± 6.7 years. Trabecular beams were identified in all 10 specimens, although distorted in 4 samples, of which 3 had a history of laser trabeculoplasty. Collagen IV staining was present in 10 of 10 samples, coating the trabecular beams. Elastin was present in 8 of 10 samples along the trabecular beams. Intraocular pressure and number of glaucoma medications decreased significantly in all cases postoperatively (P < .0001, P = .035, respectively). CONCLUSIONS: This pilot study demonstrates that tissue obtained during KDB goniotomy has a high yield of containing TM compared to reported yield of TM in specimens collected from traditional ab externo trabeculectomy (71% vs 20%, respectively). These goniotomy specimens possess sufficient anatomic preservation to be studied histologically. Trabecular meshwork obtained with this procedure may provide a novel modality to study TM dysfunction in open-angle glaucomas.
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Glaucoma/cirugía , Malla Trabecular/patología , Trabeculectomía/instrumentación , Anciano , Anciano de 80 o más Años , Humanos , Inmunohistoquímica , Proyectos Piloto , Estudios RetrospectivosRESUMEN
BACKGROUND: Sturge-Weber syndrome is a neurocutaneous disorder associated with port-wine birthmark, leptomeningeal capillary malformations, and glaucoma. It is associated with an unpredictable clinical course. Because of its rarity and complexity, many physicians are unaware of the disease and its complications. A major focus moving ahead will be to turn knowledge gaps and unmet needs into new research directions. METHODS: On October 1-3, 2017, the Sturge-Weber Foundation assembled clinicians from the Clinical Care Network with patients from the Patient Engagement Network of the Sturge-Weber Foundation to identify our current state of knowledge, knowledge gaps, and unmet needs. RESULTS: One clear unmet need is a need for consensus guidelines on care and surveillance. It was strongly recommended that patients be followed by multidisciplinary clinical teams with life-long follow-up for children and adults to monitor disease progression in the skin, eye, and brain. Standardized neuroimaging modalities at specified time points are needed together with a stronger clinicopathologic understanding. Uniform tissue banking and clinical data acquisition strategies are needed with cross-center, longitudinal studies that will set the stage for new clinical trials. A better understanding of the pathogenic roles of cerebral calcifications and stroke-like symptoms is a clear unmet need with potentially devastating consequences. CONCLUSIONS: Biomarkers capable of predicting disease progression will be needed to advance new therapeutic strategies. Importantly, how to deal with the emotional and psychological effects of Sturge-Weber syndrome and its impact on quality of life is a clear unmet need.
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Consenso , Grupo de Atención al Paciente , Guías de Práctica Clínica como Asunto , Síndrome de Sturge-Weber/diagnóstico , Síndrome de Sturge-Weber/terapia , Niño , Humanos , LactanteRESUMEN
Astrocytes respond to environmental cues, including changes in temperatures. Increased deimination, observed in many progressive neurological diseases, is thought to be contributed by astrocytes. We determined the level of deimination and expression of peptidyl arginine deiminase 2 (PAD2) in isolated primary astrocytes in response to changes on either side (31°C and 41°C) of the optimal temperature (37°C). We investigated changes in the astrocytes by using a number of established markers and accounted for cell death with the CellTiter-Blue assay. We found increased expression of glial fibrillary acidic protein, ALDH1L1, and J1-31, resulting from increased incubation temperature and increased expression of TSP1, S100ß, and AQP4, resulting from decreased incubation temperature vs. optimal temperature, suggesting activation of different biochemical pathways in astrocytes associated with different incubation temperatures. Mass spectrometric analyses support such trends. The PAD2 level was increased only as a result of increased incubation temperature with a commensurate increased level of deimination. Actin cytoskeleton and iso[4]LGE, a lipid peroxidase modification, also showed an increase with higher incubation temperature. Altogether, these results suggest that temperature, as an environmental cue, activates astrocytes in a different manner on either side of the optimal temperature and that increase in deimination is associated only with the higher temperature side of the spectrum.
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Astrocitos/metabolismo , Hidrolasas/metabolismo , Temperatura , Aldehído Deshidrogenasa/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Acuaporina 4/metabolismo , Astrocitos/efectos de los fármacos , Biofisica , Bucladesina/farmacología , Supervivencia Celular , Células Cultivadas , Corteza Cerebral/citología , Ensayo de Inmunoadsorción Enzimática , Proteína Ácida Fibrilar de la Glía , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina Proteica , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismoRESUMEN
Central and peripheral nervous systems are lipid rich tissues. Lipids, in the context of lipid-protein complexes, surround neurons and provide electrical insulation for transmission of signals allowing neurons to remain embedded within a conducting environment. Lipids play a key role in vesicle formation and fusion in synapses. They provide means of rapid signaling, cell motility and migration for astrocytes and other cell types that surround and play supporting roles neurons. Unlike many other signaling molecules, lipids are capable of multiple signaling events based on the different fragments generated from a single precursor during each event. Lipidomics, until recently suffered from two major disadvantages: (1) level of expertise required an overwhelming amount of chemical detail to correctly identify a vast number of different lipids which could be close in their chemical reactivity; and (2) high amount of purified compounds needed by analytical techniques to determine their structures. Advances in mass spectrometry have enabled overcoming these two limitations. Mass spectrometry offers a great degree of simplicity in identification and quantification of lipids directly extracted from complex biological mixtures. Mass spectrometers can be regarded to as mass analyzers. There are those that separate and analyze the product ion fragments in space (spatial) and those which separate product ions in time in the same space (temporal). Databases and standardized instrument parameters have further aided the capabilities of the spatial instruments while recent advances in bioinformatics have made the identification and quantification possible using temporal instruments.
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Progressive loss of visual function frequently accompanies demyelinating diseases such as multiple sclerosis (MS) and is hypothesized to be the result of damage to the axons and soma of neurons. Here, we show that dendritic impairment is also involved in these diseases. Deimination, a posttranslational modification, was reduced in the retinal ganglion cell layer of MS patients and in a transgenic mouse model of MS (ND4 mice). Reduced deimination accompanied a decrease in inner retinal function in ND4 mice, indicating loss of vision. Local restoration of deimination dramatically improved retinal function and elongation of neurites in isolated neurons. Further, neurite length was decreased by downregulation of deimination or siRNA knockdown of the export-binding protein REF, a primary target for deimination in these cells. REF localized to dendrites and bound selective mRNAs and translation machinery to promote protein synthesis. Thus, protein deimination and dendritic outgrowth play key roles in visual function and may be a general feature of demyelinating diseases.
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Enfermedades Desmielinizantes/complicaciones , Enfermedades Desmielinizantes/fisiopatología , Retina/fisiopatología , Trastornos de la Visión/etiología , Trastornos de la Visión/fisiopatología , Anciano , Secuencia de Aminoácidos , Animales , Enfermedades Desmielinizantes/genética , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/fisiopatología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Retina/patología , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/fisiología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Trastornos de la Visión/genética , Visión OcularRESUMEN
Deimination refers to conversion of protein-bound arginine into citrulline. An mRNA carrier, RNA binding export factor (REF), present on mitochondria undergoes loss of deimination with impaired ATP5b mRNA transport in ND4 mice (model of multiple sclerosis) compared with the controls. We present evidence of (1) reduced ATP5b mRNA binding strength of non-deiminated REF compared with deiminated REF, (2) impaired ATP5b mRNA transport in ND4 mice and (3) reduced mitochondrial ATP synthase activity on inhibition of deimination in PC12 cells. Impaired deimination of REF and defect in mitochondrial mRNA transport are critical factors in mitochondrial dysfunction in ND4 mice.
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ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , ARN Mensajero/metabolismo , Complejos de ATP Sintetasa/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Línea Celular , Citrulina/análisis , Modelos Animales de Enfermedad , Activación Enzimática , Expresión Génica , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Transporte de Proteínas , Transporte de ARN , Proteínas de Unión al ARN/metabolismo , Ratas , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: To evaluate the ND4 transgenic mouse model of multiple sclerosis using noninvasive methods. METHODS: Assessment of neurologic/behavioral abnormalities was made using pattern electroretinogram (PERG), magnetic resonance imaging (MRI), optic coherence tomography (OCT), and end point histologic analysis. RESULTS: Electrophysiologic (PERG) recordings demonstrated functional deficits in vision commensurate with neurologic/behavioral abnormalities. In ND4 mice, the authors found PERG abnormalities preceded neurologic/gait abnormalities. MRI demonstrated subtle structural changes that progressed over time in correlation with behavioral abnormalities. CONCLUSIONS: The ND4 mouse model has been evaluated using well-defined parameters of noninvasive methods (PERG, MRI, and OCT), enabling objective identification of functional and structural deficits and their correlation with neurologic/gait abnormality.