RESUMEN
GEMIN5 exerts key biological functions regulating pre-mRNAs intron removal to generate mature mRNAs. A series of patients were reported harboring mutations in GEMIN5. No treatments are currently available for this disease. We treated two of these patients with oral Coenzyme Q10 (CoQ10), which resulted in neurological improvements, although MRI abnormalities remained. Whole Exome Sequencing demonstrated compound heterozygosity at the GEMIN5 gene in both cases: Case one: p.Lys742* and p.Arg1016Cys; Case two: p.Arg1016Cys and p.Ser411Hisfs*6. Functional studies in fibroblasts revealed a decrease in CoQ10 biosynthesis compared to controls. Supplementation with exogenous CoQ10 restored it to control intracellular CoQ10 levels. Mitochondrial function was compromised, as indicated by the decrease in oxygen consumption, restored by CoQ10 supplementation. Transcriptomic analysis of GEMIN5 patients compared with controls showed general repression of genes involved in CoQ10 biosynthesis. In the rigor mortis defective flies, CoQ10 levels were decreased, and CoQ10 supplementation led to an improvement in the adult climbing assay performance, a reduction in the number of motionless flies, and partial restoration of survival. Overall, we report the association between GEMIN5 dysfunction and CoQ10 deficiency for the first time. This association opens the possibility of oral CoQ10 therapy, which is safe and has no observed side effects after long-term therapy.
Asunto(s)
Ataxia , Enfermedades Mitocondriales , Debilidad Muscular , Ubiquinona , Ubiquinona/deficiencia , Adulto , Humanos , Ubiquinona/genética , Ubiquinona/uso terapéutico , Ubiquinona/metabolismo , Estudios de Seguimiento , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Mitocondriales/genética , Mutación , Proteínas del Complejo SMN/genéticaRESUMEN
Spinal muscular atrophy (SMA), the main genetic cause of infant death, is a neurodegenerative disease characterized by the selective loss of motor neurons in the anterior horn of the spinal cord, accompanied by muscle wasting. Pathomechanically, SMA is caused by low levels of the survival motor neuron protein (SMN) resulting from the loss of the SMN1 gene. However, emerging research extends the pathogenic effect of SMN deficiency beyond motor neurons. A variety of metabolic abnormalities, especially altered fatty acid metabolism and impaired glucose tolerance, has been described in isolated cases of SMA; therefore, the impact of SMN deficiency in metabolic abnormalities has been speculated. Although the life expectancy of these patients has increased due to novel disease-modifying therapies and standardization of care, understanding of the involvement of metabolism and nutrition in SMA is still limited. Optimal nutrition support and metabolic monitoring are essential for patients with SMA, and a comprehensive nutritional assessment can guide personalized nutritional therapy for this vulnerable population. It has recently been suggested that metabolomics studies before and after the onset of SMA in patients can provide valuable information about the direct or indirect effects of SMN deficiency on metabolic abnormalities. Furthermore, identifying and quantifying the specific metabolites in SMA patients may serve as an authentic biomarker or therapeutic target for SMA. Here, we review the main epidemiological and mechanistic findings that link metabolic changes to SMA and further discuss the principles of metabolomics as a novel approach to seek biomarkers and therapeutic insights in SMA.
Asunto(s)
Atrofia Muscular Espinal/metabolismo , Terapia Nutricional/métodos , Fenómenos Fisiológicos de la Nutrición/genética , Proteínas del Complejo SMN/deficiencia , Proteína 1 para la Supervivencia de la Neurona Motora , Biomarcadores/metabolismo , Humanos , Metaboloma , Metabolómica/métodos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Evaluación NutricionalRESUMEN
Disappearance of TAR-DNA-binding protein 43 kDa (TDP-43) from the nucleus contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS), but the nuclear function of TDP-43 is not yet fully understood. TDP-43 associates with nuclear bodies including Gemini of coiled bodies (GEMs). GEMs contribute to the biogenesis of uridine-rich small nuclear RNA (U snRNA), a component of splicing machinery. The number of GEMs and a subset of U snRNAs decrease in spinal muscular atrophy, a lower motor neuron disease, suggesting that alteration of U snRNAs may also underlie the molecular pathogenesis of ALS. Here, we investigated the number of GEMs and U11/12-type small nuclear ribonucleoproteins (snRNP) by immunohistochemistry and the level of U snRNAs using real-time quantitative RT-PCR in ALS tissues. GEMs decreased in both TDP-43-depleted HeLa cells and spinal motor neurons in ALS patients. Levels of several U snRNAs decreased in TDP-43-depleted SH-SY5Y and U87-MG cells. The level of U12 snRNA was decreased in tissues affected by ALS (spinal cord, motor cortex and thalamus) but not in tissues unaffected by ALS (cerebellum, kidney and muscle). Immunohistochemical analysis revealed the decrease in U11/12-type snRNP in spinal motor neurons of ALS patients. These findings suggest that loss of TDP-43 function decreases the number of GEMs, which is followed by a disturbance of pre-mRNA splicing by the U11/U12 spliceosome in tissues affected by ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Proteínas de Unión al ADN/genética , Gemini de los Cuerpos Enrollados/metabolismo , Neuronas Motoras/patología , ARN Nuclear Pequeño/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Corteza Motora/metabolismo , Corteza Motora/patología , Neuronas Motoras/metabolismo , Empalme del ARN , ARN Nuclear Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleoproteínas Nucleares Pequeñas/genética , Proteínas del Complejo SMN/genética , Proteínas del Complejo SMN/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Tálamo/metabolismo , Tálamo/patologíaRESUMEN
Small nuclear ribonucleoproteins (snRNPs) play a fundamental role in pre-mRNA processing in the nucleus. The biogenesis of snRNPs involves a sequence of events that occurs in both the nucleus and cytoplasm. Despite the wealth of biochemical information about the cytoplasmic assembly of snRNPs, little is known about the spatial organization of snRNPs in the cytoplasm. In the cytoplasm of larch microsporocytes, a cyclic appearance of bodies containing small nuclear RNA (snRNA) and Sm proteins was observed during anther meiosis. We observed a correlation between the occurrence of cytoplasmic snRNP bodies, the levels of Sm proteins, and the dynamic formation of Cajal bodies. Larch microsporocytes were used for these studies. This model is characterized by natural fluctuations in the level of RNA metabolism, in which periods of high transcriptional activity are separated from periods of low transcriptional activity. In designing experiments, the authors considered the differences between the nuclear and cytoplasmic phases of snRNP maturation and generated a hypothesis about the direct participation of Sm proteins in a molecular switch triggering the formation of Cajal bodies.
Asunto(s)
Núcleo Celular/metabolismo , Cuerpos Enrollados/metabolismo , Citoplasma/metabolismo , Larix/citología , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN/metabolismo , Flores/metabolismo , Larix/fisiología , Meiosis/fisiología , Periodicidad , Fenómenos Fisiológicos de las Plantas , Proteínas de Plantas/metabolismo , Polen/metabolismo , Complejo Sinaptonémico/metabolismoRESUMEN
Nematode (Heterodera schachtii) resistance in sugar beet (Beta vulgaris) is controlled by a single dominant resistance gene, Hs1(pro-1). BvGLP-1 was cloned from resistant sugar beet. The BvGLP-1 messenger (m)RNA is highly upregulated in the resistant plants after nematode infection, suggesting its role in the Hs1(pro-1) mediated resistance. BvGLP-1 exhibits sequence homology to a set of plant germin-like proteins (GLP), from which several have proved to be functional in plant basal or defense resistance against fungal pathogens. To test whether BvGLP-1 is also involved in the plant-fungus interaction, we transferred BvGLP-1 into Arabidopsis and challenged the transgenic plants with the pathogenic fungi Verticillium longisporum and Rhizoctonia solani as well as with the beneficial endophytic fungus Piriformospora indica. The expression of BvGLP-1 in Arabidopsis elevated the H(2)O(2) content and conferred significant resistance to V. longisporum and R. solani but did not affect the beneficial interaction with P. indica in seedlings. Microscopic observations revealed a dramatic reduction in the amount of hyphae of the pathogenic fungi on the root surface as well as of fungal mycelium developed inside the roots of transgenic Arabidopsis compared with wild-type plants. Molecular analysis demonstrated that the BvGLP-1 expression in Arabidopsis constitutively activates the expression of a subset of plant defense-related proteins such as PR-1 to PR-4 and PDF1.2 but not PDF2.1 and PDF2.3. In contrast, the PDF2.1 mRNA level was downregulated. These data suggest an important role of BvGLP-1 in establishment of plant defense responses, which follow specific signaling routes that diverge from those induced by the beneficial fungus.
Asunto(s)
Arabidopsis/genética , Beta vulgaris/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Proteínas del Complejo SMN/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiología , Beta vulgaris/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Filogenia , Enfermedades de las Plantas/microbiología , Hojas de la Planta/citología , Hojas de la Planta/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas del Complejo SMN/genéticaRESUMEN
Spinal muscular atrophy (SMA) is characterized by selective loss of alpha-motor neurons and is caused by homozygous loss or mutation in the survival motor neuron (SMN1) gene. Loss of SMN1 is partially compensated by the copy gene, SMN2. Currently, there are no specific treatments for SMA. Key features of SMA are modeled in mice by deletion of murine Smn, and insertion of both full length human SMN2 gene and the major aberrant splice isoform of the SMN2 gene (SMNDelta7; [Le, T.T., Pham, L.T., Butchbach, M.E., Zhang, H.L., Monani, U.R., Coovert, D.D., Gavrilina, T.O., Xing, L., Bassell, G.J., and Burghes, A.H. 2005. SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN. Hum Mol Genet 14: 845-857]). The present study identified moderate-throughput, quantitative behavioral tests in neonatal SMN2(+/+);SMNDelta7(+/+);Smn(-/-) mice. It also addresses methodological approaches and common interpretational challenges in a neonatal model with motor deficiencies and frequent deaths. Animals were assessed daily for body weight and survival, and every other day for neonatal well-being indices and tests of motor function such as performance on the hind-limb suspension test (a.k.a. tube test) and geotaxis. The tube test is a novel non-invasive motor function test specifically designed for neonatal rodents. We found progressive deterioration in SMA model mice for most measures studied particularly body weight, survival, body temperature and motor function with differences appearing as early as P3. Power analysis showed that body weight, survival, righting reflex, geotaxis and tube test had highest predictive power for drug efficacy studies. This multi-functional component battery of tests provides a rapid and efficient means to identify, evaluate and develop candidate therapies as a prelude to human clinical trials.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Atrofias Musculares Espinales de la Infancia/tratamiento farmacológico , Animales , Animales Recién Nacidos , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Trastornos del Movimiento/diagnóstico , Trastornos del Movimiento/tratamiento farmacológico , Trastornos del Movimiento/genética , Proteínas del Tejido Nervioso/genética , Fenotipo , Valor Predictivo de las Pruebas , Proteínas de Unión al ARN/genética , Proyectos de Investigación , Proteínas del Complejo SMN , Atrofias Musculares Espinales de la Infancia/genética , Tasa de Supervivencia , Proteína 1 para la Supervivencia de la Neurona Motora , Proteína 2 para la Supervivencia de la Neurona Motora , Resultado del TratamientoRESUMEN
INTRODUCTION: Spinal muscular atrophy (SMA) is a neurodegenerative disease of the motor neurons that results in progressive muscle weakness. It is also the leading hereditary cause of infant mortality. Homozygous loss of the survival motor neuron (SMN1) gene causes SMA, and the number of copies of the SMN2 gene modulates the severity of the disease. Increasing the expression of the SMN2 gene by pharmacological agents is one of the therapeutic approaches currently being implemented. METHODS: In this preliminary study, we investigated the effect of phenylbutyrate, a histone deacetylase (HDAC) inhibitor, on SMN2 expression in two SMA type III Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines to understand the suitability of lymphoblastoid cell lines in drug screening. These cell lines are regarded as a good source as they can easily be established from the peripheral leucocytes of patients. Quantitative analysis of SMN2 mRNA was performed on established cell lines treated with various concentrations of phenylbutyrate and for a range of incubation periods using real-time polymerase chain reaction. Western blot analysis was used to determine SMN protein levels. RESULTS: Real-time polymerase chain reaction and Western blot analysis demonstrated that the levels of SMN2 full-length (fl-SMN2) transcripts and protein were not increased in phenylbutyrate-treated cell lines compared to non-treated controls. CONCLUSION: These results suggest that EBV-transformed lymphoblastoid cell lines are not suitable for studying the effect of certain HDAC inhibitors on SMN2 gene expression.
Asunto(s)
Línea Celular Transformada , Evaluación Preclínica de Medicamentos , Atrofias Musculares Espinales de la Infancia/patología , Adulto , Niño , Herpesvirus Humano 4 , Inhibidores de Histona Desacetilasas , Humanos , Recién Nacido , Masculino , Fenilbutiratos/farmacología , ARN Mensajero/biosíntesis , Proteínas del Complejo SMN/biosíntesis , Proteínas del Complejo SMN/genética , Proteína 2 para la Supervivencia de la Neurona MotoraRESUMEN
Spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein. Although the SMN complex is essential for assembly of spliceosomal U small nuclear RNPs, it is still not understood why reduced levels of the SMN protein specifically cause motor neuron degeneration. SMN was recently proposed to have specific functions in mRNA transport and translation regulation in neuronal processes. The defective protein in Fragile X mental retardation syndrome (FMRP) also plays a role in transport of mRNPs and in their translation. Therefore, we examined possible relationships of SMN with FMRP. We observed granules containing both transiently expressed red fluorescent protein(RFP)-tagged SMN and green fluorescent protein(GFP)-tagged FMRP in cell bodies and processes of rat primary neurons of hypothalamus in culture. By immunoprecipitation experiments, we detected an association of FMRP with the SMN complex in human neuroblastoma SH-SY5Y cells and in murine motor neuron MN-1 cells. Then, by in vitro experiments, we demonstrated that the SMN protein is essential for this association. We showed that the COOH-terminal region of FMRP, as well as the conserved YG box and the region encoded by exon 7 of SMN, are required for the interaction. Our findings suggest a link between the SMN complex and FMRP in neuronal cells.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Hipotálamo/metabolismo , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Exones/fisiología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/patología , Humanos , Hipotálamo/patología , Ratones , Neuronas Motoras/patología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patología , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína/fisiología , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Complejo SMN , Proteína 1 para la Supervivencia de la Neurona MotoraRESUMEN
Spinal muscular atrophy is one of the most heterogeneous of the single-gene neuromuscular disorders. The broad spectrum of severity, with onset from the prenatal period to adulthood, presents unique challenges in the design and implementation of clinical trials. The clinical classification of subjects into severe (type 1), intermediate (type 2), and mild (type 3) subtypes has proved useful both in enhancing communication among clinicians internationally and in forging the collaborative development of outcome measures for clinical trials. Ideally, clinical trial design in spinal muscular atrophy must take into account the spinal muscular atrophy type, patient age, severity-of-affection status, nature of the therapeutic approach, timing of the proposed intervention relative to disease progression, and relative homogeneity of the cohort to be studied. Following is an overview of the challenges and opportunities, current and future therapeutic strategies, and progress to date in clinical trials in spinal muscular atrophy.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Atrofias Musculares Espinales de la Infancia/genética , Atrofias Musculares Espinales de la Infancia/terapia , Niño , Ensayos Clínicos como Asunto/normas , Ensayos Clínicos como Asunto/tendencias , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos/tendencias , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Factores de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso/uso terapéutico , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/genética , Proteínas del Complejo SMN , Atrofias Musculares Espinales de la Infancia/clasificaciónRESUMEN
Several mice models have been created for spinal muscular atrophy (SMA); however, there is still no standard preclinical testing system for the disease. We previously generated type III-specific SMA model mice, which might be suitable for use as a preclinical therapeutic testing system for SMA. To establish such a system and test its applicability, we first created a testing protocol and then applied it as a means to investigate the use of valproic acid (VPA) as a possible treatment for SMA. These SMA mice revealed tail/ear/foot deformity, muscle atrophy, poorer motor performances, smaller compound muscle action potential and lower spinal motoneuron density at the age of 9 to 12 months in comparison with age-matched wild-type littermate mice. In addition, VPA attenuates motoneuron death, increases spinal SMN protein level and partially normalizes motor function in SMA mice. These results suggest that the testing protocol developed here is well suited for use as a standardized preclinical therapeutic testing system for SMA.
Asunto(s)
Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/fisiopatología , Degeneración Nerviosa/diagnóstico , Degeneración Nerviosa/fisiopatología , Fármacos Neuroprotectores/farmacología , Médula Espinal/fisiopatología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Músculo Esquelético/inervación , Músculo Esquelético/fisiopatología , Atrofia Muscular Espinal/tratamiento farmacológico , Degeneración Nerviosa/tratamiento farmacológico , Proteínas del Tejido Nervioso/biosíntesis , Fármacos Neuroprotectores/uso terapéutico , Valor Predictivo de las Pruebas , Proteínas de Unión al ARN/biosíntesis , Proteínas del Complejo SMN , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Resultado del Tratamiento , Ácido Valproico/farmacología , Ácido Valproico/uso terapéuticoRESUMEN
The expression pattern of the survival motor neuron (SMN) protein has been investigated immunohistochemically in the human fetal forebrain from 14 to 38 weeks of gestation. Mutations in the SMN gene cause spinal muscular atrophy (SMA), an autosomal recessive disease characterized by degeneration of lower motor neurons in the spinal cord leading to progressive muscle wasting. SMN is a multifunctional protein and has been implicated in diverse cytoplasmic and nuclear processes. The monoclonal murine SMN antibody used in this study recognized a major band at approximately 34 kDa. In spinal cord anterior horn motor neurons at 13 weeks of gestation, the soma, proximal neurites, and nucleus were immunostained. In the nucleus, SMN immunolabeling was observed at the nuclear membrane, at the nucleolus, and at dot-like structures in the nucleoplasm likely to be coiled bodies and gems. In the fetal forebrain, SMN was immunodetected as early as 14 weeks of gestation. From 14 to 24 weeks of gestation, intense immunostaining was observed in the basal nucleus of Meynert, a major source of cholinergic afferents to the cortex. Less intensely labeled cells at lower packing density were also observed in the thalamus, reticular and perireticular nucleus, globus pallidus, hippocampus, amygdala, and enthorinal cortex. Immunolabeled cells were still detectable at 38 gestational weeks, the latest time point investigated. These findings provide an anatomical basis for future investigations of SMN functions during brain development and for the neuropathological characterization of severe SMA cases.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Prosencéfalo/metabolismo , Proteínas de Unión al ARN/metabolismo , Médula Espinal/metabolismo , Células del Asta Anterior/citología , Células del Asta Anterior/embriología , Células del Asta Anterior/metabolismo , Núcleo Basal de Meynert/citología , Núcleo Basal de Meynert/embriología , Núcleo Basal de Meynert/metabolismo , Globo Pálido/citología , Globo Pálido/embriología , Globo Pálido/metabolismo , Hipocampo/citología , Hipocampo/embriología , Hipocampo/metabolismo , Humanos , Inmunohistoquímica , Neuronas/citología , Neuronas/metabolismo , Prosencéfalo/citología , Prosencéfalo/embriología , Valores de Referencia , Proteínas del Complejo SMN , Médula Espinal/citología , Médula Espinal/embriología , Tálamo/citología , Tálamo/embriología , Tálamo/metabolismo , Distribución TisularRESUMEN
High-throughput assays generate immense quantities of data that require sophisticated data analysis tools. We have created a freely available software tool, SLIMS (Small Laboratory Information Management System), for chemical genetics which facilitates the collection and analysis of large-scale chemical screening data. Compound structures, physical locations, and raw data can be loaded into SLIMS. Raw data from high-throughput assays are normalized using flexible analysis protocols, and systematic spatial errors are automatically identified and corrected. Various computational analyses are performed on tested compounds, and dilution-series data are processed using standard or user-defined algorithms. Finally, published literature associated with active compounds is automatically retrieved from Medline and processed to yield potential mechanisms of actions. SLIMS provides a framework for analyzing high-throughput assay data both as a laboratory information management system and as a platform for experimental analysis.
Asunto(s)
Evaluación Preclínica de Medicamentos , Programas Informáticos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Bases de Datos Genéticas , Flavonas , Flavonoides/farmacología , Genes Reporteros , Humanos , Atrofia Muscular Espinal/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN , Proteínas del Complejo SMNRESUMEN
Most patients with the pediatric neurodegenerative disease spinal muscular atrophy have a homozygous deletion of the survival motor neuron 1 (SMN1) gene, but retain one or more copies of the closely related SMN2 gene. The SMN2 gene encodes the same protein (SMN) but produces it at a low efficiency compared with the SMN1 gene. We performed a high-throughput screen of approximately 47,000 compounds to identify those that increase production of an SMN2-luciferase reporter protein, but not an SMN1-luciferase reporter protein. Indoprofen, a nonsteroidal anti-inflammatory drug (NSAID) and cyclooxygenase (COX) inhibitor, selectively increased SMN2-luciferase reporter protein and endogenous SMN protein and caused a 5-fold increase in the number of nuclear gems in fibroblasts from SMA patients. No other NSAIDs or COX inhibitors tested exhibited this activity.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Indoprofeno/farmacología , Proteínas del Tejido Nervioso/biosíntesis , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Inhibidores de la Ciclooxigenasa/farmacología , Evaluación Preclínica de Medicamentos , Femenino , Fibroblastos/enzimología , Humanos , Indoprofeno/farmacocinética , Ratones , Proteínas del Tejido Nervioso/genética , Embarazo , Prostaglandina-Endoperóxido Sintasas/fisiología , Proteínas de Unión al ARN , Proteínas del Complejo SMN , Proteína 1 para la Supervivencia de la Neurona Motora , Proteína 2 para la Supervivencia de la Neurona Motora , Regulación hacia ArribaRESUMEN
Proximal spinal muscular atrophy (SMA) is a common motor neuron disorder caused by mutation of the telomeric survival of motor neuron gene SMN1. The centromeric survival of motor neuron SMN2 gene is retained in all SMA patients but does not produce sufficient SMN protein to prevent the development of clinical symptoms. The SMN1 and SMN2 genes differ functionally by a single nucleotide change. This change affects the efficiency with which exon 7 is incorporated into the mRNA transcript. Thus, SMN2 produces less full-length mRNA and protein than SMN1. We have screened a library of compounds in order to identify ones that can alter the splicing pattern of the SMN2 gene. Here, we report that the compound aclarubicin increases the retention of exon 7 into the SMN2 transcript. We show that aclarubicin effectively induces incorporation of exon 7 into SMN2 transcripts from the endogenous gene in type I SMA fibroblasts as well as into transcripts from a SMN2 minigene in the motor neuron cell line NSC34. In type I fibroblasts, treatment resulted in an increase in SMN protein and gems to normal levels. Our results suggest that alteration of splicing pattern represents a new approach to modification of gene expression in disease treatment and demonstrate the feasibility of high throughput screens to detect compounds that affect the splicing pattern of a gene.
Asunto(s)
Aclarubicina/farmacología , Proteínas del Tejido Nervioso/fisiología , Atrofias Musculares Espinales de la Infancia/tratamiento farmacológico , Empalme Alternativo/efectos de los fármacos , Animales , Western Blotting , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Exones , Estudios de Factibilidad , Fibroblastos , Humanos , Inmunohistoquímica , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas del Complejo SMN , Atrofias Musculares Espinales de la Infancia/genética , Proteína 1 para la Supervivencia de la Neurona Motora , Proteína 2 para la Supervivencia de la Neurona Motora , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
The Survival of Motor Neurons (SMN) is the disease gene of spinal muscular atrophy. We have previously established a genetic system based on the chicken pre-B cell line DT40, in which expression of SMN protein is regulated by tetracycline, to study the function of SMN in vivo. Depletion of SMN protein is lethal to these cells. Here we tested the functionality of mutant SMN proteins by determining their capacity to rescue the cells after depletion of wild-type SMN. Surprisingly, all of the spinal muscular atrophy-associated missense mutations tested were able to support cell viability and proliferation. Deletion of the amino acids encoded by exon 7 of the SMN gene resulted in a partial loss of function. A mutant SMN protein lacking both the tyrosine/glycine repeat (in exon 6) and exon 7 failed to sustain viability, indicating that the C terminus of the protein is critical for SMN activity. Interestingly, the Tudor domain of SMN, encoded by exon 3, does not appear to be essential for SMN function since a mutant deleted of this domain restored cell viability. Unexpectedly, a chicken SMN mutant (DeltaN39) lacking the N-terminal 39 amino acids that encompass the Gemin2-binding domain also rescued the lethal phenotype. Moreover, the level of Gemin2 in DeltaN39-rescued cells was significantly reduced, indicating that Gemin2 is not required for DeltaN39 to perform the essential function of SMN in DT40 cells. These findings suggest that SMN may perform a novel function in DT40 cells.
Asunto(s)
Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Animales , Supervivencia Celular , Pollos/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , ADN Complementario/metabolismo , Epítopos , Exones , Eliminación de Gen , Glicina/química , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Mutación Missense , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Fenotipo , Plásmidos/metabolismo , Mutación Puntual , Pruebas de Precipitina , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Retroviridae/genética , Proteínas del Complejo SMN , Homología de Secuencia de Aminoácido , Transcripción Genética , Tirosina/químicaRESUMEN
Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, leading to muscular paralysis with muscular atrophy. No effective treatment of this disorder is presently available. Studies of the correlation between disease severity and the amount of survival motor neuron (SMN) protein have shown an inverse relationship. We report that sodium butyrate effectively increases the amount of exon 7-containing SMN protein in SMA lymphoid cell lines by changing the alternative splicing pattern of exon 7 in the SMN2 gene. In vivo, sodium butyrate treatment of SMA-like mice resulted in increased expression of SMN protein in motor neurons of the spinal cord and resulted in significant improvement of SMA clinical symptoms. Oral administration of sodium butyrate to intercrosses of heterozygous pregnant knockout-transgenic SMA-like mice decreased the birth rate of severe types of SMA-like mice, and SMA symptoms were ameliorated for all three types of SMA-like mice. These results suggest that sodium butyrate may be an effective drug for the treatment of human SMA patients.
Asunto(s)
Empalme Alternativo/efectos de los fármacos , Butiratos/uso terapéutico , Atrofia Muscular Espinal/tratamiento farmacológico , Proteínas del Tejido Nervioso/biosíntesis , Anomalías Múltiples/genética , Animales , Línea Celular Transformada/efectos de los fármacos , Cruzamientos Genéticos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Evaluación Preclínica de Medicamentos , Elementos de Facilitación Genéticos , Inhibidores Enzimáticos/farmacología , Exones/genética , Femenino , Enfermedades Fetales/tratamiento farmacológico , Flavonoides/farmacología , Edad Gestacional , Cabello/anomalías , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Intercambio Materno-Fetal , Ratones , Ratones Noqueados , Ratones Transgénicos , Atrofia Muscular Espinal/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Ácido Ocadaico/farmacología , Fenotipo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Proteínas del Complejo SMN , Proteína 2 para la Supervivencia de la Neurona Motora , Cola (estructura animal)/anomalíasRESUMEN
Disruption of the survival motor neuron (SMN) gene leads to selective loss of spinal motor neurons, resulting in the fatal human neurodegenerative disorder spinal muscular atrophy (SMA). SMN has been shown to function in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and pre-mRNA splicing. We have demonstrated that SMN also interacts with fibrillarin, a highly conserved nucleolar protein that is associated with all Box C/D small nucleolar RNAs and functions in processing and modification of rRNA. Fibrillarin and SMN co-immunoprecipitate from HeLa cell extracts indicating that the proteins exist as a complex in vivo. Furthermore, in vitro binding studies indicate that the interaction between SMN and fibrillarin is direct and salt-stable. We show that the glycine/arginine-rich domain of fibrillarin is necessary and sufficient for SMN binding and that the region of SMN encoded by exon 3, including the Tudor domain, mediates the binding of fibrillarin. Tudor domain missense mutations, including one found in an SMA patient, impair the interaction between SMN and fibrillarin (as well as the common snRNP protein SmB). Our results suggest a function for SMN in small nucleolar RNP biogenesis (akin to its known role as an snRNP assembly factor) and reveal a potential link between small nucleolar RNP biogenesis and SMA.