RESUMEN
Neuronal migration disorders (NMDs) are a heterogeneous group of conditions caused by the abnormal migration of neuroblasts in the developing brain and nervous system, resulting in severe developmental impairment, intractable epilepsy and intellectual disability (Spalice et al. 2009). To date, many genes have been identified as the leading cause of migration defects, i.e. agyria/pachygyria, polymicrogyria, heterotopias, agenesis of the corpus callosum and agenesis of the cranial nerves (Spalice et al. 2009). Here, we present a patient with early infantile epileptic encephalopathy (Ohtahara syndrome) with seizure onset on the first dayof life, severe developmental delay and an abnormal brain MRI with excessive folding of small, fused gyri and bilateral perisylvian polymicrogyria, suggestive of neuronal migration disorder. To clarify the unknown aetiology, we conducted whole-exome sequencing, which detected a de novo missense variant (c.5308A>T; p.(Met1770Leu)) in the SCN2A gene. This is a report of SCN2A gene variant identified in a patient with neuronal migration disorder which could further expand the phenotypic spectrum of these genetic disorders.
Asunto(s)
Mutación , Canal de Sodio Activado por Voltaje NAV1.2/genética , Neuroimagen , Fenotipo , Espasmos Infantiles/diagnóstico , Espasmos Infantiles/genética , Secuencia de Aminoácidos , Diagnóstico Diferencial , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Malformaciones del Desarrollo Cortical del Grupo II/diagnóstico , Neuroimagen/métodosRESUMEN
To understand how receptors are involved in neuronal trafficking and to be able to utilize them for specific targeting via the peripheral route would be of great benefit. Here, we describe the generation of novel lentiviral vectors with tropism to motor neurons that were made by coexpressing onto the lentiviral surface a fusogenic glycoprotein (mutated sindbis G) and an antibody against a cell-surface receptor (Thy1.1, p75(NTR), or coxsackievirus and adenovirus receptor) on the presynaptic terminal of the neuromuscular junction. These vectors exhibit binding specificity and efficient transduction of receptor positive cell lines and primary motor neurons in vitro. Targeting of each of these receptors conferred to these vectors the capability of being transported retrogradely from the axonal tip, leading to transduction of motor neurons in vitro in compartmented microfluidic cultures. In vivo delivery of coxsackievirus and adenovirus receptor-targeted vectors in leg muscles of mice resulted in predicted patterns of motor neuron labeling in lumbar spinal cord. This opens up the clinical potential of these vectors for minimally invasive administration of central nervous system-targeted therapeutics in motor neuron diseases.
Asunto(s)
Vectores Genéticos/genética , Lentivirus/genética , Neuronas Motoras/metabolismo , Receptores Presinapticos/genética , Transducción Genética/métodos , Animales , Línea Celular , Línea Celular Tumoral , Humanos , Ratones , Unión Neuromuscular , Células PC12 , RatasRESUMEN
We have produced high-titre HIV-1 green fluorescent protein-expressing lentiviral (LV) vectors pseudotyped with strain 3908 Venezuelan equine encephalitis virus glycoprotein (VEEV-G) and used them to study transduction of: (1) rat embryonic motor neuron (MN) and striatal neuron primary cultures, (2) differentiated MN cell line NSC-34 and (3) adult rat striatum. In primary neuronal cultures, transduction with VEEV-G-pseudotyped LV was more efficient and more neuronal than with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped LV. In NSC-34 cells clear retrograde transport of VEEV-G vector particles was observed. In the striatum at the injection site, transduction with the VEEV-G vectors driven by cytomegalovirus or phosphoglycerate kinase promoters exhibited a distinct neuronal tropism with no microglial and only a minor astroglial component, superior to that obtained with VSV-G-pseudotyped LV, irrespective of the promoter used. Neuronal transduction efficiency increased over time. Distal to the injection site transduction of mitral cells in the olfactory bulb, thalamic neurons and dopaminergic neurons in the substantia nigra pars compacta was detected. This, together with observations of retrograde axonal trafficking in vitro indicates that these vectors also possess low level of retrograde neuronal transduction capability in vivo. In this study, we demonstrate both strong neurotropism as well as sustainability of expression and minimal host immune response in vivo, making the VEEV-G-pseudotyped LV vectors potentially useful for gene therapy of neurodegenerative diseases.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Glicoproteínas/genética , VIH-1/genética , Neuronas Motoras/citología , Enfermedades Neurodegenerativas/terapia , Animales , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/fisiología , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Lentivirus/genética , Neuronas Motoras/fisiología , Enfermedades Neurodegenerativas/genética , Ratas , Transducción GenéticaRESUMEN
Rabies virus glycoprotein (RVG) can pseudotype lentiviral vectors, although at a lower efficiency to that of vesicular stomatitis virus glycoprotein (VSVG). Transduction with VSVG-pseudotyped vectors of rodent central nervous system (CNS) leads to local neurotropic gene transfer, whereas with RVG-pseudotyped vectors additional disperse transduction of neurons located at distal efferent sites occurs via axonal retrograde transport. Attempts to produce high-titre RVG-pseudotyped lentiviral vectors for preclinical and clinical trials has to date been problematic. We have constructed several chimeric RVG/VSVG glycoproteins and found that a construct bearing the external/transmembrane domain of RVG and the cytoplasmic domain of VSVG shows increased incorporation onto HIV-1 lentiviral particles and has increased infectivity in vitro in 293T cells and in differentiated neuronal cell lines of human, rat and murine origin. Stereotactic application of vector pseudotyped with this RVG/VSVG chimera in the rat striatum resulted in efficient gene transfer at the site of injection showing both neuronal and glial tropism. Distal neuronal transduction in the substantia nigra, thalamus and olfactory bulb via retrograde axonal transport also occurs after intrastriatal administration of chimera-pseudotyped vectors at similar levels to that observed with a RVG-pseudotyped vector. This is the first report of distal transduction in the olfactory bulb. The enhanced pseudotyping with this envelope should enable easier production of higher-titre pseudotyped lentiviral vectors that exhibit efficient local and dispersed neuronal transduction in the CNS.
Asunto(s)
Antígenos Virales/genética , Vectores Genéticos , Glicoproteínas/genética , VIH-1/genética , Glicoproteínas de Membrana/genética , Transducción Genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
The identification of genes differentially regulated by ischemia will lead to an improved understanding of cell death pathways such as those involved in the neuronal loss observed following a stroke. Furthermore, the characterization of such pathways could facilitate the identification of novel targets for stroke therapy. We have used a novel approach to amplify differential gene expression patterns in a primary neuronal model of stroke by employing a lentiviral vector system to specifically bias the transcriptional activation of hypoxically regulated genes. Overexpression of the hypoxia-induced transcription factor subunits HIF-1 alpha and HIF-2 alpha elevated hypoxia-mediated transcription of many known HIF-regulated genes well above control levels. Furthermore, many potentially novel HIF-regulated genes were discovered that were not previously identified as hypoxically regulated. Most of the novel genes identified were activated by a combination of HIF-2 alpha overexpression and hypoxic insult. These included several genes with particular importance in cell survival pathways and of potential therapeutic value. Hypoxic induction of HIF-2 alpha may therefore be a critical factor in mediating protective responses against ischemic injury. Further investigation of the genes identified in this study may provide increased understanding of the neuronal response to hypoxia and may uncover novel therapeutic targets for the treatment of cerebral ischemia.
Asunto(s)
Hipoxia de la Célula , Perfilación de la Expresión Génica/métodos , Vectores Genéticos , Neuronas/fisiología , Accidente Cerebrovascular/fisiopatología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Cultivadas , Corteza Cerebral/citología , Embrión de Mamíferos/anatomía & histología , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Lentivirus/genética , Neuronas/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
A number of potential gene therapy applications in the adult nervous system include neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. During the last five years, lentiviral vectors have developed into extremely efficient gene transfer vehicles to the nervous system, revealing a wide range of possibilities for the treatment or such disorders. This review describes the most important and recent advances in the development of lentiviral vectors as well as the demonstration of proof-of-principle in animal models of human neurodegenerative diseases.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos , Lentivirus/genética , Enfermedades Neurodegenerativas/terapia , Enfermedad de Alzheimer/terapia , Esclerosis Amiotrófica Lateral/terapia , Animales , Terapia Genética/efectos adversos , Terapia Genética/tendencias , Humanos , Enfermedad de Huntington/terapia , Sistema Nervioso/metabolismo , Trastornos Parkinsonianos/terapia , SeguridadRESUMEN
In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease.
Asunto(s)
Antígenos Virales , Transporte Axonal/fisiología , Glicoproteínas/genética , Virus de la Anemia Infecciosa Equina/fisiología , Glicoproteínas de Membrana , Sistema Nervioso/virología , Virus de la Rabia/fisiología , Rabia/virología , Proteínas del Envoltorio Viral/genética , Animales , Células Cultivadas , Cuerpo Estriado/virología , Cartilla de ADN/química , ADN Viral/análisis , Técnicas de Transferencia de Gen , Vectores Genéticos , Técnicas para Inmunoenzimas , Operón Lac/fisiología , Masculino , Ratones , Reacción en Cadena de la Polimerasa , Ratas , Ratas EndogámicasRESUMEN
Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain alpha-keto acid dehydrogenase that can result in neurodegenerative sequelae in human infants. In the present study, increased concentrations of MSUD metabolites, in particular alpha-keto isocaproic acid, specifically induced apoptosis in glial and neuronal cells in culture. Apoptosis was associated with a reduction in cell respiration but without impairment of respiratory chain function, without early changes in mitochondrial membrane potential and without cytochrome c release into the cytosol. Significantly, alpha-keto isocaproic acid also triggered neuronal apoptosis in vivo after intracerebral injection into the developing rat brain. These findings suggest that MSUD neurodegeneration may result, at least in part, from an accumulation of branched chain amino acids and their alpha-keto acid derivatives that trigger apoptosis through a cytochrome c-independent pathway.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Grupo Citocromo c/metabolismo , Enfermedad de la Orina de Jarabe de Arce/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , Respiración de la Célula/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Fragmentación del ADN , Activación Enzimática , Humanos , Membranas Intracelulares/metabolismo , Cetoácidos/metabolismo , Cetoácidos/farmacología , Leucina/metabolismo , Leucina/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuronas/patología , Ratas , Ratas WistarRESUMEN
Transient unilateral forebrain hypoxia-ischaemia (HI) in 14-day-old rats produces infarction and delayed neuronal death in the frontal cortex. Cell death can also be observed in regions distant from the primary injury, a phenomenon known as diaschisis. While apoptosis is involved in selective neuronal death, its role in infarction and diaschisis remains poorly understood. Here, we have investigated the proteolytic cleavage of poly(ADP ribose) polymerase (PARP) and the occurrence of apoptosis in the hippocampus and the cerebellum following either HI or traumatic brain injury. We demonstrate that: (i) in vitro, PARP is cleaved during apoptosis but not necrosis in cultured neuronal (N1E) cells and Swiss 3T3 fibroblasts; (ii) following HI, apoptotic cells can be detected by 4 h after injury in the hippocampus; (iii) in the ipsilateral hippocampus the appearance of cells with apoptotic morphology is preceded by a dramatic increase in PARP cleavage in the same region, starting immediately following HI and persisting for 24 h; (iv) HI also induces apoptosis in the cerebellum and, as in the hippocampus, the appearance of cells with apoptotic morphology is preceded by PARP cleavage that is greater on the side ipsilateral to forebrain injury; and (v) similarly, traumatic brain injury to the forebrain leads to PARP cleavage and apoptosis in the cerebellum. We conclude that HI injury or traumatic injury to the developing rat forebrain leads to PARP cleavage in directly affected areas and in sites distant from the primary injury that precedes the appearance of cells with apoptotic morphology. Our results are consistent with a role for apoptotic cell death in infarction and in diaschisis resulting from forebrain injury to the developing brain.
Asunto(s)
Isquemia Encefálica/metabolismo , Hipocampo/lesiones , Neuronas/citología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Prosencéfalo/lesiones , Células 3T3 , Factores de Edad , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting , Caspasas/metabolismo , Cerebelo/citología , Cerebelo/metabolismo , Fragmentación del ADN , Inhibidores Enzimáticos/farmacología , Hipocampo/irrigación sanguínea , Hipocampo/citología , Ratones , Necrosis , Agujas , Neuroblastoma , Neuronas/enzimología , Neuronas/patología , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/análisis , Prosencéfalo/irrigación sanguínea , Prosencéfalo/citología , Ratas , Ratas Wistar , Azida Sódica/farmacología , Estaurosporina/farmacología , Azul de Tripano , Células Tumorales Cultivadas , Heridas Punzantes/metabolismoAsunto(s)
Apoptosis , Grupo Citocromo c/metabolismo , Enfermedad de la Orina de Jarabe de Arce/metabolismo , Potenciales de la Membrana , Mitocondrias/fisiología , Neuronas/patología , Humanos , Enfermedad de la Orina de Jarabe de Arce/enzimología , Enfermedad de la Orina de Jarabe de Arce/patología , Mitocondrias/enzimologíaRESUMEN
We have investigated the effect of nitric oxide (NO) on apoptosis in Swiss 3T3 fibroblasts and compared it to the effect of the nitrosonium cation (NO+). Both species induced apoptosis, confirmed by electron microscopy, propidium iodide staining, DNA laddering and activation of caspases. The kinetics of triggering apoptosis were different for the two redox species: NO+ required only a 2 hour exposure, whereas NO required 24 hours. Three sources of NO were used: aqueous solutions of NO and two NO donors, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine. The time course of apoptosis induced by these two S-nitrosothiols correlated with their rate of decomposition to NO. The apoptotic effect of NO was reduced in the presence of the NO scavenger oxyhaemoglobin, or the antioxidants N-acetylcysteine and ascorbic acid, whereas in the case of NO+ these antioxidants potentiated apoptosis. Glutathione also had a potentiating effect on the cytotoxicity of NO+. This suggests that cellular antioxidants may play a role in protecting the cell from NO-induced apoptosis while NO+ may trigger apoptosis independently of oxidative stress mechanisms.
Asunto(s)
Células 3T3/citología , Apoptosis/fisiología , Cationes/metabolismo , Óxido Nítrico/metabolismo , Proteínas de Transporte Vesicular , Células 3T3/metabolismo , Células 3T3/ultraestructura , Acetilcisteína/farmacología , Animales , Antídotos/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Ácido Ascórbico/farmacología , Proteínas Portadoras/farmacología , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/farmacología , Glutatión/análogos & derivados , Glutatión/farmacología , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/farmacología , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Mitocondrias/química , Mitocondrias/metabolismo , Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacología , Oxihemoglobinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , S-Nitrosoglutatión , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Factores de TiempoRESUMEN
BACKGROUND: Animal models of carcinogenesis have been produced in transgenic mice by directing the expression of oncogenes such as SV40 T antigen and myc to different tissues by creating fusions with promoter/enhancer elements of various mammalian or viral genes. RESULTS: A transgenic mouse line was created in which SV40 T antigen is under the control of the mouse nerve growth factor (NGF) promoter. While the oncogene is expressed in a wide range of NGF producing tissues, it specifically causes the development of either neurofibromas or neurofibrosarcomas similar to those found in the human disease neurofibromatosis type 1 (NF1). These tumours are completely penetrant and appear after a mean latency of about 8 months. In contrast to the previously reported neurofibromatosis mouse model HTLV-1 tax, the tumours in these transgenic mice arise in Schwann cells rather than perineural fibroblasts and have a very restricted tissue distribution. In a cell line cloned from a neurofibroma from these mice, NGF was detected in the culture medium at levels similar to those produced by cultured primary Schwann cells. CONCLUSION: As all animal model for a heritable neurocristopathy resembling NF1, this mouse should allow study of the pathology and treatment of this disease.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Factores de Crecimiento Nervioso/genética , Neurofibromatosis 1/genética , Animales , Línea Celular , Clonación Molecular , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Ratones Transgénicos , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología , ARN/genética , ARN/metabolismoRESUMEN
We differentially screened a pool of mouse testis clones in order to identify genes important in germ cell development. One of the isolated clones was found to be expressed only in the male germ line where it is first detected at around the pachytene spermatocyte stage. This gene maps to a subregion of the t-complex in the distal inversion near, but not within, the tw18 and the th20 deletions. A comparison of the t and wild forms of the gene reveals a high degree of sequence conservation. This gene is associated with a CpG-rich island at its 5' end. It encodes a novel protein with extensive alpha-helical structure indicative of coiled-coil interactions.