RESUMEN
Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Péptidos/metabolismo , Factores de Transcripción/metabolismo , Ubiquitinas/metabolismo , Actinas/metabolismo , Anciano , Línea Celular , Tamaño de la Célula , Cuerpo Estriado/citología , Genes Reporteros , Humanos , Proteína Huntingtina , Etiquetado Corte-Fin in Situ , Microscopía Fluorescente , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Péptidos/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteína de Unión a TATA-Box , TransfecciónRESUMEN
Expanded polyglutamine (polyQ) tracts have been linked to a new class of human disease characterized by psychiatric/motor syndromes associated with specific patterns of neurodegeneration. We have used a direct viral approach to locally express expanded polyglutamine tracts fused to the green fluorescent protein (97Q-GFP) in the adult rat brain. We show that intrastriatal expression of 97Q-GFP causes the rapid formation of fibrillar, cytoplasmic, and ubiquitinated nuclear aggregates in neurons. 97Q-GFP expression also results in a specific temporal pattern of cell death in the striatum. Co-infection studies suggest that high level 97Q-GFP-expressing cells die during the first month, whereas low level 97Q-GFP-expressing neurons persist for up to 6 months after infection. These data indicate that cumulative expression of polyQ repeats throughout the life of the animal is not required to induce neuronal death, but rather acute overexpression of polyQ is toxic to adult neurons in vivo.
Asunto(s)
Degeneración Nerviosa/patología , Neuronas/citología , Péptidos/genética , Adenoviridae , Infecciones por Adenoviridae , Factores de Edad , Animales , Anticuerpos , Apoptosis/genética , Encéfalo/citología , Agregación Celular/fisiología , Núcleo Celular/patología , Cuerpo Estriado/patología , Cuerpo Estriado/fisiopatología , Citoplasma/patología , Modelos Animales de Enfermedad , Femenino , Regulación Viral de la Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros , Proteínas Fluorescentes Verdes , Enfermedad de Huntington/patología , Enfermedad de Huntington/fisiopatología , Etiquetado Corte-Fin in Situ , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/inmunología , Microinyecciones , Degeneración Nerviosa/fisiopatología , Neuronas/virología , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Sustancia Negra/patología , Sustancia Negra/fisiopatología , Transgenes/genética , Transgenes/inmunología , Ubiquitinas/metabolismoRESUMEN
We have purified and characterized a factor, from the conditioned medium of neural stem cell cultures, which is required for fibroblast growth factor 2's (FGF-2) mitogenic activity on neural stem cells. This autocrine/paracrine cofactor is a glycosylated form of cystatin C (CCg), whose N-glycosylation is required for its activity. We further demonstrated that, both in vitro and in vivo, neural stem cells undergoing cell division are immunopositive for cystatin C. Finally, we showed in vivo functional activity of CCg by demonstrating that the combined delivery of FGF-2 and CCg to the adult dentate gyrus stimulated neurogenesis. We propose that the process of neurogenesis is controlled by the cooperation between trophic factors and autocrine/paracrine cofactors, of which CCg is a prototype.
Asunto(s)
Cistatinas/química , Cistatinas/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Neuronas/metabolismo , Células Madre/metabolismo , Animales , Comunicación Autocrina/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/química , Cistatina C , Cistatinas/genética , Cistatinas/farmacología , Giro Dentado/citología , Giro Dentado/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Glicosilación , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Neuronas/citología , Neuronas/efectos de los fármacos , Comunicación Paracrina/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Ratas , Análisis de Secuencia de Proteína , Trasplante de Células Madre , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
Only a handful of the hundreds of known vertebrate retroviruses have been deliberately subverted for use as carriers of recombinant genetic material. Retroviruses receive their name from the fact that their genome undergoes conversion from RNA to DNA following infection of a host cell. Also characteristic of retroviruses and uncommon for most other types of viruses is that the genome of the retrovirus integrates itself permanently into the DNA of the host cell. Once integrated into the host genome, the inserted provirus acts as a factory for producing more retroviral RNA genomes and expressing retroviral packaging proteins. Both components combine to form viral particles that bud from the surface of the infected cells.
Asunto(s)
Enfermedades del Sistema Nervioso Central/terapia , Terapia Genética/métodos , Recombinación Genética , Retroviridae/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Técnicas de Cultivo de Célula/métodos , Enfermedades del Sistema Nervioso Central/genética , Técnicas de Transferencia de Gen , Humanos , Neuronas/citologíaRESUMEN
Our studies of the Bombyx mori ecdysone receptor (BE) revealed that, unlike the Drosophila melanogaster ecdysone receptor (DE), treatment of BE with the ecdysone agonist tebufenozide stimulated high level transactivation in mammalian cells without adding an exogenous heterodimer partner. Gel mobility shift and transfection assays with both the ultraspiracle gene product (Usp) and retinoid X receptor heterodimer partners indicated that this property of BE stems from significantly augmented heterodimer complex formation and concomitant DNA binding. We have mapped this "gain of function" to determinants within the D and E domains of BE and demonstrated that, although the D domain determinant is sufficient for high affinity heterodimerization with Usp, both determinants are necessary for high affinity interaction with retinoid X receptor. Modified BE receptors alone used as replication-defective retroviruses potently stimulated separate "reporter" viruses in all cell types examined, suggesting that BE has potentially broad utility in the modulation of transgene expression in mammalian cells.
Asunto(s)
Receptores de Ácido Retinoico/genética , Receptores de Esteroides/genética , Factores de Transcripción/genética , Activación Transcripcional , Animales , Bombyx , Línea Celular , Dimerización , Ecdisona/metabolismo , Expresión Génica , Receptores de Ácido Retinoico/metabolismo , Receptores de Esteroides/metabolismo , Receptores X Retinoide , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
The delivery of biologically active factors to the developing mammalian embryo by in utero gene transfer has generated considerable interest but limited success. The chorioallantoic placenta is a potential alternative target for providing therapeutic transgenes to the fetus during gestation. We demonstrate that somatic gene transfer to the midgestation rat placenta may be efficiently accomplished in situ through the implantation of a variety of genetically modified cells with different antigenic and growth properties. Ex vivo-modified cells survived and retained transgene expression until term. Proteins secreted from the transplanted cells were detectable within the fetal trunk blood. These studies suggest that gene transfer to the placenta may be a useful tool for answering questions of both embryonic and placental development and providing therapeutic proteins during gestation for amelioration of diseases with onset during embryonic life.
Asunto(s)
Feto , Técnicas de Transferencia de Gen , Placenta , Animales , Línea Celular , Trasplante de Células , Desarrollo Embrionario y Fetal , Femenino , Enfermedades Fetales/terapia , Feto/citología , Feto/fisiología , Edad Gestacional , Hormona de Crecimiento Humana/biosíntesis , Hormona de Crecimiento Humana/genética , Humanos , Operón Lac , Placenta/citología , Placenta/fisiología , Placentación , Embarazo , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Retroviridae/genéticaRESUMEN
To investigate the molecular mechanisms of cholinergic sprouting in the hippocampus after removal of entorhinal cortical inputs, we evaluated trophic factor gene expression in the denervated hippocampus. Despite the proposed role for nerve growth factor (NGF) in this sprouting, we observed no change in NGF mRNA or protein at several postlesion time points. In contrast, FGF-2 mRNA was increased within 16 hr. FGF-2 immunoreactivity was localized within GFAP-positive hypertrophic astrocytes distributed specifically within the denervated outer molecular layer after the lesion. To address the functional significance of this increase in FGF-2, we assessed the magnitude of cholinergic sprouting in animals receiving chronic intracerebroventricular infusions of neutralizing antibodies specific for FGF-2 and compared it with that observed in lesioned animals receiving infusate controls. Animals given FGF-2 antibodies displayed a marked reduction in cholinergic sprouting as compared with controls. In fact, many of these animals exhibited virtually no sprouting at all despite histological verification of complete lesions. These results suggest that endogenous FGF-2 promotes cholinergic axonal sprouting in the injured adult brain. Furthermore, immunocytochemical localization of receptors for FGF-2 (i.e., FGFR1) on projecting basal forebrain cholinergic neurons suggests that FGF-2 acts directly on these neurons to induce the lesion-induced sprouting response.
Asunto(s)
Astrocitos/fisiología , Factor 2 de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Hipocampo/fisiología , Factores de Crecimiento Nervioso/genética , Neuronas/fisiología , Vía Perforante/fisiología , Receptores de Factor de Crecimiento Nervioso/genética , Transcripción Genética , Animales , Desnervación , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Factores de Crecimiento Nervioso/análisis , Biosíntesis de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoAsunto(s)
Cuerpo Estriado/cirugía , Rechazo de Injerto/prevención & control , Células de Sertoli/trasplante , Trasplante Heterólogo/inmunología , Trasplante Heterotópico/inmunología , Animales , Encéfalo , Bovinos , Células Cromafines/inmunología , Células Cromafines/trasplante , Masculino , Enfermedad de Parkinson/terapia , Ratas , Células de Sertoli/inmunología , Receptor fas/inmunologíaRESUMEN
The dentate gyrus of the hippocampus is one of the few areas of the adult brain that undergoes neurogenesis. In the present study, cells capable of proliferation and neurogenesis were isolated and cultured from the adult rat hippocampus. In defined medium containing basic fibroblast growth factor (FGF-2), cells can survive, proliferate, and express neuronal and glial markers. Cells have been maintained in culture for 1 year through multiple passages. These cultured adult cells were labeled in vitro with bromodeoxyuridine and adenovirus expressing beta-galactosidase and were transplanted to the adult rat hippocampus. Surviving cells were evident through 3 months postimplantation with no evidence of tumor formation. Within 2 months postgrafting, labeled cells were found in the dentate gyrus, where they differentiated into neurons only in the intact region of the granule cell layer. Our results indicate that FGF-2 responsive progenitors can be isolated from the adult hippocampus and that these cells retain the capacity to generate mature neurons when grafted into the adult rat brain.
Asunto(s)
Trasplante de Tejido Encefálico , Técnicas de Cultivo/métodos , Hipocampo/citología , Hipocampo/cirugía , Neuronas/trasplante , Trasplante de Células Madre , Animales , Biomarcadores , Diferenciación Celular , Supervivencia Celular , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Técnica del Anticuerpo Fluorescente , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Ratas , Ratas Endogámicas F344 , Células Madre/efectos de los fármacos , Células Madre/ultraestructuraRESUMEN
Neurotrophin 3 (NT3) belongs to the neurotrophin family, which also includes nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 4/5. NT3 mRNA is widely expressed in the rodent nervous system, but the physiological function of the native protein is still unclear. Genetically modified cell lines that produce physiological amounts of NT3 can provide a useful tool in the elucidation of the NT3 effects in the adult central nervous system (CNS). Genetically modified rat primary skin fibroblasts expressing and secreting human NT3 (hNT3) were prepared and characterized. In vitro, cell lines derived from different retroviral constructs expressed hNT3 mRNA, as determined by PCR and RNA blot analysis. Secretion of biologically active hNT3 was confirmed by specific elicitation of neurite outgrowth from cultured chick primary sympathetic and sensory neurons and from rat fetal locus coeruleus neurons in the presence of hNT3-producing cell conditioned media. In vivo, implanted fibroblasts survived well up to the maximal experimental time points of 6 weeks (brain) and 4 weeks (spinal cord) and continued to express hNT3 mRNA in vivo. As early as 2 weeks postgrafting, specific sprouting of host sensory neurites in response to hNT3-producing grafts was observed in the spinal cord. In contrast, hNT3-producing cerebral grafts did not induce a sprouting response different from that observed with control grafts. These findings establish the existence of a regionally different responsiveness of the CNS axons to local hNT3 overexpression.
Asunto(s)
Fibroblastos/fisiología , Factores de Crecimiento Nervioso/genética , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/fisiología , Femenino , Fibroblastos/trasplante , Ganglios Simpáticos/metabolismo , Ganglios Simpáticos/fisiología , Hipocampo/metabolismo , Hipocampo/fisiología , Humanos , Locus Coeruleus/metabolismo , Locus Coeruleus/fisiología , Sondas Moleculares , Datos de Secuencia Molecular , Neurotrofina 3 , Ratas , Médula Espinal/metabolismo , Médula Espinal/fisiologíaRESUMEN
The cholinergic system plays a crucial role in learning and memory. Lesions of cholinergic nuclei, pharmacological manipulations of cholinergic systems, intracerebral transplantation of fetal tissue and anatomical changes in cholinergic pathways during ageing have all been correlated with altered cognitive behaviour. However, it has not been proved that regional acetylcholine is causally required for learning and memory. Here we describe how we achieved a permanent and selective impairment of learning and memory by damaging the nucleus basalis magnocellularis, a nucleus that provides the major cholinergic innervation of the neocortex, in adult rats. To test the hypothesis that acetylcholine is essential for restoration of cognitive function, we implanted genetically modified cells that produce acetylcholine into denervated neocortical target regions. After grafting, rats with increased neocortical acetylcholine levels showed a significant improvement in a spatial navigation task. Acetylcholine is thus not only necessary for learning and memory, as previously argued, but its presence within the neocortex is also sufficient to ameliorate learning deficits and restore memory following damage to the nucleus basalis.
Asunto(s)
Acetilcolina/fisiología , Corteza Cerebral/fisiología , Memoria/fisiología , Percepción Espacial/fisiología , Acetilcolina/genética , Animales , Secuencia de Bases , Conducta Animal , Cartilla de ADN , Desnervación , Fibroblastos/metabolismo , Fibroblastos/trasplante , Aprendizaje/fisiología , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas F344 , Médula Espinal/fisiologíaRESUMEN
The molecular characterization of GHRH and the GHRH receptor provides a framework for understanding the hypothalamic regulation of pituitary somatotroph function. The signaling events discerned from our investigation of GHRH receptor structure and function form the basis of a model for GHRH action, which is shown in Fig. 20. GHRH interaction with its seven transmembrane domain Gs-coupled receptor on the somatotroph (step 1) leads to the release of growth hormone from secretory granules (step 2), which is likely to involve a G protein-mediated interaction with ion channels, and to a stimulation of intracellular cAMP accumulation (step 3) (Mayo, 1992; Lin et al., 1992; Gaylinn et al., 1993). In several cell types tested, elevated cAMP leads to the phosphorylation and activation of the transcription factor CREB by protein kinase A (Gonzalez and Montminy, 1989; Sheng et al., 1991), and one target gene for CREB action is the pituitary-specific transcription factor Pit-1 or GHF-1 (step 4) (Bodner et al., 1988; Ingraham et al., 1988; McCormick et al., 1990). Pit-1 is a prototypic POU domain protein that is required for the appropriate regulation of the growth hormone gene in somatotroph cells, thus providing a pathway by which a GHRH signal can lead to increased growth hormone synthesis in the pituitary (step 5). In addition, Pit-1 is likely to directly regulate the synthesis of the GHRH receptor (step 6), in that the receptor is not expressed in the pituitary of dw/dw mice that lack functional Pit-1 (Lin et al., 1992), and a cotransfected Pit-1 expression construct can activate the GHRH receptor promoter in transiently transfected CV1 cells (Lin et al., 1993). It remains to be determined whether additional direct regulation of the GHRH receptor gene in response to the cAMP signaling pathway occurs (step 7). The inhibitory peptide somatostatin presumably interacts with this same signaling pathway through G protein-mediated suppression of the cAMP pathway (Tallent and Reisine, 1992; Bell and Reisine, 1993). In agreement with the importance of this signaling system for normal growth, a transgene encoding a nonphosphorylatable mutant CREB protein, which blocks the function of the endogenous CREB protein, is able to cause somatotroph hypoplasia and dwarfism in mice when its expression is targeted to pituitary somatotrophs (Struthers et al., 1991). Several steps in the signaling pathway leading to growth hormone secretion are subject to disruption, resulting in growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/biosíntesis , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Cartilla de ADN/genética , Femenino , Expresión Génica , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/fisiología , Humanos , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Hipófisis/fisiología , Embarazo , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/fisiología , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/fisiología , Homología de Secuencia de Aminoácido , Transducción de Señal , Distribución TisularRESUMEN
Gene therapy is a potentially potent new method of treating a number of neurologic disorders previously considered refractory to current conventional therapeutic treatments. Numerous advances have been made in the construction of expression vectors, cellular and viral transgene carriers, and the characterization of target cells for neuronal gene therapy. Two primary approaches to nervous system gene transfer have emerged as a result of these advances. The in vivo approach concentrates on direct transfer of genetic material to cells in vivo using viral and chemical agents. The ex vivo approach relies on genetic transfer to cultured cells that are subsequently implanted into a host organism. Both of these methods have been used in preliminary experiments designed to test the efficacy of gene transfer strategies in the amelioration of nervous system dysfunction.
Asunto(s)
Terapia Genética , Enfermedades del Sistema Nervioso/terapia , Adenoviridae/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/terapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , División Celular , ADN/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Técnicas de Transferencia de Gen , Marcadores Genéticos , Vectores Genéticos , Heterocigoto , Humanos , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/terapia , Enfermedades del Sistema Nervioso/genética , Neuronas/citología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/terapia , Regiones Promotoras Genéticas , Retroviridae/genética , Moldes GenéticosRESUMEN
To pursue questions concerning the regulation of somatic growth in a species amenable to both genetics and germ-line manipulation, we have isolated and characterized a full-length cDNA clone encoding mouse GH-releasing hormone (mGHRH). A GHRH cDNA clone isolated from a mouse placental library contains an open-reading frame of 309 basepairs that predicts a 103 amino acid mouse GHRH precursor protein. The mature mouse GHRH is predicted to be 42 amino acids with a free carboxyl-terminus. Although the mGHRH precursor sequence is clearly related to those determined for rat and human, the mature mGHRH peptide differs at seven of its 42 positions from all previously characterized GHRH peptides. RNA blot analysis of mouse tissues indicates that the mature 750 nucleotide mGHRH mRNA is found in hypothalamus and placenta, while testis contains a larger GHRH-related transcript. In situ hybridization analysis of GHRH gene expression in the mouse brain indicates that GHRH mRNA is localized predominantly to the arcuate nucleus of the hypothalamus. In the placenta, GHRH mRNA levels are developmentally regulated and peak on days 16-17 of gestation. GHRH mRNA is localized predominantly to trophoblast giant cells and to cytotrophoblasts of the placental labyrinth.