RESUMEN
Purpose: Vitronectin, a cell adhesion and spreading factor, is suspected to play a role in the pathogenesis of age-related macular degeneration (AMD), as it is a major component of AMD-specific extracellular deposits (e.g., soft drusen, subretinal drusenoid deposits). The present study addressed the impact of AMD-associated non-synonymous variant rs704 in the vitronectin-encoding gene VTN on vitronectin functionality. Methods: Effects of rs704 on vitronectin expression and processing were analyzed by semi-quantitative sequencing of VTN transcripts from retinal pigment epithelium (RPE) cells generated from human induced pluripotent stem cells (hiPSCs) and from human neural retina, as well as by western blot analyses on heterologously expressed vitronectin isoforms. Binding of vitronectin isoforms to retinal and endothelial cells was analyzed by western blot. Immunofluorescence staining followed extracellular matrix (ECM) deposition in cultured RPE cells heterologously expressing the vitronectin isoforms. Adhesion of fluorescently labeled RPE or endothelial cells in dependence of recombinant vitronectin or vitronectin-containing ECM was investigated fluorometrically or microscopically. Tube formation and migration assays addressed effects of vitronectin on angiogenesis-related processes. Results: Variant rs704 affected expression, secretion, and processing but not oligomerization of vitronectin. Cell binding and influence on RPE-mediated ECM deposition differed between AMD-risk-associated and non-AMD-risk-associated protein isoforms. Finally, vitronectin affected adhesion and endothelial tube formation. Conclusions: The AMD-risk-associated vitronectin isoform exhibits increased expression and altered functionality in cellular processes related to the sub-RPE aspects of AMD pathology. Although further research is required to address the subretinal disease aspects, this initial study supports an involvement of vitronectin in AMD pathogenesis.
Asunto(s)
Degeneración Macular/genética , Vitronectina/genética , Western Blotting , Encapsulación Celular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Variación Genética/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Degeneración Macular/patología , Isoformas de Proteínas , Proteínas Recombinantes , Retina/citología , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Vitronectina/metabolismoRESUMEN
BACKGROUND: During developmental and adult neurogenesis, doublecortin is an early neuronal marker expressed when neural stem cells assume a neuronal cell fate. To understand mechanisms involved in early processes of neuronal fate decision, we investigated cell lines for their capacity to induce expression of doublecortin upon neuronal differentiation and develop in vitro reporter models using doublecortin promoter sequences. RESULTS: Among various cell lines investigated, the human teratocarcinoma cell line NTERA-2 was found to fulfill our criteria. Following induction of differentiation using retinoic acid treatment, we observed a 16-fold increase in doublecortin mRNA expression, as well as strong induction of doublecortin polypeptide expression. The acquisition of a neuronal precursor phenotype was also substantiated by the establishment of a multipolar neuronal morphology and expression of additional neuronal markers, such as Map2, betaIII-tubulin and neuron-specific enolase. Moreover, stable transfection in NTERA-2 cells of reporter constructs encoding fluorescent or luminescent genes under the control of the doublecortin promoter allowed us to directly detect induction of neuronal differentiation in cell culture, such as following retinoic acid treatment or mouse Ngn2 transient overexpression. CONCLUSION: Induction of doublecortin expression in differentiating NTERA-2 cells suggests that these cells accurately recapitulate some of the very early events of neuronal determination. Hence, the use of reporter genes under the control of the doublecortin promoter in NTERA-2 cells will help us to investigate factors involved early in the course of neuronal differentiation processes. Moreover the ease to detect the induction of a neuronal program in this model will permit to perform high throughput screening for compounds acting on the early neuronal differentiation mechanisms.
Asunto(s)
Genes Reporteros/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/citología , Neuropéptidos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Proteínas de Dominio Doblecortina , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas del Tejido Nervioso/metabolismo , Tretinoina/farmacologíaRESUMEN
Progress in the field of neurogenesis is limited by the lack of animal models allowing direct detection and analysis of living cells participating in neurogenesis. We engineered a transgenic mouse model that expresses the fluorescent reporter proteins enhanced green fluorescent protein or Discoma sp. reef coral red fluorescent protein under the control of the doublecortin (DCX) promoter, a gene specifically and transiently active in neuronal precursors and young neurons. The expression of the reporter proteins correlated with expression of the endogenous DCX protein, and with developmental and adult neurogenesis. Neurogenesis was unaffected by the presence of the fluorescent proteins. The transgenic mice allowed direct identification of the very few newly generated neurons present in the aged brain. We performed electrophysiological analysis and established that newly generated hippocampal granule cells in aged and young mice shared identical physiological properties. Hence, although the rate of neurogenesis tapers with ageing, a population of highly excitable young neurons indistinguishable to those found in younger animals is continuously generated. Therefore, maintenance of the fundamental properties of neuronal precursors even at advanced age suggests that stimulation of neurogenesis may constitute a valid strategy to counteract age-related neuronal loss and cognitive declines.
Asunto(s)
Expresión Génica/fisiología , Hipocampo/citología , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/fisiología , Neuropéptidos/metabolismo , Factores de Edad , Animales , Western Blotting/métodos , Bromodesoxiuridina/metabolismo , Carbocianinas/metabolismo , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Inmunohistoquímica/métodos , Técnicas In Vitro , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas Luminiscentes/metabolismo , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuropéptidos/genética , Técnicas de Placa-Clamp/métodos , Regiones Promotoras Genéticas/fisiologíaRESUMEN
The doublecortin (DCX) gene encodes a 40-kDa microtubule-associated protein specifically expressed in neuronal precursors of the developing and adult CNS. Due to its specific expression pattern, attention was drawn to DCX as a marker for neuronal precursors and neurogenesis, thereby underscoring the importance of its promoter identification and promoter analysis. Here, we analysed the human DCX regulatory sequence and confined it to a 3.5-kb fragment upstream of the ATG start codon. We demonstrate by transient transfection experiments that this fragment is sufficient and specific to drive expression of reporter genes in embryonic and adult neuronal precursors. The activity of this regulatory fragment overlapped with the expression of endogenous DCX and with the young neuronal markers class III beta-tubulin isotype and microtubule-associated protein Map2ab but not with glial or oligodendroglial markers. Electrophysiological data further confirmed the immature neuronal nature of these cells. Deletions within the 3.5-kb region demonstrated the relevance of specific regions containing transcription factor-binding sites. Moreover, application of neurogenesis-related growth factors in the neuronal precursor cultures suggested the lack of direct signalling of these factors on the DCX promoter construct.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Neuronas/metabolismo , Neuropéptidos/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Células Madre/metabolismo , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Western Blotting , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Linaje de la Célula/genética , Células Cultivadas , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Regulación de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Hipocampo/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/biosíntesis , Datos de Secuencia Molecular , Neuropéptidos/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos/efectos de los fármacos , Análisis de Secuencia de ADN , Eliminación de Secuencia , Células Madre/efectos de los fármacos , TransfecciónRESUMEN
Mutations in doublecortin ( DCX) affect the migration of neuronal precursor cells and cause subcortical band heterotopia and lissencephaly. DCX is known to bind and bundle microtubules; however, the impact of mutation on DCX function and its relation to the manifestation of DCX-associated disorders is still unclear. We analyzed the impact of DCX mutants on COS7 cell microtubule networks. We found that both mutant and wild type DCX are able to bind and bundle microtubules; however, mutants possess a decreased ability to perturb the mitotic machinery, to cause abnormal spindle orientation, and to impair mitotic progression. The magnitude of this decrease is proportional to the severity of the mutation-associated clinical symptoms, thereby providing a cell-based assay for the prognosis of DCX-associated neuronal migration disorders.
Asunto(s)
Corteza Cerebral/anomalías , Corteza Cerebral/patología , Proteínas Asociadas a Microtúbulos/genética , Mitosis/genética , Neuropéptidos/genética , Animales , Células COS , Movimiento Celular , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Neuronas/patología , Neuropéptidos/metabolismo , Linaje , FenotipoRESUMEN
Neural stem cells (NSCs) from the adult central nervous system are currently being investigated for their potential use in autologous cell replacement strategies. High expansion rates of NSCs in culture are crucial for the generation of a sufficient amount of cells needed for transplantation. Here, we describe efficient growth of adult NSCs in Neurobasal medium containing B27 supplement under clonal and low-density conditions in the absence of serum or conditioned medium. Expansion of up to 15-fold within 1 week was achieved on low-density NSC cultures derived from the lateral ventricle wall, the hippocampal formation, and the spinal cord of adult rats. A 27% single-cell cloning efficiency in Neurobasal/B27 combination further demonstrates its growth-promoting ability. Multipotency and nontumorgenicity of NSCs were retained despite the high rate of culture expansion. In addition, increased cell survival was obtained when Accutase, instead of trypsin, was used for enzymatic dissociation of NSC cultures. This work provides an important step toward the development of standardized protocols for highly efficient in vitro expansion of NSCs from the adult central nervous system to move more closely to the clinical use of NSCs.