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1.
Int J Mol Sci ; 24(19)2023 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-37834345

RESUMEN

Neurological diseases, including neurodegenerative and neurodevelopmental disorders, affect nearly one in six of the world's population. The burden of the resulting deaths and disability is set to rise during the next few decades as a consequence of an aging population. To address this, zebrafish have become increasingly prominent as a model for studying human neurological diseases and exploring potential therapies. Zebrafish offer numerous benefits, such as genetic homology and brain similarities, complementing traditional mammalian models and serving as a valuable tool for genetic screening and drug discovery. In this comprehensive review, we highlight various drug delivery techniques and systems employed for therapeutic interventions of neurological diseases in zebrafish, and evaluate their suitability. We also discuss the challenges encountered during this process and present potential advancements in innovative techniques.


Asunto(s)
Enfermedades del Sistema Nervioso , Trastornos del Neurodesarrollo , Animales , Humanos , Anciano , Pez Cebra/genética , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Encéfalo , Modelos Animales de Enfermedad , Mamíferos
2.
Int J Mol Sci ; 23(19)2022 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-36232902

RESUMEN

Traumatic brain injury (TBI) has the highest mortality rates worldwide, yet effective treatment remains unavailable. TBI causes inflammatory responses, endoplasmic reticulum stress, disruption of the blood-brain barrier and neurodegeneration that lead to loss of cognition, memory and motor skills. Saffron (Crocus sativus L.) is known for its anti-inflammatory and neuroprotective effects, which makes it a potential candidate for TBI treatment. Zebrafish (Danio rerio) shares a high degree of genetic homology and cell signaling pathways with mammals. Its active neuro-regenerative function makes it an excellent model organism for TBI therapeutic drug identification. The objective of this study was to assess the effect of saffron administration to a TBI zebrafish model by investigating behavioral outcomes such as anxiety, fear and memory skills using a series of behavioral tests. Saffron exhibited anxiolytic effect on anxiety-like behaviors, and showed prevention of fear inhibition observed after TBI. It improved learning and enhanced memory performance. These results suggest that saffron could be a novel therapeutic enhancer for neural repair and regeneration of networks post-TBI.


Asunto(s)
Ansiolíticos , Lesiones Traumáticas del Encéfalo , Crocus , Fármacos Neuroprotectores , Animales , Ansiolíticos/farmacología , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Cognición , Mamíferos , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Pez Cebra
3.
Ann Neurol ; 86(5): 729-742, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31393621

RESUMEN

OBJECTIVE: CLN3 disease is the commonest of the neuronal ceroid lipofuscinoses, a group of pediatric neurodegenerative disorders. Functions of the CLN3 protein include antiapoptotic properties and facilitating anterograde transport of galactosylceramide from Golgi to lipid rafts. This study confirms the beneficial effects of long-term exogenous galactosylceramide supplementation on longevity, neurobehavioral parameters, neuronal cell counts, astrogliosis, and diminution in brain and serum ceramide levels in Cln3 Δex7/8 knock-in mice. Additionally, the impact of galactosylceramide on ceramide synthesis enzymes is documented. METHODS: A group of 72 mice received galactosylceramide or vehicle for 40 weeks. The effect of galactosylceramide supplementation on Cln3 Δex7/8 mice was determined by performing behavioral tests, measuring ceramide in brains and serum, and assessing impact on longevity, subunit C storage, astrogliosis, and neuronal cell counts. RESULTS: Galactosylceramide resulted in enhanced grip strength of forelimbs in male and female mice, better balance on the accelerating rotarod in females, and improved motor coordination during pole climbing in male mice. Brain and serum ceramide levels as well as apoptosis rates were lower in galactosylceramide-treated Cln3 Δex7/8 mice. Galactosylceramide also increased neuronal cell counts significantly in male and female mice and tended to decrease subunit C storage in specific brain regions. Astrogliosis dropped in females compared to a slight increase in males after galactosylceramide. Galactosylceramide increased the lifespan of affected mice. INTERPRETATION: Galactosylceramide improved behavioral, neuropathological, and biochemical parameters in Cln3 Δex7/8 mice, paving the way for effective therapy for CLN3 disease and use of serum ceramide as a potential biomarker to track impact of therapies. ANN NEUROL 2019;86:729-742.


Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/patología , Galactosilceramidas/farmacología , Lipofuscinosis Ceroideas Neuronales/patología , Animales , Conducta Animal/efectos de los fármacos , Femenino , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL
4.
Genes Dev ; 25(8): 831-44, 2011 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-21498572

RESUMEN

Neural stem cells (NSCs) are slowly dividing astrocytes that are intimately associated with capillary endothelial cells in the subventricular zone (SVZ) of the brain. Functionally, members of the vascular endothelial growth factor (VEGF) family can stimulate neurogenesis as well as angiogenesis, but it has been unclear whether they act directly via VEGF receptors (VEGFRs) expressed by neural cells, or indirectly via the release of growth factors from angiogenic capillaries. Here, we show that VEGFR-3, a receptor required for lymphangiogenesis, is expressed by NSCs and is directly required for neurogenesis. Vegfr3:YFP reporter mice show VEGFR-3 expression in multipotent NSCs, which are capable of self-renewal and are activated by the VEGFR-3 ligand VEGF-C in vitro. Overexpression of VEGF-C stimulates VEGFR-3-expressing NSCs and neurogenesis in the SVZ without affecting angiogenesis. Conversely, conditional deletion of Vegfr3 in neural cells, inducible deletion in subventricular astrocytes, and blocking of VEGFR-3 signaling with antibodies reduce SVZ neurogenesis. Therefore, VEGF-C/VEGFR-3 signaling acts directly on NSCs and regulates adult neurogenesis, opening potential approaches for treatment of neurodegenerative diseases.


Asunto(s)
Neurogénesis/fisiología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Linfangiogénesis/genética , Linfangiogénesis/fisiología , Ratones , Ratones Mutantes , Microscopía Electrónica de Transmisión , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética
5.
Circ Res ; 111(4): 437-45, 2012 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-22723296

RESUMEN

RATIONALE: The lymphatic vasculature plays a major role in fluid homeostasis, absorption of dietary lipids, and immune surveillance. Fluid transport depends on the presence of intraluminal valves within lymphatic collectors. Defective formation of lymphatic valves leads to lymphedema, a progressive and debilitating condition for which curative treatments are currently unavailable. How lymphatic valve formation is regulated remains largely unknown. OBJECTIVE: We investigated if the repulsive axon guidance molecule Semaphorin3A (Sema3A) plays a role in lymphatic valve formation. METHODS AND RESULTS: We show that Sema3A mRNA is expressed in lymphatic vessels and that Sema3A protein binds to lymphatic valves expressing the Neuropilin-1 (Nrp1) and PlexinA1 receptors. Using mouse knockout models, we show that Sema3A is selectively required for lymphatic valve formation, via interaction with Nrp1 and PlexinA1. Sema3a(-/-) mice exhibit defects in lymphatic valve formation, which are not due to abnormal lymphatic patterning or sprouting, and mice carrying a mutation in the Sema3A binding site of Nrp1, or deficient for Plxna1, develop lymphatic valve defects similar to those seen in Sema3a(-/-) mice. CONCLUSIONS: Our data demonstrate an essential direct function of Sema3A-Nrp1-PlexinA1 signaling in lymphatic valve formation.


Asunto(s)
Vasos Linfáticos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1/metabolismo , Receptores de Superficie Celular/metabolismo , Semaforina-3A/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos Neutralizantes/administración & dosificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Edad Gestacional , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Vasos Linfáticos/embriología , Ratones , Ratones Noqueados , Ratones Transgénicos , Morfogénesis , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Neuropilina-1/deficiencia , Neuropilina-1/genética , Neuropilina-1/inmunología , Fenotipo , ARN Mensajero/metabolismo , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Semaforina-3A/deficiencia , Semaforina-3A/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo
6.
Transl Psychiatry ; 13(1): 404, 2023 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-38129378

RESUMEN

The landscape of autism spectrum disorder (ASD) in Lebanon is unique because of high rates of consanguinity, shared ancestry, and increased remote consanguinity. ASD prevalence in Lebanon is 1 in 68 with a male-to-female ratio of 2:1. This study aims to investigate the impact of an inherited deletion in UBLCP1 (Ubiquitin-Like Domain-Containing CTD Phosphatase 1) on the ubiquitin-proteasome system (UPS) and proteolysis. Whole exome sequencing in a Lebanese family with ASD without pathogenic copy number variations (CNVs) uncovered a deletion in UBLCP1. Functional evaluation of the identified variant is described in fibroblasts from the affected. The deletion in UBLCP1 exon 10 (g.158,710,261CAAAG > C) generates a premature stop codon interrupting the phosphatase domain and is predicted as pathogenic. It is absent from databases of normal variation worldwide and in Lebanon. Wild-type UBLCP1 is widely expressed in mouse brains. The mutation results in decreased UBLCP1 protein expression in patient-derived fibroblasts from the autistic patient compared to controls. The truncated UBLCP1 protein results in increased proteasome activity decreased ubiquitinated protein levels, and downregulation in expression of other proteasome subunits in samples from the affected compared to controls. Inhibition of the proteasome by using MG132 in proband cells reverses alterations in gene expression due to the restoration of protein levels of the common transcription factor, NRF1. Finally, treatment with gentamicin, which promotes premature termination codon read-through, restores UBLCP1 expression and function. Discovery of an ASD-linked mutation in UBLCP1 leading to overactivation of cell proteolysis is reported. This, in turn, leads to dysregulation of proteasome subunit transcript levels as a compensatory response.


Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Animales , Femenino , Humanos , Masculino , Ratones , Trastorno del Espectro Autista/genética , Variaciones en el Número de Copia de ADN , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinas/genética
7.
PLoS One ; 15(10): e0239537, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33006978

RESUMEN

BACKGROUND: CLN3 disease is caused by mutations in the CLN3 gene. The purpose of this study is to discern global expression patterns reflecting therapeutic targets in CLN3 disease. METHODS: Differential gene expression in vehicle-exposed mouse brain was determined after intraperitoneal vehicle/Galactosylceramide (GalCer) injections for 40 weeks with GeneChip Mouse Genome 430 2.0 arrays. RESULTS: Analysis identified 66 genes in male and 30 in female brains differentially expressed in GalCer-treated versus vehicle-exposed Cln3Δex7/8 mice. Gene ontology revealed aberrations of biological function including developmental, cellular, and behavioral processes. GalCer treatment altered pathways of long-term potentiation/depression, estrogen signaling, synaptic vesicle cycle, ErbB signaling, and prion diseases in males, but prolactin signaling, selenium compound metabolism and steroid biosynthesis in females. Gene-gene network analysis highlighted networks functionally pertinent to GalCer treatment encompassing motor dysfunction, neurodegeneration, memory disorder, inflammation and astrogliosis in males, and, cataracts, inflammation, astrogliosis, and anxiety in females. CONCLUSIONS: This study sheds light on global expression patterns following GalCer treatment of Cln3Δex7/8 mice. Understanding molecular effects of GalCer on mouse brain gene expression, paves the way for personalized strategies for treating this debilitating disease in humans.


Asunto(s)
Galactosilceramidas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Caracteres Sexuales , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Femenino , Ontología de Genes , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos
8.
Front Neurol ; 10: 128, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30837943

RESUMEN

CLN3 disease is a neurodevelopmental disease leading to early visual failure, motor decline, and death. CLN3 pathogenesis has been linked to dysregulation of ceramide, a key intracellular messenger impacting various biological functions. Ceramide is upregulated in brains of CLN3 patients and activates apoptosis. Ceramide levels over the lifespan of WT and Cln3 Δex7/8 mice were measured using the DGK assay. Ceramide subspecies were determined by LC-MS. Ceramide synthesis enzymes and pre- and post-synaptic mRNA expression was measured in Cln3 Δex7/8 and normal mouse brains. Neuronal cell death was established by PARP cleavage and Caspases 3/6/9 and cytochrome C mRNA expression in Cln3 Δex7/8 and normal mouse brains. In WT mouse, a ceramide peak was noted at 3 weeks of age. The absence of this peak in Cln3 Δex7/8 mice might be related to early disease pathogenesis. Increase of ceramide in Cln3 Δex7/8 mouse brain at 24 weeks of age precedes neuronal apoptosis. The correlation between serum and brain ceramide in WT mice, and dysregulation of ceramide in serum and brain of Cln3 Δex7/8 mice, and the significant increase in ceramide in Cln3 Δex7/8 mouse brains and sera argue for use of easily accessible serum ceramide levels to track response to novel therapies in human CLN3 disease.

9.
Front Neurol ; 9: 895, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30405520

RESUMEN

Traumatic brain injury (TBI) is a major cause of death worldwide. Depending on the severity of the injury, TBI can reflect a broad range of consequences such as speech impairment, memory disturbances, and premature death. In this study, embryonic neural stem cells (ENSC) were isolated from E14 mouse embryos and cultured to produce neurospheres which were induced to generate differentiated cells (DC). As a cell replacement treatment option, we aimed to transplant ENSC or DC into the adult injured C57BL/6 mouse cortex controlled cortical impact (CCI) model, 7 days post-trauma, in comparison to saline injection (control). The effect of grafted cells on neuroinflammation and neurogenesis was investigated at 1 and 4 weeks post-transplantation. Results showed that microglia were activated following mild CCI, but not enhanced after engraftment of ENSC or DC. Indeed, ipsilateral lesioned somatosensory area expressed high levels of Iba-1+ microglia within the different groups after 1 and 4 weeks. On the other hand, treatment with ENSC or DC demonstrated a significant reduction in astrogliosis. The levels of GFAP expressing astrocytes started decreasing early (1 week) in the ENSC group and then were similarly low at 4 weeks in both ENSC and DC. Moreover, neurogenesis was significantly enhanced in ENSC and DC groups. Indeed, a significant increase in the number of DCX expressing progenitor cells was observed at 1 week in the ENSC group, and in DC and ENSC groups at 4 weeks. Furthermore, the number of mature neuronal cells (NeuN+) significantly increased in DC group at 4 weeks whereas they decreased in ENSC group at 1 week. Therefore, injection of ENSC or DC post-CCI caused decreased astrogliosis and suggested an increased neurogenesis via inducing neural progenitor proliferation and expression rather than neuronal maturation. Thus, ENSC may play a role in replacing lost cells and brain repair following TBI by improving neurogenesis and reducing neuroinflammation, reflecting an optimal environment for transplanted and newly born cells.

10.
Behav Brain Res ; 340: 1-13, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29126932

RESUMEN

Traumatic Brain Injury (TBI) is a major cause of death and disability worldwide with 1.5 million people inflicted yearly. Several neurotherapeutic interventions have been proposed including drug administration as well as cellular therapy involving neural stem cells (NSCs). Among the proposed drugs is docosahexaenoic acid (DHA), a polyunsaturated fatty acid, exhibiting neuroprotective properties. In this study, we utilized an innovative intervention of neonatal NSCs transplantation in combination with DHA injections in order to ameliorate brain damage and promote functional recovery in an experimental model of TBI. Thus, NSCs derived from the subventricular zone of neonatal pups were cultured into neurospheres and transplanted in the cortex of an experimentally controlled cortical impact mouse model of TBI. The effect of NSC transplantation was assessed alone and/or in combination with DHA administration. Motor deficits were evaluated using pole climbing and rotarod tests. Using immunohistochemistry, the effect of transplanted NSCs and DHA treatment was used to assess astrocytic (Glial fibrillary acidic protein, GFAP) and microglial (ionized calcium binding adaptor molecule-1, IBA-1) activity. In addition, we quantified neuroblasts (doublecortin; DCX) and dopaminergic neurons (tyrosine hydroxylase; TH) expression levels. Combined NSC transplantation and DHA injections significantly attenuated TBI-induced motor function deficits (pole climbing test), promoted neurogenesis, coupled with an increase in glial reactivity at the cortical site of injury. In addition, the number of tyrosine hydroxylase positive neurons was found to increase markedly in the ventral tegmental area and substantia nigra in the combination therapy group. Immunoblotting analysis indicated that DHA+NSCs treated animals showed decreased levels of 38kDa GFAP-BDP (breakdown product) and 145kDa αII-spectrin SBDP indicative of attenuated calpain/caspase activation. These data demonstrate that prior treatment with DHA may be a desirable strategy to improve the therapeutic efficacy of NSC transplantation in TBI.


Asunto(s)
Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/cirugía , Ácidos Docosahexaenoicos/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/trasplante , Fármacos Neuroprotectores/farmacología , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Encéfalo/fisiopatología , Encéfalo/cirugía , Lesiones Traumáticas del Encéfalo/patología , Lesiones Traumáticas del Encéfalo/fisiopatología , Células Cultivadas , Terapia Combinada , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Neuronas Dopaminérgicas/fisiología , Proteína Doblecortina , Masculino , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Células-Madre Neurales/patología , Células-Madre Neurales/fisiología , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Neuroglía/efectos de los fármacos , Neuroglía/patología , Neuroglía/fisiología , Distribución Aleatoria , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Nicho de Células Madre , Trasplante de Células Madre/métodos
11.
Sci Rep ; 7: 45336, 2017 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-28358038

RESUMEN

Autism spectrum disorder (ASD) is characterized by ritualistic-repetitive behaviors and impaired verbal/non-verbal communication. Many ASD susceptibility genes implicated in neuronal pathways/brain development have been identified. The Lebanese population is ideal for uncovering recessive genes because of shared ancestry and a high rate of consanguineous marriages. Aims here are to analyze for published ASD genes and uncover novel inherited ASD susceptibility genes specific to the Lebanese. We recruited 36 ASD families (ASD: 37, unaffected parents: 36, unaffected siblings: 33) and 100 unaffected Lebanese controls. Cytogenetics 2.7 M Microarrays/CytoScan™ HD arrays allowed mapping of homozygous regions of the genome. The CNTNAP2 gene was screened by Sanger sequencing. Homozygosity mapping uncovered DPP4, TRHR, and MLF1 as novel candidate susceptibility genes for ASD in the Lebanese. Sequencing of hot spot exons in CNTNAP2 led to discovery of a 5 bp insertion in 23/37 ASD patients. This mutation was present in unaffected family members and unaffected Lebanese controls. Although a slight increase in number was observed in ASD patients and family members compared to controls, there were no significant differences in allele frequencies between affecteds and controls (C/TTCTG: γ2 value = 0.014; p = 0.904). The CNTNAP2 polymorphism identified in this population, hence, is not linked to the ASD phenotype.


Asunto(s)
Trastorno del Espectro Autista/genética , Dipeptidil Peptidasa 4/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas/genética , Receptores de Hormona Liberadora de Tirotropina/genética , Adolescente , Proteínas de Ciclo Celular , Niño , Preescolar , Consanguinidad , Proteínas de Unión al ADN , Femenino , Predisposición Genética a la Enfermedad , Herencia , Homocigoto , Humanos , Líbano , Masculino , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Linaje , Análisis de Secuencia de ADN , Adulto Joven
12.
Sci Rep ; 6: 19088, 2016 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-26742492

RESUMEN

Autism Spectrum Disorders (ASDs) are a group of neurodevelopmental disorders characterized by ritualistic-repetitive behaviors and impaired verbal and non-verbal communication. Objectives were to determine the contribution of genetic variation to ASDs in the Lebanese. Affymetrix Cytogenetics Whole-Genome 2.7 M and CytoScan(™) HD Arrays were used to detect CNVs in 41 Lebanese autistic children and 35 non-autistic, developmentally delayed and intellectually disabled patients. 33 normal participants were used as controls. 16 de novo CNVs and 57 inherited CNVs, including recognized pathogenic 16p11.2 duplications and 2p16.3 deletions were identified. A duplication at 1q43 classified as likely pathogenic encompasses RYR2 as a potential ASD candidate gene. This previously identified CNV has been classified as both pathogenic, and, of uncertain significance. A duplication of unknown significance at 10q11.22, proposed as a modulator for phenotypic disease expression in Rett syndrome, was also identified. The novel potential autism susceptibility genes PTDSS1 and AREG were uncovered and warrant further genetic and functional analyses. Previously described and novel genetic targets in ASD were identified in Lebanese families with autism. These findings may lead to improved diagnosis of ASDs and informed genetic counseling, and may also lead to untapped therapeutic targets applicable to Lebanese and non-Lebanese patients.


Asunto(s)
Anfirregulina/genética , Trastorno del Espectro Autista/genética , Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN , Discapacidades del Desarrollo/genética , Transferasas de Grupos Nitrogenados/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Adolescente , Anfirregulina/deficiencia , Trastorno del Espectro Autista/fisiopatología , Estudios de Casos y Controles , Niño , Preescolar , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 10 , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 2 , Discapacidades del Desarrollo/fisiopatología , Femenino , Eliminación de Gen , Genoma Humano , Humanos , Líbano , Masculino , Transferasas de Grupos Nitrogenados/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/deficiencia
13.
Methods Mol Biol ; 1462: 689-710, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27604746

RESUMEN

Traumatic brain injury (TBI) is one of the leading causes of death and disabilities worldwide. It affects approximately 1.5 million people each year and is associated with severe post-TBI symptoms such as sensory and motor deficits. Several neuro-therapeutic approaches ranging from cell therapy interventions such as the use of neural stem cells (NSCs) to drug-based therapies have been proposed for TBI management. Successful cell-based therapies are tightly dependent on reproducible preclinical animal models to ensure safety and optimal therapeutic benefits. In this chapter, we describe the isolation of NSCs from neonatal mouse brain using the neurosphere assay in culture. Subsequently, dissociated neurosphere-derived cells are used for transplantation into the ipsilateral cortex of a controlled cortical impact (CCI) TBI model in C57BL/6 mice. Following intra-cardiac perfusion and brain removal, the success of NSC transplantation is then evaluated using immunofluorescence in order to assess neurogenesis along with gliosis in the ipsilateral coronal brain sections. Behavioral tests including rotarod and pole climbing are conducted to evaluate the motor activity post-treatment intervention.


Asunto(s)
Lesiones Traumáticas del Encéfalo/etiología , Lesiones Traumáticas del Encéfalo/terapia , Células-Madre Neurales/citología , Trasplante de Células Madre , Animales , Conducta Animal , Biomarcadores , Lesiones Traumáticas del Encéfalo/diagnóstico , Lesiones Traumáticas del Encéfalo/fisiopatología , Técnicas de Cultivo de Célula , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Ratones , Células-Madre Neurales/metabolismo , Recuperación de la Función , Prueba de Desempeño de Rotación con Aceleración Constante , Resultado del Tratamiento
14.
PLoS One ; 10(6): e0128601, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26047500

RESUMEN

Traumatic Brain Injury (TBI) is the result of a mechanical impact on the brain provoking mild, moderate or severe symptoms. It is acknowledged that TBI leads to apoptotic and necrotic cell death; however, the exact mechanism by which brain trauma leads to neural injury is not fully elucidated. Some studies have highlighted the pivotal role of the Kallikrein-Kinin System (KKS) in brain trauma but the results are still controversial and inconclusive. In this study, we investigated both the expression and the role of Bradykinin 1 and 2 receptors (B1R and B2R), in mediating neuronal injury under chemical neurotoxicity paradigm in PC12 cell lines. The neuronal cell line PC12 was treated with the apoptotic drug Staurosporine (STS) to induce cell death. Intracellular calcium release was evaluated by Fluo 4-AM staining and showed that inhibition of the B2R prevented calcium release following STS treatment. Differential analyses utilizing immunofluorescence, Western blot and Real-time Polymerase Chain Reaction revealed an upregulation of both bradykinin receptors occurring at 3h and 12h post-STS treatment, but with a higher induction of B2R compared to B1R. This implies that STS-mediated apoptosis in PC12 cells is mainly conducted through B2R and partly via B1R. Finally, a neuroproteomics approach was conducted to find relevant proteins associated to STS and KKS in PC12 cells. Neuroproteomics results confirmed the presence of an inflammatory response leading to cell death during apoptosis-mediated STS treatment; however, a "survival" capacity was shown following inhibition of B2R coupled with STS treatment. Our data suggest that B2R is a key player in the inflammatory pathway following STS-mediated apoptosis in PC12 cells and its inhibition may represent a potential therapeutic tool in TBI.


Asunto(s)
Inhibidores Enzimáticos/toxicidad , Sistema Calicreína-Quinina/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Estaurosporina/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Neuronas/inmunología , Neuronas/metabolismo , Células PC12 , Proteómica , Ratas , Receptor de Bradiquinina B1/análisis , Receptor de Bradiquinina B1/genética , Receptor de Bradiquinina B1/inmunología , Receptor de Bradiquinina B2/análisis , Receptor de Bradiquinina B2/genética , Receptor de Bradiquinina B2/inmunología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional
15.
PLoS One ; 7(6): e39895, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22761925

RESUMEN

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.


Asunto(s)
Daño del ADN , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Células Madre Embrionarias/metabolismo , Recombinación Homóloga , Oxidorreductasas Intramoleculares/genética , Alelos , Animales , Secuencia de Bases , Cartilla de ADN , Marcación de Gen , Ratones , Reacción en Cadena de la Polimerasa
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