RESUMEN
The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1 in postnatal cochlear supporting cells (SCs) in vivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K+ current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3, and Atoh1 enhances the efficiency of HC generation and promotes the functional maturation of new HCs.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al ADN/genética , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodominio/genética , Células Laberínticas de Soporte/metabolismo , Organogénesis/genética , Factor de Transcripción Brn-3C/genética , Factores de Transcripción/genética , Potenciales de Acción/fisiología , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Calbindinas/genética , Calbindinas/metabolismo , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Proteínas de Homeodominio/metabolismo , Transporte Iónico , Células Laberínticas de Soporte/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Miosina VIIa/genética , Miosina VIIa/metabolismo , Neuritas/metabolismo , Neuritas/ultraestructura , Parvalbúminas/genética , Parvalbúminas/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Transducción de Señal , Factor de Transcripción Brn-3C/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Neurotoxicity is a common side effect of chemotherapeutics that often leads to the development of chemotherapy-induced peripheral neuropathy (CIPN). The peptide Prokineticin 2 (PK2) has a key role in experimental models of CIPN and can be considered an insult-inducible endangering mediator. Since primary afferent sensory neurons are highly sensitive to anticancer drugs, giving rise to dysesthesias, the aim of our study was to evaluate the alterations induced by vincristine (VCR) and bortezomib (BTZ) exposure in sensory neuron cultures and the possible preventive effect of blocking PK2 signaling. Both VCR and BTZ induced a concentration-dependent reduction of total neurite length that was prevented by the PK receptor antagonist PC1. Antagonizing the PK system also reduced the upregulation of PK2, PK-R1, TLR4, IL-6, and IL-10 expression induced by chemotherapeutic drugs. In conclusion, inhibition of PK signaling with PC1 prevented the neurotoxic effects of chemotherapeutics, suggesting a promising strategy for neuroprotective therapies against the sensory neuron damage induced by exposure to these drugs.
Asunto(s)
Antineoplásicos/toxicidad , Bortezomib/toxicidad , Hormonas Gastrointestinales/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neuropéptidos/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Síndromes de Neurotoxicidad/prevención & control , Células Receptoras Sensoriales/efectos de los fármacos , Triazinas/farmacología , Vincristina/toxicidad , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Evaluación Preclínica de Medicamentos , Hormonas Gastrointestinales/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/fisiología , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Neuroinmunomodulación/efectos de los fármacos , Neuropéptidos/fisiología , Fármacos Neuroprotectores/uso terapéutico , ARN Mensajero/biosíntesis , Células Receptoras Sensoriales/fisiología , Células Receptoras Sensoriales/ultraestructura , Triazinas/uso terapéuticoRESUMEN
This study investigated micro-patterned, high-density complementary metal-oxide-semiconductor (CMOS) electrode array to be used as biologically permissive environment for organization, guidance and electrical stimulation of spiral ganglion neurons (SGN). SGNs extracted and isolated from cochleae of P5-P7 rat pups and adult guinea pigs were cultured 1, 4 and 7 days in vitro on glass coverslips (control) and CMOS electrode array. The cultures were analyzed visually and immunohistochemically for SGN presence, outgrowth, neurite alignment, neurite length, neurite asymmetry as well as the contact of a neuronal soma and neurites with the micro-electrodes. Our findings indicate that topographical environment of CMOS chip with micro-patterned pillars enhanced growth, survival, morphology, neural orientation and alignment of SGNs in vitro compared to control. Smaller spacing (0.8-1.6 µm) between protruding pillars on CMOS led SGNs to develop structured and guided neurites oriented along three topographical axes separated by 60°. We found morphological basis for positioning of the micro-electrodes on the chip that was appropriate for direct contact of SGNs with them. This configuration allowed CMOS electrode array to electrically stimulate the SGN whose responses were observed with live Fluo 4 calcium imaging.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Estimulación Eléctrica/instrumentación , Dispositivos Laboratorio en un Chip , Neuronas/citología , Semiconductores , Ganglio Espiral de la Cóclea/citología , Animales , Orientación del Axón , Células Cultivadas , Electrodos , Diseño de Equipo , Cobayas , Metales/química , Neuritas/metabolismo , Neuritas/ultraestructura , Neurogénesis , Neuronas/metabolismo , Neuronas/ultraestructura , Óxidos/química , Ratas , Ganglio Espiral de la Cóclea/metabolismo , Ganglio Espiral de la Cóclea/ultraestructuraRESUMEN
Prostaglandins (PGs) play important roles in diverse physiological processes in the central nervous system. PGD2 is the most abundant PG in the brain and acts through specific receptors, DP1 and CRTH2. We investigated the effects of PGD2 on the morphology of the hypothalamic cell line mHypoE-N37 (N37). In N37 cells, serum starvation induced neurite outgrowth and PGD2 elicited neurite retraction, although we failed to detect transcripts for DP1 and CRTH2. Such an effect of PGD2 was efficiently mimicked by its metabolite, 15-deoxy-Δ(12,14)-prostaglandin J2. N-acetyl cysteine completely abolished the effect of PGD2, and reactive oxygen species (ROS) were considered to be important. Notably, neurite outgrowth was restored by PGD2 removal. These results suggest that PGD2 induces reversible neurite retraction in a ROS-mediated mechanism that does not involve any known receptor.
Asunto(s)
Aumento de la Célula/efectos de los fármacos , Hipotálamo/citología , Hipotálamo/metabolismo , Neuritas/fisiología , Prostaglandina D2/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Hipotálamo/efectos de los fármacos , Ratones , Neuritas/efectos de los fármacos , Neuritas/ultraestructuraRESUMEN
Docosahexaenoic acid (DHA) is an ω-3 polyunsaturated fatty acid that is essential in brain development and has structural and signaling roles. Acute DHA administration is neuroprotective and promotes functional recovery in animal models of adult spinal cord injury (SCI). However, the mechanisms underlying this recovery have not been fully characterized. Here we investigated the effects of an acute intravenous bolus of DHA delivered after SCI and characterized DHA-induced neuroplasticity within the adult injured spinal cord. We found robust sprouting of uninjured corticospinal and serotonergic fibers in a rat cervical hemisection SCI model. A mouse pyramidotomy model was used to confirm that this robust sprouting was not species or injury model specific. Furthermore, we demonstrated that corticospinal fibers sprouting to the denervated side of the cord following pyramidotomy contact V2a interneurons. We also demonstrated increased serotonin fibers and synaptophysin in direct contact with motor neurons. DHA also increased synaptophysin in rat cortical cell cultures. A reduction in phosphatase and tensin homolog (PTEN) has been shown to be involved in axonal regeneration and synaptic plasticity. We showed that DHA significantly upregulates miR-21 and downregulates PTEN in corticospinal neurons. Downregulation of PTEN and upregulation of phosphorylated AKT by DHA were also seen in primary cortical neuron cultures and were accompanied by increased neurite outgrowth. In summary, acute DHA induces anatomical and synaptic plasticity in adult injured spinal cord. This study shows that DHA has therapeutic potential in cervical SCI and provides evidence that DHA could exert its beneficial effects in SCI via enhancement of neuroplasticity. SIGNIFICANCE STATEMENT: In this study, we show that an acute intravenous injection of docosahexaenoic acid (DHA) 30 min after spinal cord injury induces neuroplasticity. We found robust sprouting of uninjured corticospinal and serotonergic fibers in a rat hemisection spinal cord injury model. A mouse pyramidotomy model was used to confirm that the robust sprouting involved V2a interneurons. We show that DHA significantly upregulates miR-21 and phosphorylated AKT, and downregulates phosphatase and tensin homolog (PTEN), which is involved in suppressing anatomical plasticity, in corticospinal neurons and in primary cortical neuron cultures. We conclude that acute DHA can induce anatomical and synaptic plasticity. This provides direct evidence that DHA could exert its beneficial effects in spinal cord injury via neuroplasticity enhancement.
Asunto(s)
Ácidos Docosahexaenoicos/uso terapéutico , Interneuronas/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Regeneración Nerviosa/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Tractos Piramidales/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Animales , Células Cultivadas , Vértebras Cervicales , Ácidos Docosahexaenoicos/administración & dosificación , Ácidos Docosahexaenoicos/farmacología , Evaluación Preclínica de Medicamentos , Conducta Exploratoria/efectos de los fármacos , Femenino , Trastornos Neurológicos de la Marcha/tratamiento farmacológico , Trastornos Neurológicos de la Marcha/etiología , Regulación de la Expresión Génica/efectos de los fármacos , Inyecciones Intravenosas , Interneuronas/fisiología , Ratones , MicroARNs/biosíntesis , MicroARNs/genética , Neuronas Motoras/fisiología , Regeneración Nerviosa/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Plasticidad Neuronal/fisiología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacología , Fosfohidrolasa PTEN/biosíntesis , Fosfohidrolasa PTEN/genética , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tractos Piramidales/lesiones , Tractos Piramidales/patología , Tractos Piramidales/fisiología , Ratas , Ratas Sprague-Dawley , Neuronas Serotoninérgicas/fisiología , Neuronas Serotoninérgicas/ultraestructura , Médula Espinal/fisiología , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Células-Madre Neurales/efectos de los fármacos , Péptidos/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Humanos , Indicadores y Reactivos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratones , Neuritas/efectos de los fármacos , Neuritas/ultraestructuraRESUMEN
In this study, we demonstrate capabilities to selectively manipulate dissociated co-cultures of neurons plated in dual-compartment devices. Synaptic receptor antagonists and tetrodotoxin solutions were used to selectively control and study the network-wide burst propagation and cell firing in cortical-cortical and cortical-thalamic co-culture systems. The results show that in cortical-thalamic dissociated co-cultures, burst events initiate in the cortical region and propagate to the thalamic region and the burst events in thalamic region can be controlled by blocking the synaptic receptors in the cortical region. Whereas, in cortical-cortical co-culture system, one of the region acts as a site of burst initiation and facilitate propagation of bursts in the entire network. Tetrodotoxin, a sodium channel blocker, when applied to either of the regions blocks the firing of neurons in that particular region with significant influence on the firing of neurons in the other region. The results demonstrate selective pharmacological manipulation capabilities of co-cultures in a dual compartment device and helps understand the effects of neuroactive compounds on networks derived from specific CNS tissues and the dynamic interaction between them.
Asunto(s)
Corteza Cerebral/citología , Técnicas de Cocultivo/instrumentación , Técnicas Analíticas Microfluídicas , Neuronas/efectos de los fármacos , Tálamo/citología , Potenciales de Acción/efectos de los fármacos , Animales , Comunicación Celular/efectos de los fármacos , Corteza Cerebral/embriología , Dimetilpolisiloxanos , Diseño de Equipo , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Microelectrodos , Red Nerviosa/fisiología , Neuritas/ultraestructura , Neuronas/citología , Neurotoxinas/farmacología , Nylons , Cultivo Primario de Células/métodos , Ratas , Ratas Wistar , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/fisiología , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Tálamo/embriologíaRESUMEN
The serotonin 5-HT(7) receptor has been linked to various psychiatric disorders, including schizophrenia, anxiety and depression, and is antagonized by antipsychotics such as risperidone, clozapine and lurasidone. In this study, we examined whether inhibiting the 5-HT(7) receptor could reverse behavioral abnormalities in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP), an experimental mouse model for psychiatric disorders such as schizophrenia. The selective 5-HT(7) antagonist SB-269970 effectively suppressed abnormal jumping behavior in PACAP-deficient mice. SB-269970 tended to alleviate the higher immobility in the forced swim test in PACAP-deficient mice, although SB-269970 reduced the immobility also in wild-type mice. In addition, we found that mutant mice had impaired performance in the Y-maze test, which was reversed by SB-269970. In the mutant mouse brain, 5-HT(7) protein expression did not differ from wild-type mice. In primary embryonic hippocampal neurons, the 5-HT(7) agonist AS19 increased neurite length and number. Furthermore, SB-269970 significantly inhibited the increase in neurite extension mediated by the 5-HT(1A/7) agonist 8-OH-DPAT. These results indicate that 5-HT(7) receptor blockade ameliorates psychomotor and cognitive deficits in PACAP-deficient mice, providing additional evidence that the 5-HT(7) receptor is a rational target for the treatment of psychiatric disorders.
Asunto(s)
Antipsicóticos/uso terapéutico , Hipocampo/citología , Proteínas del Tejido Nervioso/fisiología , Neuritas/efectos de los fármacos , Fenoles/uso terapéutico , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/deficiencia , Receptores de Serotonina/fisiología , Antagonistas de la Serotonina/uso terapéutico , Sulfonamidas/uso terapéutico , 8-Hidroxi-2-(di-n-propilamino)tetralin/antagonistas & inhibidores , 8-Hidroxi-2-(di-n-propilamino)tetralin/toxicidad , Animales , Antipsicóticos/farmacología , Recuento de Células , Células Cultivadas/efectos de los fármacos , Células Cultivadas/ultraestructura , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Conducta Exploratoria/efectos de los fármacos , Reacción Cataléptica de Congelación/efectos de los fármacos , Hipocampo/embriología , Hipercinesia/tratamiento farmacológico , Hipercinesia/fisiopatología , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Neuritas/ultraestructura , Fenoles/farmacología , Resistencia Física/efectos de los fármacos , Pirazoles/farmacología , Pirazoles/uso terapéutico , Receptores de Serotonina/biosíntesis , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/genética , Serotonina/fisiología , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Agonistas de Receptores de Serotonina/toxicidad , Sulfonamidas/farmacología , Tetrahidronaftalenos/farmacología , Tetrahidronaftalenos/uso terapéuticoRESUMEN
Pituitary adenylate cyclase activating peptide (PACAP), a potent neuropeptide which crosses the blood-brain barrier, is known to provide neuroprotection in rat stroke models of middle cerebral artery occlusion (MCAO) by mechanism(s) which deserve clarification. We confirmed that following i.v. injection of 30 ng/kg of PACAP38 in rats exposed to 2 h of MCAO focal cerebral ischemia and 48 h reoxygenation, 50 % neuroprotection was measured by reduced caspase-3 activity and volume of cerebral infarction. Similar neuroprotective effects were measured upon PACAP38 treatment of oxygen-glucose deprivation and reoxygenation of brain cortical neurons. The neuroprotection was temporally associated with increased expression of brain-derived neurotrophic factor, phosphorylation of its receptor-tropomyosin-related kinase receptor type B (trkB), activation of phosphoinositide 3-kinase and Akt, and reduction of extracellular signal-regulated kinases 1/2 phosphorylation. PACAP38 increased expression of neuronal markers beta-tubulin III, microtubule-associated protein-2, and growth-associated protein-43. PACAP38 induced stimulation of Rac and suppression of Rho GTPase activities. PACAP38 downregulated the nerve growth factor receptor (p75(NTR)) and associated Nogo-(Neurite outgrowth-A) receptor. Collectively, these in vitro and in vivo results propose that PACAP exhibits neuroprotective effects in cerebral ischemia by three mechanisms: a direct one, mediated by PACAP receptors, and two indirect, induced by neurotrophin release, activation of the trkB receptors and attenuation of neuronal growth inhibitory signaling molecules p75(NTR) and Nogo receptor.
Asunto(s)
Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/uso terapéutico , Animales , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipoxia de la Célula , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Corteza Cerebral/citología , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Inyecciones Intravenosas , Masculino , Proteínas del Tejido Nervioso/genética , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacología , Fosforilación/efectos de los fármacos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/administración & dosificación , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Wistar , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/efectos de los fármacos , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Daño por Reperfusión/prevención & control , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: To date, some of the most useful and physiologically relevant neuronal cell culture systems, such as high density co-cultures of astrocytes and primary hippocampal neurons, or differentiated stem cell-derived cultures, are characterized by high cell density and partially overlapping cellular structures. Efficient analytical strategies are required to enable rapid, reliable, quantitative analysis of neuronal morphology in these valuable model systems. RESULTS: Here we present the development and validation of a novel bioinformatics pipeline called NeuriteQuant. This tool enables fully automated morphological analysis of large-scale image data from neuronal cultures or brain sections that display a high degree of complexity and overlap of neuronal outgrowths. It also provides an efficient web-based tool to review and evaluate the analysis process. In addition to its built-in functionality, NeuriteQuant can be readily extended based on the rich toolset offered by ImageJ and its associated community of developers. As proof of concept we performed automated screens for modulators of neuronal development in cultures of primary neurons and neuronally differentiated P19 stem cells, which demonstrated specific dose-dependent effects on neuronal morphology. CONCLUSIONS: NeuriteQuant is a freely available open-source tool for the automated analysis and effective review of large-scale high-content screens. It is especially well suited to quantify the effect of experimental manipulations on physiologically relevant neuronal cultures or brain sections that display a high degree of complexity and overlap among neurites or other cellular structures.
Asunto(s)
Citometría de Imagen/métodos , Neuritas/ultraestructura , Neurogénesis/fisiología , Validación de Programas de Computación , Programas Informáticos/normas , Algoritmos , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Biología Computacional/métodos , Evaluación Preclínica de Medicamentos/métodos , Difusión de la Información/métodos , Ratones , Factores de Crecimiento Nervioso/fisiología , Neuritas/fisiologíaRESUMEN
OBJECTIVES: To observe the protective and therapeutic effect of schisandrol A on the Abeta damaged PC12 cells. METHODS: PC12 cells were damaged by Abeta in vitro. Morphological changes were observed and the number of cells with neurite was analyzed by phase contrast microscope. The cell viability of PC12 cells was determined by the MTT method. RESULTS: Many dispirited cells with atrophied or fragmented neurites in the Abeta damaged PC12 cells were observed under the microscope. More vital cells with longer neurites were observed in the schisandrol A treated PC12 cells and the number of cells with neurite increased. The difference of cell viability between the two groups was statistical significant. CONCLUSION: Schisandrol A can antagonize the neurotoxicity of Abeta and has protective and therapeutic efficacy on Abeta damaged PC12 cells.
Asunto(s)
Enfermedad de Alzheimer/prevención & control , Supervivencia Celular/efectos de los fármacos , Ciclooctanos/farmacología , Lignanos/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Schisandra/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/toxicidad , Animales , Diferenciación Celular/efectos de los fármacos , Ciclooctanos/administración & dosificación , Relación Dosis-Respuesta a Droga , Lignanos/administración & dosificación , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Neuronas/ultraestructura , Células PC12 , RatasRESUMEN
Ellagic acid, a polyphenolic compound found in berries, fruits and nuts, has been shown to possess growth-inhibiting and apoptosis promoting activities in cancer cell lines in vitro. The objective of this study was to investigate the effect of ellagic acid in human neuroblastoma SH-SY5Y cells. In cultures of SH-SY5Y cells incubated with ellagic acid, time- and concentration-dependent inhibitory effects on cell number were demonstrated. Ellagic acid induced cell detachment, decreased cell viability and induced apoptosis as measured by DNA strand breaks. Ellagic acid-induced alterations in cell cycle were also observed. Simultaneous treatment with all-trans retinoic acid did not rescue the cells from ellagic acid effects. Furthermore, the results suggested that pre-treatment with all-trans retinoic acid to induce differentiation and cell cycle arrest did not rescue the cells from ellagic acid-induced cell death.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Ácido Elágico/farmacología , Inhibidores de Crecimiento/farmacología , Neuroblastoma/patología , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular , División Celular/efectos de los fármacos , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Neuritas/ultraestructura , Tretinoina/farmacología , Ensayo de Tumor de Célula MadreRESUMEN
We examined the development of the nervous system in Aurelia (Cnidaria, Scyphozoa) from the early planula to the polyp stage using confocal and transmission electron microscopy. Fluorescently labeled anti-FMRFamide, antitaurine, and antityrosinated tubulin antibodies were used to visualize the nervous system. The first detectable FMRFamide-like immunoreactivity occurs in a narrow circumferential belt toward the anterior/aboral end of the ectoderm in the early planula. As the planula matures, the FMRFamide-immunoreactive cells send horizontal processes (i.e., neurites) basally along the longitudinal axis. Neurites extend both anteriorly/aborally and posteriorly/orally, but the preference is for anterior neurite extension, and neurites converge to form a plexus at the aboral/anterior end at the base of the ectoderm. In the mature planula, a subset of cells in the apical organ at the anterior/aboral pole begins to show FMRFamide-like and taurine-like immunoreactivity, suggesting a sensory function of the apical organ. During metamorphosis, FMRFamide-like immunoreactivity diminishes in the ectoderm but begins to occur in the degenerating primary endoderm, indicating that degenerating FMRFamide-immunoreactive neurons are taken up by the primary endoderm. FMRFamide-like expression reappears in the ectoderm of the oral disc and the tentacle anlagen of the growing polyp, indicating metamorphosis-associated restructuring of the nervous system. These observations are discussed in the context of metazoan nervous system evolution.
Asunto(s)
Tipificación del Cuerpo , Estadios del Ciclo de Vida/fisiología , Sistema Nervioso/embriología , Escifozoos/embriología , Escifozoos/crecimiento & desarrollo , Animales , Ectodermo/citología , Ectodermo/ultraestructura , Embrión no Mamífero/citología , Embrión no Mamífero/ultraestructura , Epitelio/ultraestructura , FMRFamida/metabolismo , Larva/citología , Larva/ultraestructura , Metamorfosis Biológica , Degeneración Nerviosa , Sistema Nervioso/ultraestructura , Neuritas/ultraestructura , Escifozoos/citología , Escifozoos/ultraestructura , Células Receptoras Sensoriales/patología , Células Receptoras Sensoriales/ultraestructura , Taurina/metabolismoRESUMEN
Epidermal fatty acid-binding protein (E-FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E-FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E-FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n-3 docosahexaenoic acid (DHA), C20:5n-3 eicosapentaenoic acid (EPA), and C20:4n-6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E-FABP expression that is blocked by mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor-differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12-AS) which exhibit low E-FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12-S). Replenishing NGFDPC12-AS cells with biotinylated recombinant E-FABP (biotin-E-FABP) protein restores normal neurite outgrowth. Cellular localization of biotin-E-FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E-FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12-S than in NGFDPC12-AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E-FABP.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas del Ojo/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-6/metabolismo , Metabolismo de los Lípidos/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Animales , Compartimento Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/metabolismo , Ácido Eicosapentaenoico/farmacología , Proteínas del Ojo/genética , Proteínas de Unión a Ácidos Grasos/genética , Ácidos Grasos Omega-3/farmacología , Ácidos Grasos Omega-6/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/genética , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Oligodesoxirribonucleótidos Antisentido/farmacología , Células PC12 , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Following spinal cord injury, a variety of inhibitory molecules hinder the success of axon regeneration. The motile tip of the axon, the growth cone, shares a similar cytoskeletal array as a migrating cell, and in general the cytoskeleton is regulated by a conserved set of signaling pathways that act downstream of guidance cue and growth factor receptors. We exploit these similarities by using migrating cells as a model system to screen for extracts that promote axon outgrowth. The screen is a high-throughput wound-healing assay performed by a 96-pin tool Biogrid robot where positive candidates are identified as extracts that stimulate complete wound healing. Testing of positive candidates on chick DRG explants has lead to the identification of extracts that promote neurite outgrowth on permissive and inhibitory substrates. Extracts can be fractionated to purity, identifying novel compounds that promote neurite outgrowth on inhibitory substrates.
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Ensayos de Migración Celular/métodos , Movimiento Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Conos de Crecimiento/efectos de los fármacos , Factores de Crecimiento Nervioso/aislamiento & purificación , Neuritas/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Aumento de la Célula/efectos de los fármacos , Extractos Celulares/aislamiento & purificación , Extractos Celulares/farmacología , Ensayos de Migración Celular/instrumentación , Movimiento Celular/fisiología , Células Cultivadas , Embrión de Pollo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Evaluación Preclínica de Medicamentos/instrumentación , Conos de Crecimiento/fisiología , Conos de Crecimiento/ultraestructura , Inhibidores de Crecimiento/farmacología , Humanos , Factores de Crecimiento Nervioso/farmacología , Neuritas/fisiología , Neuritas/ultraestructura , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
High-throughput screening (HTS) of cell-based assays has recently emerged as an important tool of drug discovery. The analysis and modeling of HTS microscopy neuron images, however, is particularly challenging. In this paper we present a novel algorithm for extraction and quantification of neurite segments from HTS neuron images. The algorithm is designed to be able to detect and link neurites even with complex neuronal structures and of poor imaging quality. Our proposed algorithm automatically detects initial seed points on a set of grid lines and estimates the ending points of the neurite by iteratively tracing the centerline points along the line path representing the neurite segment. The live-wire method is then applied to link the seed points and the corresponding ending points using dynamic programming techniques, thus enabling the extraction of the centerlines of the neurite segments accurately and robustly against noise, discontinuity, and other image artifacts. A fast implementation of our algorithm using dynamic programming is also provided in the paper. Any thin neurite and its segments with low intensity contrast can be well preserved by detecting the starting and ending points of the neurite. All these properties make the proposed algorithm attractive for high-throughput screening of neuron-based assays.
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Evaluación Preclínica de Medicamentos/métodos , Neuritas/efectos de los fármacos , Algoritmos , Automatización , Evaluación Preclínica de Medicamentos/estadística & datos numéricos , Procesamiento de Imagen Asistido por Computador/métodos , Neuritas/ultraestructura , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
Folate deficiency contributes to a variety of age-related neurological and psychological disorders including amyotrophic lateral sclerosis (ALS). The environmental neurotoxin arsenic has recently been linked with decreased neurofilament (NF) content in peripheral nerve. We examined herein, whether or not folate deprivation potentiated the impact of arsenic on NF dynamics. Arsenic inhibited translocation of NFs into axonal neurites in culture and increased perikaryal NF phosphoepitopes. Folate deprivation potentiated the impact of arsenic on these phenomena. Supplementation with S-adenosyl methionine (SAM) attenuated the impact of folate deprivation on arsenic neurotoxicity, consistent with the decrease in SAM following folate deprivation and the requirement for SAM-mediated methylation for arsenic bioelimination. These findings demonstrate how key nutritional deficiencies can potentiate the impact of enrivonmental neurotoxins.
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Arsénico/toxicidad , Ácido Fólico/administración & dosificación , Enfermedades del Sistema Nervioso/inducido químicamente , S-Adenosilmetionina/administración & dosificación , Esclerosis Amiotrófica Lateral/inducido químicamente , Axones/ultraestructura , Bucladesina/farmacología , Línea Celular , Citoesqueleto/efectos de los fármacos , Ácido Fólico/fisiología , Proteínas Fluorescentes Verdes/genética , Metilación , Enfermedades del Sistema Nervioso/prevención & control , Neuritas/fisiología , Neuritas/ultraestructura , Proteínas de Neurofilamentos/análisis , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Fosforilación , Proteínas Recombinantes de Fusión , TransfecciónRESUMEN
Improved sensory and motor prostheses for the central nervous system will require large numbers of electrodes with low electrical thresholds for neural excitation. With the eventual goal of reducing stimulation thresholds, we have investigated the use of biodegradable, neurotrophin-eluting hydrogels (i.e., poly(ethylene glycol)-poly(lactic acid), PEGPLA) as a means of attracting neurites to the surface of stimulating electrodes. PEGPLA hydrogels with release rates ranging from 1.5 to 3 weeks were synthesized. These hydrogels were applied to multielectrode arrays with sputtered iridium oxide charge-injection sites. The coatings had little impact on the iridium oxide electrochemical properties, including charge storage capacity, impedance, and voltage transients during current pulsing. Additionally, we quantitatively examined the ability of neurotrophin-eluting, PEGPLA hydrogels to promote neurite extension in vitro using a PC12 cell culture model. Hydrogels released neurotrophin (nerve growth factor, NGF) for at least 1 week, with neurite extension near that of an NGF positive control and much higher than extension seen from sham, bovine serum albumin-releasing boluses, and a negative control. These results show that neurotrophin-eluting hydrogels can be applied to multielectrode arrays, and suggest a method to improve neuron-electrode proximity, which could result in lowered electrical stimulation thresholds. Reduced thresholds support the creation of smaller electrode structures and high density electrode prostheses, greatly enhancing prosthesis control and function.
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Sistema Nervioso Central/fisiología , Materiales Biocompatibles Revestidos , Terapia por Estimulación Eléctrica , Factores de Crecimiento Nervioso/administración & dosificación , Animales , Estimulación Eléctrica , Electrodos , Hidrogeles , Técnicas In Vitro , Lactatos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuritas/ultraestructura , Células PC12 , Polietilenglicoles , RatasRESUMEN
Mutant huntingtin can affect vesicular and receptor trafficking via its abnormal protein interactions, suggesting that impairment of intracellular trafficking may contribute to Huntington's disease. There is growing evidence that huntingtin-associated protein-1 (HAP1) also interacts with microtubule-dependent transporters and is involved in intracellular trafficking. However, it remains unclear how the trafficking of HAP1 is regulated and contributes to neuronal function. Here we report that phosphorylation of HAP1 decreases its association with microtubule-dependent transport proteins dynactin p150 and kinesin light chain and reduces its localization in neurite tips. Suppressing HAP1 expression by RNA interference reduces neurite outgrowth and the level of tropomyosin-related kinase A receptor tyrosine kinase (TrkA), a nerve growth factor receptor whose internalization and trafficking are required for neurite outgrowth. HAP1 maintains the normal level of membrane TrkA by preventing the degradation of internalized TrkA. Mutant huntingtin also reduces the association of HAP1 with dynactin p150 and kinesin light chain and thereby decreases the intracellular level of TrkA. These findings suggest that HAP1 trafficking is critical for the stability of TrkA and neurite function, both of which can be attenuated by mutant huntingtin.
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Proteínas del Tejido Nervioso/metabolismo , Neuritas/fisiología , Neuronas/fisiología , Receptor trkA/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Genes Reporteros , Enfermedad de Huntington , Hipotálamo , Microscopía Confocal , Neuritas/ultraestructura , Neuronas/citología , Células PC12 , Feocromocitoma , ARN Interferente Pequeño/genética , Ratas , Ganglio Cervical Superior/fisiologíaRESUMEN
BACKGROUND: Genetically manipulated embryonic stem (ES) cell derived neurons (ESNs) provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK), which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice. RESULTS: Mouse ES cells carrying homozygous null mutations (FAK-/-) were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases. CONCLUSION: FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.