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1.
Neuronal Signal ; 4(1): NS20190148, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32714599

RESUMEN

Episodes of hypoxia and hypoxia/reoxygenation during foetal development have been associated with increased risk of neurodevelopmental conditions presenting in later life. The mechanism for this is not understood; however, several authors have suggested that the placenta plays an important role. Previously we found both placentas from a maternal hypoxia model and pre-eclamptic placentas from patients release factors lead to a loss of dendrite complexity in rodent neurons. Here to further explore the nature and origin of these secretions we exposed a simple in vitro model of the placental barrier, consisting of a barrier of human cytotrophoblasts, to hypoxia or hypoxia/reoxygenation. We then exposed cortical cultures from embryonic rat brains to the conditioned media (CM) from below these exposed barriers and examined changes in cell morphology, number, and receptor presentation. The barriers released factors that reduced dendrite and astrocyte process lengths, decreased GABAB1 staining, and increased astrocyte number. The changes in astrocytes required the presence of neurons and were prevented by inhibition of the SMAD pathway and by neutralising Bone Morphogenetic Proteins (BMPs) 2/4. Barriers exposed to hypoxia/reoxygenation also released factors that reduced dendrite lengths but increased GABAB1 staining. Both oxygen changes caused barriers to release factors that decreased GluN1, GABAAα1 staining and increased GluN3a staining. We find that hypoxia in particular will elicit the release of factors that increase astrocyte number and decrease process length as well as causing changes in the intensity of glutamate and GABA receptor staining. There is some evidence that BMPs are released and contribute to these changes.

2.
Exp Neurol ; 261: 386-95, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24818543

RESUMEN

Some psychiatric diseases in children and young adults are thought to originate from adverse exposures during foetal life, including hypoxia and hypoxia/reoxygenation. The mechanism is not understood. Several authors have emphasised that the placenta is likely to play an important role as the key interface between mother and foetus. Here we have explored whether a first trimester human placenta or model barrier of primary human cytotrophoblasts might secrete factors, in response to hypoxia or hypoxia/reoxygenation, that could damage neurones. We find that the secretions in conditioned media caused an increase of [Ca(2+)]i and mitochondrial free radicals and a decrease of dendritic lengths, branching complexity, spine density and synaptic activity in dissociated neurones from embryonic rat cerebral cortex. There was altered staining of glutamate and GABA receptors. We identify glutamate as an active factor within the conditioned media and demonstrate a specific release of glutamate from the placenta/cytotrophoblast barriers invitro after hypoxia or hypoxia/reoxygenation. Injection of conditioned media into developing brains of P4 rats reduced the numerical density of parvalbumin-containing neurones in cortex, hippocampus and reticular nucleus, reduced immunostaining of glutamate receptors and altered cellular turnover. These results show that the placenta is able to release factors, in response to altered oxygen, that can damage developing neurones under experimental conditions.


Asunto(s)
Encéfalo , Medios de Cultivo Condicionados/efectos adversos , Hipoxia , Neuronas/efectos de los fármacos , Oxígeno/farmacología , Placenta/química , Animales , Animales Recién Nacidos , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/patología , Hipoxia de la Célula/fisiología , Células Cultivadas , Corteza Cerebral/citología , Medios de Cultivo Condicionados/química , Dendritas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Femenino , Feto , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Hipoxia/tratamiento farmacológico , Hipoxia/patología , Hipoxia/fisiopatología , Potenciales de la Membrana/efectos de los fármacos , Neuronas/citología , Neuronas/fisiología , Placenta/citología , Embarazo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Técnicas de Cultivo de Tejidos
3.
J Biol Chem ; 289(2): 895-908, 2014 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-24275654

RESUMEN

Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and ß-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three ß-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of ß-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.


Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Tubulina (Proteína)/metabolismo , Acetilación , Alanina/química , Alanina/genética , Alanina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Lisina/química , Lisina/genética , Lisina/metabolismo , Ratones , Ratones Noqueados , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
4.
Exp Neurol ; 239: 82-90, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23022459

RESUMEN

Pharmacological inhibitors of epidermal growth factor receptor (ErbB1) attenuate the ability of CNS myelin to inhibit axonal regeneration. However, it has been claimed that such effects are mediated by off-target interactions. We have tested the role of ErbB1 in axonal regeneration by culturing neurons from ErbB1 knockout mice in the presence of various inhibitors of axonal regeneration: CNS myelin, chondroitin sulfate proteoglycans (CSPG), fibrinogen or polyinosinic:polycytidylic acid (poly I:C). We confirmed that ErbB1 was activated in cultures of cerebellar granule cells exposed to inhibitors of axonal regeneration and that ErbB1 kinase inhibitors promoted neurite outgrowth under these conditions. In the presence of myelin, fibrinogen, CSPG and poly I:C ErbB1 -/- neurons grew longer neurites than neurons expressing ErbB1. Furthermore, inhibitors of ErbB1 kinase did not improve neurite outgrowth from ErbB1 -/- neurons, ruling out an off-target mechanism of action. ErbB1 kinase activity is therefore a valid target for promoting axonal elongation in the presence of many of the molecules believed to contribute to the failure of axonal regeneration in the injured CNS.


Asunto(s)
Axones/efectos de los fármacos , Genes erbB-1/efectos de los fármacos , Regeneración Nerviosa/efectos de los fármacos , Animales , Barrera Hematoencefálica/efectos de los fármacos , Señalización del Calcio/fisiología , Cerebelo/citología , Sulfatos de Condroitina/farmacología , Gránulos Citoplasmáticos , Fibrinógeno/farmacología , Ratones , Ratones Noqueados , Vaina de Mielina/fisiología , Fosforilación , Poli I-C/farmacología , Proteoglicanos/farmacología , Quinazolinas/farmacología , ARN/metabolismo , ARN Bicatenario/farmacología , Células Receptoras Sensoriales/efectos de los fármacos , Receptor Toll-Like 3/efectos de los fármacos
5.
Biomaterials ; 32(33): 8538-47, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21824652

RESUMEN

The CD95/CD95L receptor-ligand system is mainly recognised in the induction of apoptosis. However, it has also been shown that CD95L is over-expressed in many cancer types where it modulates immune-evasion and together with its receptor CD95 promotes tumour growth. Here, we show that CD95 surface modification of relatively large microparticles >0.5 µm in diameter, including those made from biodegradable polylactic-co-glycolic acid (PLGA), enhances intracellular uptake by a range of CD95L expressing cells in a process akin to phagocytosis. Using this approach we describe the intracellular uptake of microparticles and agent delivery in neurons, medulloblastoma, breast and ovarian cancer cells in vitro. CD95 modified paclitaxel-loaded PLGA microparticles are shown to be significantly more effective compared to conventional paclitaxel therapy (Taxol) at the same dose in subcutaneous medulloblastoma (∗∗∗P < 0.0001) and orthotopic ovarian cancer xenograft models where a >65-fold reduction in tumour bioluminescence was measured after treatment (∗P = 0.012). This drug delivery platform represents a new way of manipulating the normally advantageous tumour CD95L over-expression towards a therapeutic strategy. CD95 functionalised drug carriers could contribute to the improved function of cytotoxics in cancer, potentially increasing drug targeting and efficacy whilst reducing toxicity.


Asunto(s)
Antineoplásicos Fitogénicos/farmacocinética , Ácido Láctico , Microesferas , Paclitaxel/farmacocinética , Ácido Poliglicólico , Receptor fas/química , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/química , Línea Celular Tumoral , Portadores de Fármacos , Citometría de Flujo , Humanos , Paclitaxel/administración & dosificación , Paclitaxel/química , Fagocitosis , Copolímero de Ácido Poliláctico-Ácido Poliglicólico
6.
Stem Cells ; 29(4): 700-12, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21305672

RESUMEN

The Polycomb group protein Bmi1 is a key regulator of self-renewal of embryonic and adult central nervous system stem cells, and its overexpression has been shown to occur in several types of brain tumors. In a Cre/LoxP-based conditional transgenic mouse model, we show that fine-tuning of Bmi1 expression in embryonic neural stem cell (NSC) is sufficient to increase their proliferation and self-renewal potential both in vitro and in vivo. This is linked to downregulation of both the ink4a/ARF and the p21/Foxg1 axes. However, increased and ectopic proliferation induced by overexpression of Bmi1 in progenitors committed toward a neuronal lineage during embryonic cortical development, triggers apoptosis through a survivin-mediated mechanism and leads to reduced brain size. Postnatally, however, increased self-renewal capacity of neural stem/progenitor cells (NSPC) is independent of Foxg1 and resistance to apoptosis is observed in neural progenitors derived from NSC-overexpressing Bmi1. Neoplastic transformation is absent in mice-overexpressing Bmi1 aged up to 20 months. These studies provide strong evidence that fine tuning of Bmi1 expression is a viable tool to increase self-renewal capacity of NSCs both in vitro and in vivo without eliciting neoplastic transformation of these cells.


Asunto(s)
Apoptosis , Diferenciación Celular , Células-Madre Neurales/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción Activador 2/metabolismo , Animales , Células Cultivadas , Sistema Nervioso Central/embriología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Factores de Transcripción Forkhead/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/citología , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Survivin , Proteína p14ARF Supresora de Tumor/genética
7.
Mol Cell Neurosci ; 40(2): 207-16, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19049877

RESUMEN

Peripheral nerve transections cause much more neuronal death in embryonic and neonatal dorsal root ganglia (DRG) than in adult DRG. Here we used transgenic approaches to examine the hypothesis that NF-kappaB is an important intrinsic factor of adult DRG neurons for their in vivo capacity to survive after nerve injury. We generated transgenic mice expressing the NF-kappaB super-inhibitor (IkappaBalpha-SI), a multi-mutant form of IkappaBalpha, specifically in adult neurons. Adult DRG neurons in these transgenic animals are not abnormally susceptible to apoptosis after peripheral nerve injury, although there is a significant inhibition in the ability of NF-kappaB to translocate into their nucleus. We investigated the observed lack of NF-kappaB neuroprotective function at the level of NF-kappaB transcriptional activity using transgenic NF-kappaB/LacZ reporter mice. We show that the expression of the NF-kappaB reporter transgene is restricted in naïve and injured DRG neurons. However, NF-kappaB transcriptional activity in adult DRG neurons is evident upon exposure to Trichostatin A (TSA) which is a specific inhibitor of histone deacetylases. Taken together our results illustrate that the functions of NF-kappaB are limited in adult primary sensory neurons due to a transcriptional repression mechanism mediated by histone deacetylases, and that intrinsic neuroprotective factors other than NF-kappaB are responsible for the resistance of adult DRG neurons to apoptosis in response to nerve injury.


Asunto(s)
Ganglios Espinales/citología , FN-kappa B/metabolismo , Neuronas Aferentes/patología , Neuronas Aferentes/fisiología , Animales , Apoptosis/fisiología , Axotomía , Supervivencia Celular , Células Cultivadas , Ganglios Espinales/fisiología , Regulación de la Expresión Génica , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Masculino , Ratones , Ratones Transgénicos , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Neuronas Aferentes/citología
8.
Neurosci Lett ; 433(3): 231-4, 2008 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-18280043

RESUMEN

Flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate nick end-labelling (TUNEL) immunohistochemistry have been used to assess cell death in the dorsal root ganglia (DRG) or spinal cord 1, 2 or 14 days after multiple lumbar dorsal root rhizotomy or dorsal root avulsion injury in adult rats. Neither injury induced significant cell death in the DRG compared to sham-operated or naïve animals at any time point. In the spinal cord, a significant increase in death was seen at 1-2 days, but not 14 days, post injury by both methods. TUNEL staining revealed that more apoptotic cells were present in the dorsal columns and dorsal horn of avulsion animals compared to rhizotomised animals. This suggests that avulsion injury, which can often partially damage the spinal cord, has more severe effects on cell survival than rhizotomy, a surgical lesion which does not affect the spinal cord. The location of TUNEL positive cells suggests that both neuronal and non-neuronal cells are dying.


Asunto(s)
Ganglios Espinales/fisiopatología , Degeneración Nerviosa/fisiopatología , Células del Asta Posterior/fisiopatología , Rizotomía/efectos adversos , Raíces Nerviosas Espinales/lesiones , Raíces Nerviosas Espinales/fisiopatología , Vías Aferentes/patología , Vías Aferentes/fisiopatología , Animales , Apoptosis/fisiología , Axones/patología , Muerte Celular/fisiología , Ganglios Espinales/patología , Etiquetado Corte-Fin in Situ , Masculino , Degeneración Nerviosa/patología , Neuronas Aferentes/patología , Células del Asta Posterior/patología , Radiculopatía/patología , Radiculopatía/fisiopatología , Ratas , Ratas Wistar , Raíces Nerviosas Espinales/patología , Sobrevida/fisiología , Factores de Tiempo , Degeneración Walleriana/patología , Degeneración Walleriana/fisiopatología
9.
Eur J Neurosci ; 24(12): 3343-53, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17229083

RESUMEN

This study explored the effects of riluzole administration on cell survival and neurite growth in adult and neonatal rat dorsal root ganglion (DRG) neurones in vitro. Neuronal survival was assessed by comparing numbers of remaining neurones in vehicle- and riluzole-treated cultures. A single dose of 0.1 microm riluzole was sufficient to promote neuronal survival in neonatal DRG cultures, whereas repeated riluzole administration was necessary in adult cultures. However, a single administration of riluzole was sufficient to induce neuritogenesis, promote neurite branching and enhance neurite outgrowth in both neonatal and adult DRG cultures. The effects of a single dose of riluzole on adult DRG neurones after peripheral nerve or dorsal root injury were also studied in vitro at 48 h. For both types of injury, riluzole enhanced neurite outgrowth in terms of number, length and branch pattern significantly more on the injured side as compared with the contralateral side. No effect was seen on cell survival. The results suggest that, in addition to its cell survival effects, riluzole has novel growth-promoting effects on sensory neurones in vitro and that riluzole may offer a new way to promote sensory afferent regeneration following peripheral injury.


Asunto(s)
Neuritas/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Riluzol/administración & dosificación , Análisis de Varianza , Animales , Animales Recién Nacidos , Recuento de Células/métodos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Esquema de Medicación , Lateralidad Funcional , Ganglios Espinales/citología , Inmunohistoquímica/métodos , Regeneración Nerviosa/efectos de los fármacos , Neuronas Aferentes/citología , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/patología , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Tubulina (Proteína)/metabolismo
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