RESUMEN

Brain-derived neurotrophic factor (BDNF) and its tropomyosin receptor kinase B (TrkB) are important signaling proteins that regulate dendritic growth and maintenance in the central nervous system (CNS). After binding of BDNF, TrkB is endocytosed into endosomes and continues signaling within the cell soma, dendrites, and axon. In previous studies, we showed that BDNF signaling initiated in axons triggers long-distance signaling, inducing dendritic arborization in a CREB-dependent manner in cell bodies, processes that depend on axonal dynein and TrkB activities. The binding of BDNF to TrkB triggers the activation of different signaling pathways, including the ERK, PLC-γ and PI3K-mTOR pathways, to induce dendritic growth and synaptic plasticity. How TrkB downstream pathways regulate long-distance signaling is unclear. Here, we studied the role of PLC-γ-Ca2+ in BDNF-induced long-distance signaling using compartmentalized microfluidic cultures. We found that dendritic branching and CREB phosphorylation induced by axonal BDNF stimulation require the activation of PLC-γ in the axons of cortical neurons. Locally, in axons, BDNF increases PLC-γ phosphorylation and induces intracellular Ca2+ waves in a PLC-γ-dependent manner. In parallel, we observed that BDNF-containing signaling endosomes transport to the cell body was dependent on PLC-γ activity and intracellular Ca2+ stores. Furthermore, the activity of PLC-γ is required for BDNF-dependent TrkB endocytosis, suggesting a role for the TrkB/PLC-γ signaling pathway in axonal signaling endosome formation.

RESUMEN

Neuromodulators, such as antidepressants, may contribute to neuroprotection by modulating growth factor expression to exert anti-inflammatory effects and to support neuronal plasticity after stroke. Our objective was to study whether early treatment with venlafaxine, a serotonin-norepinephrine reuptake inhibitor, modulates growth factor expression and positively contributes to reducing the volume of infarcted brain tissue resulting in increased functional recovery. We studied the expression of BDNF, FGF2 and TGF-ß1 by examining their mRNA and protein levels and cellular distribution using quantitative confocal microscopy at 5 days after venlafaxine treatment in control and infarcted brains. Venlafaxine treatment did not change the expression of these growth factors in sham rats. In infarcted rats, BDNF mRNA and protein levels were reduced, while the mRNA and protein levels of FGF2 and TGF-ß1 were increased. Venlafaxine treatment potentiated all of the changes that were induced by cortical stroke alone. In particular, increased levels of FGF2 and TGF-ß1 were observed in astrocytes at 5 days after stroke induction, and these increases were correlated with decreased astrogliosis (measured by GFAP) and increased synaptophysin immunostaining at twenty-one days after stroke in venlafaxine-treated rats. Finally, we show that venlafaxine reduced infarct volume after stroke resulting in increased functional recovery, which was measured using ladder rung motor tests, at 21 days after stroke. Our results indicate that the early oral administration of venlafaxine positively contributes to neuroprotection during the acute and late events that follow stroke.

Asunto(s)

Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Fármacos Neuroprotectores/farmacología , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/metabolismo , Clorhidrato de Venlafaxina/farmacología , Animales , Antidepresivos/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/patología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Endotelina-1 , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Gliosis/tratamiento farmacológico , Gliosis/metabolismo , Gliosis/patología , Masculino , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Accidente Cerebrovascular/patología , Factor de Crecimiento Transformador beta1/metabolismo

RESUMEN

Dendritic arborization of neurons is regulated by brain-derived neurotrophic factor (BDNF) together with its receptor, TrkB. Endocytosis is required for dendritic branching and regulates TrkB signaling, but how postendocytic trafficking determines the neuronal response to BDNF is not well understood. The monomeric GTPase Rab11 regulates the dynamics of recycling endosomes and local delivery of receptors to specific dendritic compartments. We investigated whether Rab11-dependent trafficking of TrkB in dendrites regulates BDNF-induced dendritic branching in rat hippocampal neurons. We report that TrkB in dendrites is a cargo for Rab11 endosomes and that both Rab11 and its effector, MyoVb, are required for BDNF/TrkB-induced dendritic branching. In addition, BDNF induces the accumulation of Rab11-positive endosomes and GTP-bound Rab11 in dendrites and the expression of a constitutively active mutant of Rab11 is sufficient to increase dendritic branching by increasing TrkB localization in dendrites and enhancing sensitization to endogenous BDNF. We propose that Rab11-dependent dendritic recycling provides a mechanism to retain TrkB in dendrites and to increase local signaling to regulate arborization.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/farmacología , Dendritas/efectos de los fármacos , Endosomas/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Neuronas/citología , Análisis de Varianza , Animales , Anticuerpos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Carbazoles/farmacología , Células Cultivadas , Dendritas/fisiología , Dendritas/ultraestructura , Embrión de Mamíferos , Endocitosis/efectos de los fármacos , Endosomas/ultraestructura , Inhibidores Enzimáticos/farmacología , Femenino , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Guanosina Trifosfato/metabolismo , Hipocampo/citología , Alcaloides Indólicos/farmacología , Masculino , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación/genética , Miosinas/metabolismo , Neuronas/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Ratas , Receptor trkB/metabolismo , Tiazolidinas/farmacología , Transfección

RESUMEN

BACKGROUND: Neurotrophins and their receptors regulate several aspects of the developing and mature nervous system, including neuronal morphology and survival. Neurotrophin receptors are active in signaling endosomes, which are organelles that propagate neurotrophin signaling along neuronal processes. Defects in the Npc1 gene are associated with the accumulation of cholesterol and lipids in late endosomes and lysosomes, leading to neurodegeneration and Niemann-Pick type C (NPC) disease. The aim of this work was to assess whether the endosomal and lysosomal alterations observed in NPC disease disrupt neurotrophin signaling. As models, we used i) NPC1-deficient mice to evaluate the central cholinergic septo-hippocampal pathway and its response to nerve growth factor (NGF) after axotomy and ii) PC12 cells treated with U18666A, a pharmacological cellular model of NPC, stimulated with NGF. RESULTS: NPC1-deficient cholinergic cells respond to NGF after axotomy and exhibit increased levels of choline acetyl transferase (ChAT), whose gene is under the control of NGF signaling, compared to wild type cholinergic neurons. This finding was correlated with increased ChAT and phosphorylated Akt in basal forebrain homogenates. In addition, we found that cholinergic neurons from NPC1-deficient mice had disrupted neuronal morphology, suggesting early signs of neurodegeneration. Consistently, PC12 cells treated with U18666A presented a clear NPC cellular phenotype with a prominent endocytic dysfunction that includes an increased size of TrkA-containing endosomes and reduced recycling of the receptor. This result correlates with increased sensitivity to NGF, and, in particular, with up-regulation of the Akt and PLC-γ signaling pathways, increased neurite extension, increased phosphorylation of tau protein and cell death when PC12 cells are differentiated and treated with U18666A. CONCLUSIONS: Our results suggest that the NPC cellular phenotype causes neuronal dysfunction through the abnormal up-regulation of survival pathways, which causes the perturbation of signaling cascades and anomalous phosphorylation of the cytoskeleton.

Asunto(s)

Encéfalo/metabolismo , Fibras Colinérgicas/metabolismo , Endosomas/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Transducción de Señal/fisiología , Animales , Fibras Colinérgicas/patología , Modelos Animales de Enfermedad , Endosomas/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Microscopía Confocal , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Enfermedad de Niemann-Pick Tipo C/patología , Células PC12 , Ratas , Regulación hacia Arriba

RESUMEN

BACKGROUND: Septal cholinergic neurons account for most of the cholinergic innervations of the hippocampus, playing a key role in the regulation of hippocampal synaptic activity. Disruption of the septo-hippocampal pathway by an experimental transection of the fimbria-fornix drastically reduces the target-derived trophic support received by cholinergic septal neurons, mainly nerve growth factor (NGF) from the hippocampus. Axotomy of cholinergic neurons induces a reduction in the number of neurons positive for cholinergic markers in the medial septum. In several studies, the reduction of cholinergic markers has been interpreted as analogous to the neurodegeneration of cholinergic cells, ruling out the possibility that neurons lose their cholinergic phenotype without dying. Understanding the mechanism of cholinergic neurodegeneration after axotomy is relevant, since this paradigm has been extensively explored as an animal model of the cholinergic impairment observed in neuropathologies such as Alzheimer's disease.The principal aim of this study was to evaluate, using modern quantitative confocal microscopy, neurodegenerative changes in septal cholinergic neurons after axotomy and to assess their response to delayed infusion of NGF in rats. RESULTS: We found that there is a slow reduction of cholinergic cells labeled by ChAT and p75 after axotomy. However, this phenomenon is not accompanied by neurodegenerative changes or by a decrease in total neuronal number in the medial septum. Although the remaining axotomized-neurons appear healthy, they are unable to respond to delayed NGF infusion. CONCLUSIONS: Our results demonstrate that at 3 weeks, axotomized cholinergic neurons lose their cholinergic phenotype without dying and down-regulate their NGF-receptors, precluding the possibility of a response to NGF. Therefore, the physiological role of NGF in the adult septal cholinergic system is to support phenotypic differentiation and not survival of neurons. This evidence raises questions about the relationship between transcriptional regulation of the cholinergic phenotype by retrograde-derived trophic signaling and the transcriptional changes experienced when retrograde transport is impaired due to neuropathological conditions.