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1.
Front Mol Neurosci ; 17: 1009404, 2024.
Article in English | MEDLINE | ID: mdl-38660384

ABSTRACT

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.

3.
J Neurosci ; 40(42): 8042-8054, 2020 10 14.
Article in English | MEDLINE | ID: mdl-32928890

ABSTRACT

Brain-derived neurotrophic factor (BDNF) is a key regulator of the morphology and connectivity of central neurons. We have previously shown that BDNF/TrkB signaling regulates the activity and mobility of the GTPases Rab5 and Rab11, which in turn determine the postendocytic sorting of signaling TrkB receptors. Moreover, decreased Rab5 or Rab11 activity inhibits BDNF-induced dendritic branching. Whether Rab5 or Rab11 activity is important for local events only or for regulating nuclear signaling and gene expression is unknown. Here, we investigated, in rat hippocampal neuronal cultures derived from embryos of unknown sex, whether BDNF-induced signaling cascades are altered when early and recycling endosomes are disrupted by the expression of dominant-negative mutants of Rab5 and Rab11. The activity of both Rab5 and Rab11 was required for sustained activity of Erk1/2 and nuclear CREB phosphorylation, and increased transcription of a BDNF-dependent program of gene expression containing CRE binding sites, which includes activity-regulated genes such as Arc, Dusp1, c-fos, Egr1, and Egr2, and growth and survival genes such as Atf3 and Gem Based on our results, we propose that early and recycling endosomes provide a platform for the integration of neurotrophic signaling from the plasma membrane to the nucleus in neurons, and that this mechanism is likely to regulate neuronal plasticity and survival.SIGNIFICANCE STATEMENT BDNF is a neurotrophic factor that regulates plastic changes in the brain, including dendritic growth. The cellular and molecular mechanisms underlying this process are not completely understood. Our results uncover the cellular requirements that central neurons possess to integrate the plasma membrane into nuclear signaling in neurons. Our results indicate that the endosomal pathway is required for the signaling cascade initiated by BDNF and its receptors at the plasma membrane to modulate BDNF-dependent gene expression and neuronal dendritic growth mediated by the CREB transcription factor. CREB is a key transcription factor regulating circuit development and learning and memory.


Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Cyclic AMP Response Element-Binding Protein/biosynthesis , Hippocampus/metabolism , Neurons/metabolism , Signal Transduction/physiology , rab GTP-Binding Proteins/physiology , rab5 GTP-Binding Proteins/physiology , Animals , Cyclic AMP Response Element-Binding Protein/genetics , Dendrites/drug effects , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , MAP Kinase Signaling System/physiology , Phosphorylation , Primary Cell Culture , Rats
4.
Traffic ; 21(1): 13-33, 2020 01.
Article in English | MEDLINE | ID: mdl-31670447

ABSTRACT

Neurons are highly polarized cells that critically depend on long-range, bidirectional transport between the cell body and synapse for their function. This continual and highly coordinated trafficking process, which takes place via the axon, has fascinated researchers since the early 20th century. Ramon y Cajal first proposed the existence of axonal trafficking of biological material after observing that dissociation of the axon from the cell body led to neuronal degeneration. Since these first indirect observations, the field has come a long way in its understanding of this fundamental process. However, these advances in our knowledge have been aided by breakthroughs in other scientific disciplines, as well as the parallel development of novel tools, techniques and model systems. In this review, we summarize the evolution of tools used to study axonal transport and discuss how their deployment has refined our understanding of this process. We also highlight innovative tools currently being developed and how their addition to the available axonal transport toolkit might help to address key outstanding questions.


Subject(s)
Axonal Transport , Kinesins , Animals , Axons/metabolism , Humans , Kinesins/metabolism , Models, Biological , Neurons/metabolism
5.
Neuropharmacology ; 107: 131-145, 2016 08.
Article in English | MEDLINE | ID: mdl-26965219

ABSTRACT

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.


Subject(s)
Brain/drug effects , Brain/metabolism , Neuroprotective Agents/pharmacology , Stroke/drug therapy , Stroke/metabolism , Venlafaxine Hydrochloride/pharmacology , Animals , Antidepressive Agents/pharmacology , Astrocytes/drug effects , Astrocytes/metabolism , Astrocytes/pathology , Brain/pathology , Brain-Derived Neurotrophic Factor/metabolism , Disease Models, Animal , Drug Evaluation, Preclinical , Endothelin-1 , Fibroblast Growth Factor 2/metabolism , Gliosis/drug therapy , Gliosis/metabolism , Gliosis/pathology , Male , RNA, Messenger/metabolism , Random Allocation , Rats, Sprague-Dawley , Recovery of Function/drug effects , Recovery of Function/physiology , Stroke/pathology , Transforming Growth Factor beta1/metabolism
6.
J Neurosci ; 33(14): 6112-22, 2013 Apr 03.
Article in English | MEDLINE | ID: mdl-23554492

ABSTRACT

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.


Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Dendrites/drug effects , Endosomes/drug effects , GTP-Binding Proteins/metabolism , Neurons/cytology , Analysis of Variance , Animals , Antibodies/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carbazoles/pharmacology , Cells, Cultured , Dendrites/physiology , Dendrites/ultrastructure , Embryo, Mammalian , Endocytosis/drug effects , Endosomes/ultrastructure , Enzyme Inhibitors/pharmacology , Female , GTP-Binding Proteins/genetics , GTP-Binding Proteins/immunology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Guanosine Triphosphate/metabolism , Hippocampus/cytology , Indole Alkaloids/pharmacology , Male , Microscopy, Confocal , Microtubule-Associated Proteins/metabolism , Mutation/genetics , Myosins/metabolism , Neurons/drug effects , RNA, Small Interfering/pharmacology , Rats , Receptor, trkB/metabolism , Thiazolidines/pharmacology , Transfection
7.
Mol Neurodegener ; 7: 11, 2012 Mar 29.
Article in English | MEDLINE | ID: mdl-22458984

ABSTRACT

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.


Subject(s)
Brain/metabolism , Cholinergic Fibers/metabolism , Endosomes/metabolism , Nerve Growth Factor/metabolism , Niemann-Pick Disease, Type C/metabolism , Signal Transduction/physiology , Animals , Cholinergic Fibers/pathology , Disease Models, Animal , Endosomes/pathology , Immunohistochemistry , Mice , Mice, Inbred BALB C , Mice, Knockout , Microscopy, Confocal , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Niemann-Pick Disease, Type C/pathology , PC12 Cells , Rats , Up-Regulation
8.
Mol Neurodegener ; 5: 5, 2010 Jan 19.
Article in English | MEDLINE | ID: mdl-20205865

ABSTRACT

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.

9.
Dev Neurobiol ; 70(3): 150-64, 2010 Feb 15.
Article in English | MEDLINE | ID: mdl-19953569

ABSTRACT

The D5 domain of TrkC receptors is a docking site for Neurotrophin-3 (NT-3), but other domains may be relevant for function or harmonizing signals with p75(NTR) coreceptors. We report a monoclonal antibody (mAb) 2B7 targeting the juxtamembrane domain of TrkC. mAb 2B7 binds to murine and human TrkC receptors and is a functional agonist that affords activation of TrkC, AKT, and MAPK. These signals result in cell survival but not in cellular differentiation. Monomeric 2B7 Fabs also affords cell survival. Binding of 2B7 mAb and 2B7 Fabs to TrkC are blocked by NT-3 in a dose-dependent manner but not by pro-NT-3. Expression of p75(NTR) coreceptors on the cell surface block the binding and function of mAb 2B7, whereas NT-3 binding and function are enhanced. mAb 2B7 defines a previously unknown neurotrophin receptor functional hot spot; that exclusively generates survival signals; that can be activated by non-dimeric ligands; and potentially unmasks a site for p75-TrkC interactions.


Subject(s)
Receptor, trkC/metabolism , Receptors, Nerve Growth Factor/metabolism , Animals , Antibodies, Monoclonal/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Cell Differentiation/physiology , Cell Line, Tumor , Cell Membrane/physiology , Cell Survival/physiology , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/metabolism , Hippocampus/physiology , Humans , Mice , Nerve Growth Factors/metabolism , Nerve Tissue Proteins , Neurons/physiology , Proto-Oncogene Proteins c-akt/metabolism , Rats , Receptor, trkC/agonists , Receptors, Growth Factor
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