Differential effects of transient and sustained activation of BDNF‐TrkB signaling

Brain‐derived neurotrophic factor (BDNF) serves a pleiotropic role in the central nervous system, ranging from promoting neuronal survival and differentiation during development and synaptic modulation in the adult. An important, yet unanswered question is how BDNF could serve such diverse functions, sometimes in the same cell. At least two modes of BDNF actions have been elucidated so far based on BDNF signaling kinetics and/or the activity status of the responding neurons. Acute and gradual increases in extracellular BDNF concentrations elicit, respectively, transient and sustained activation of TrkB receptor and its downstream signaling, leading to differential molecular and cellular functions. In cultured neurons, sustained TrkB activation promotes neuronal dendritic arborization and spinogenesis, whereas transient TrkB activation facilitates dendritic growth and spine morphogenesis. In hippocampal slices, slow delivery of BDNF facilitates LTP, whereas fast application of BDNF enhances basal synaptic transmission in schaffer collateral synapses. High‐frequency stimulation of neurons converts BDNF‐induced TrkB signaling from a transient to a sustained mode. These initial insights lay the foundation for future investigation of the BDNF‐TrkB pathway, and analogous signaling pathways to gain a comprehensive understanding to enable translational research. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 647–659, 2018     

Clathrin-dependent endocytosis is required for TrkB-dependent Akt-mediated neuronal protection and dendritic growth

Endocytosis of Trk (tropomyosin-related kinase) receptors is critical for neurotrophin signal transduction and biological functions. However, the mechanism governing endocytosis of TrkB (tropomyosin-related kinase B) and the specific contributions of TrkB endocytosis to downstream signaling are unknown. In this study, we report that blocking clathrin, dynamin, or AP2 in cultured neurons of the central nervous system inhibited brain-derived neurotrophic factor (BDNF)-induced activation of Akt but not ERK. Treating neurons with the clathrin inhibitor monodansylcadaverine or a peptide that blocks dynamin function specifically abrogated Akt pathway activation in response to BDNF but did not affect the response of other downstream effectors or the up-regulation of immediate early genes neuropeptide Y and activity-regulated cytoskeleton-associated protein. Similar effects were found in neurons expressing small interfering RNA to silence AP2 or a dominant negative form of dynamin that inhibits clathrin-mediated endocytosis. In PC12 cells, ERK but not Akt activation required TrkA endocytosis following stimulation with nerve growth factor, whereas the opposite was true when TrkA-expressing neurons were stimulated with nerve growth factor in the central nervous system. Thus, the specific effects of internalized Trk receptors probably depend on the presence of cell type-specific modulators of neurotrophin signaling and not on differences inherent to Trk receptors themselves. Endocytosis-dependent activation of Akt in neurons was found to be critical for BDNF-supported survival and dendrite outgrowth. Together, these results demonstrate the functional requirement of clathrin- and dynamin-dependent endocytosis in generating the full intracellular response of neurons to BDNF in the central nervous system.     

BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition

The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling can protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.     

Induction of a Distinct Morphology and Signal Transduction in TrkB/PC12 Cells by Nerve Growth Factor and Brain-Derived Neurotrophic Factor

Abstract: A clonal cell line stably expressing trkB (TrkB/PC12) was established from rat pheochromocytoma PC12 cells. Brain-derived neurotrophic factor (BDNF), as well as nerve growth factor (NGF), stimulates neurite outgrowth in TrkB/PC12 cells. However, the morphology of BDNF-differentiated cells was clearly different from NGF-differentiated cells. BDNF treatment brought about longer and thicker neurites and induced a flattened soma and an increase in somatic size. This is not explained enough by the quantitative difference in the strength between TrkA and TrkB stimulation, because the level of BDNF-stimulated tyrosine phosphorylation of TrkB was similar to that of TrkA stimulated with NGF in PC12/TrkB cells. There was no difference in major tyrosine phosphorylated proteins induced by NGF and BDNF. Signal proteins such as phosphatidylinositol 3-kinase, phospholipase C-γ1, Shc, and mitogen-activated protein kinase seem to be involved in both TrkA- and TrkB-mediated signaling pathways. However, a tyrosine-phosphorylated 38-kDa protein (pp38) was detected in anti-pan-Trk immunoprecipitation only after NGF stimulation. Immunoprecipitation using three distinct anti-pan-Trk antibodies suggests that pp38 is not a fragment of TrkA. These data indicate that TrkA has a unique signal transduction pathway that is not stimulated through TrkB in TrkB/PC12 cells and suggest distinct functions among neurotrophin receptors.     

HAP1 Is Required for Endocytosis and Signalling of BDNF and Its Receptors in Neurons

When BDNF binds to its receptors, TrkB and p75^NTR, the BDNF-receptor complex is endocytosed and trafficked to the cell body for downstream signal transduction, which plays a critical role in neuronal functions. Huntingtin-associated protein 1 (HAP1) is involved in trafficking of vesicles intracellularly and also interacts with several membrane proteins including TrkB. Although it has been known that HAP1 has functions in vesicular trafficking and receptor stabilisation, it is not yet established whether HAP1 has a role in BDNF and its receptor endocytosis. In the present study, we found that HAP1 is in an interacting complex with p75^NTR, TrkB and BDNF, especially newly endocytosed BDNF. BDNF and TrkB internalisation is abolished in HAP1 knock-out (KO) cortical neurons. TrkB downstream signalling pathways such as ERK, Akt and PLCγ-1 are also impaired in HAP1 KO cortical neurons upon BDNF stimulation. Proliferation of cerebellar granule cells is also impaired in cell culture and cerebellum of HAP1 KO mice. Our findings suggest that HAP1 may play a key role in BDNF and its receptor endocytosis and may promote neuronal survival and proliferation.     

Truncated TrkB: beyond a dominant negative receptor

BDNF activates trkB receptors to regulate neuronal survival, differentiation, and proliferation. Mutations in the BDNF gene, altered BDNF expression, and altered trkB expression are associated with degenerative and psychiatric disorders. The full-length trkB receptor (trkB.tk(+)) undergoes autophosphorylation to activate intracellular signaling pathways. The truncated trkB receptor (trkB.t1) is abundantly expressed in the brain but lacks the catalytic tyrosine kinase domain. TrkB.t1 is a dominant-negative receptor that inhibits trkB.tk(+) signaling. While this is an important function of trkB.t1, it is only one of its many functions. TrkB.t1 sequesters and translocate BDNF, induces filopodia and neurite outgrowth, stimulates intracellular signaling cascades, regulates Rho GTPase signaling, and modifies cytoskeletal structures. TrkB.t1 is an active signaling molecule with regulatory effects on neurons and astrocytes.     

BDNF modulates heart contraction force and long-term homeostasis through truncated TrkB.T1 receptor activation

Brain-derived neurotrophic factor (BDNF) is critical for mammalian development and plasticity of neuronal circuitries affecting memory, mood, anxiety, pain sensitivity, and energy homeostasis. Here we report a novel unexpected role of BDNF in regulating the cardiac contraction force independent of the nervous system innervation. This function is mediated by the truncated TrkB.T1 receptor expressed in cardiomyocytes. Loss of TrkB.T1 in these cells impairs calcium signaling and causes cardiomyopathy. TrkB.T1 is activated by BDNF produced by cardiomyocytes, suggesting an autocrine/paracrine loop. These findings unveil a novel signaling mechanism in the heart that is activated by BDNF and provide evidence for a global role of this neurotrophin in the homeostasis of the organism by signaling through different TrkB receptor isoforms.     

Mutual Regulation of Src Family Kinases and the Neurotrophin Receptor TrkB

The neurotrophin receptor tyrosine kinase TrkB is critical to diverse biological processes. We investigated the interplay of Src family kinases (SFKs) and TrkB to better understand mechanisms of TrkB signaling in physiological and pathological conditions. We compared and contrasted the role of SFKs in TrkB signaling following activation of TrkB by two mechanisms, its transactivation by zinc, and its activation by its prototypic neurotrophin ligand, brain-derived neurotrophic factor (BDNF). Using biochemical, pharmacological, and chemical genetic studies of cultured rodent neurons, we found that zinc promotes preferential phosphorylation of Tyr-705/Tyr-706 of TrkB by a SFK-dependent but TrkB kinase-independent mechanism, a signaling event critical for transactivation of TrkB by zinc. By contrast, SFK activity is not essential for BDNF-mediated activation of TrkB, yet SFK activity is increased as a consequence of TrkB activation by BDNF. Moreover, BDNF-induced phosphorylation of Tyr-705/Tyr-706 of TrkB was inhibited by SFK inhibitors, implicating a regulatory role of SFKs in TrkB activation by BDNF. In sum, SFKs are activated by TrkB and, in turn, SFKs can promote TrkB activation. We propose models depicting the mutual regulation of SFKs and TrkB following activation of TrkB by zinc and BDNF.     

Rapid Activation of the Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Signaling Pathway by Electroconvulsive Shock in the Rat Prefrontal Cortex Is Not Associated with TrkB Neurotrophin Receptor Activation

1. Emerging evidence indicates that brain-derived neurotrophic factor (BDNF) and its receptor TrkB play important roles in the mechanism of action of electroconvulsive shock (ECS) treatment. ECS produces a significant increase in brain BDNF synthesis together with a variety of neuroplastic changes including neurogenesis and axonal sprouting in the rodent brain, which is believed to be associated to the antidepressant effect of ECS. ERK1/2 (extracellular signal-regulated kinase-1/2) and Akt (protein kinase B), both intracellular signaling molecules being linked to neurotrophin signaling and synthesis, are important pathways triggered by TrkB autophosphorylation. 2. We have previously observed that chemical antidepressants induce a rapid activation of TrkB signaling in the rodent prefrontal cortex (PFC), which is likely a consequence of the stimulatory effect of antidepressants on BDNF synthesis. However, it is not known whether ECS triggers TrkB autophosphorylation and if any ECS-induced effect on TrkB function may be associated with the activation of the ERK1/2 and Akt pathways. 3. The present study assayed the phosphorylation levels of TrkB, ERK1/2, and Akt in the PFC of sham and ECS-treated rats. While the TrkB autophosphorylation (pTrkB) levels were decreased 30 min after both acute and chronic ECS, no change in pTrkB levels were observed at any other time points measured. In contrast, acute but not chronic ECS, transiently induced a very rapid and robust hyperphosphorylation of ERK1/2. Akt phosphorylation levels remained unchanged following acute or chronic ECS. Hence, although ECS effectively stimulates the ERK1/2 pathway in the PFC, this effect does not appear to involve upstream activation of TrkB.     

TrkB receptor signaling in the nucleus tractus solitarius mediates the food intake-suppressive effects of hindbrain BDNF and leptin

Brain-derived neurotrophic factor (BDNF) and TrkB receptor signaling contribute to the central nervous system (CNS) control of energy balance. The role of hindbrain BDNF/TrkB receptor signaling in energy balance regulation is examined here. Hindbrain ventricular BDNF suppressed body weight through reductions in overall food intake and meal size and by increasing core temperature. To localize the neurons mediating the energy balance effects of hindbrain ventricle-delivered BDNF, ventricle subthreshold doses were delivered directly to medial nucleus tractus solitarius (mNTS). mNTS BDNF administration reduced food intake significantly, and this effect was blocked by preadministration of a highly selective TrkB receptor antagonist {[N2-2-2-Oxoazepan-3-yl amino]carbonyl phenyl benzo (b)thiophene-2-carboxamide (ANA-12)}, suggesting that TrkB receptor activation mediates hindbrain BDNF's effect on food intake. Because both BDNF and leptin interact with melanocortin signaling to reduce food intake, we also examined whether the intake inhibitory effects of hindbrain leptin involve hindbrain-specific BDNF/TrkB activation. BDNF protein content within the dorsal vagal complex of the hindbrain was increased significantly by hindbrain leptin delivery. To assess if BDNF/TrkB receptor signaling acts downstream of leptin signaling in the control of energy balance, leptin and ANA-12 were coadministered into the mNTS. Administration of the TrkB receptor antagonist attenuated the intake-suppressive effects of leptin, suggesting that mNTS TrkB receptor activation contributes to the mediation of the anorexigenic effects of hindbrain leptin. Collectively, these results indicate that TrkB-mediated signaling in the mNTS negatively regulates food intake and, in part, the intake inhibitory effects of leptin administered into the NTS.     

BBS4 Is Necessary for Ciliary Localization of TrkB Receptor and Activation by BDNF

Primary cilia regulate an expanding list of signaling pathways in many different cell types. It is likely that identification of the full catalog of pathways associated with cilia will be necessary to fully understand their role in regulation of signaling and the implications for diseases associated with their dysfunction, ciliopathies. Bardet-Biedl Syndrome (BBS) is one such ciliopathy which is characterized by a spectrum of phenotypes. These include neural defects such as impaired cognitive development, centrally mediated hyperphagia and peripheral sensory defects. Here we investigate potential defects in a signaling pathway associated with neuronal function, brain derived neurotrophic factor (BDNF) signaling. Upon loss of BBS4 expression in cultured cells, we observed decreased phosphorylation and activation by BDNF of its target receptor, TrkB. Assessment of ciliary localization revealed that, TrkB localized to the axonemes or basal bodies of cilia only in the presence of BDNF. Axonemal localization, specifically, was abrogated with loss of BBS4. Finally, we present evidence that loss of the ciliary axoneme through depletion of KIF3A impedes activation of TrkB. Taken together, these data suggest the possibility of a previously uninvestigated pathway associated with perturbation of ciliary proteins.     

Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development

Increasing evidence indicates that development of embryonic central nervous system precursors is tightly regulated by extrinsic cues located in the local environment. Here, we asked whether neurotrophin-mediated signaling through Trk tyrosine kinase receptors is important for embryonic cortical precursor cell development. These studies demonstrate that inhibition of TrkB (Ntrk2) and/or TrkC (Ntrk3) signaling using dominant-negative Trk receptors, or genetic knockdown of TrkB using shRNA, caused a decrease in embryonic precursor cell proliferation both in culture and in vivo. Inhibition of TrkB/C also caused a delay in the generation of neurons, but not astrocytes, and ultimately perturbed the postnatal localization of cortical neurons in vivo. Conversely, overexpression of BDNF in cortical precursors in vivo promoted proliferation and enhanced neurogenesis. Together, these results indicate that neurotrophin-mediated Trk signaling plays an essential, cell-autonomous role in regulating the proliferation and differentiation of embryonic cortical precursors and thus controls cortical development at earlier stages than previously thought.     

Numb links extracellular cues to intracellular polarity machinery to promote chemotaxis

Cell polarization is essential throughout development for proliferation, migration, and differentiation. However, it is not known how extracellular cues correctly orient cell polarity at distinct stages of development. Here, we show that the endocytic adaptor protein Numb, previously characterized for its role in cell proliferation, subsequently plays an important role in cell migration. In neural precursors stimulated with the chemotactic factor BDNF, Numb binds to activated TrkB, the BDNF receptor, and functions both as an endocytic regulator for TrkB and as a scaffold for aPKC (aPKC). Thus, Numb promotes BDNF-dependent aPKC activation. Interestingly, Numb is also a substrate of aPKC. When phosphorylated, Numb exhibits increased efficacy in binding TrkB and in promoting a chemotactic response to BDNF. Therefore, Numb functions in a feed-forward loop to promote chemotaxis of neural precursors, linking BDNF, an extracellular cue, to aPKC, a critical component of the intrinsic polarity machinery.     

Brain-derived neurotrophic factor selectively regulates dendritogenesis of parvalbumin-containing interneurons in the main olfactory bulb through the PLCgamma pathway

Molecular mechanisms of neurotrophin signaling on dendrite development and dynamics are only partly understood. To address the role of brain-derived neurotrophic factor (BDNF) in the morphogenesis of GABAergic neurons of the main olfactory bulb, we analyzed mice lacking BDNF, mice carrying neurotrophin-3 (NT3) in the place of BDNF, and TrkB signaling mutant mice with a receptor that can activate phospholipase Cgamma (PLCgamma) but is unable to recruit the adaptors Shc/Frs2. BDNF deletion yielded a compressed olfactory bulb with a significant loss of parvalbumin (PV) immunoreactivity in GABAergic interneurons of the external plexiform layer. Dendrite development of PV-positive interneurons was selectively attenuated by BDNF since other Ca2+ -binding protein-containing neuron populations appeared unaffected. The deficit in PV-positive neurons could be rescued by the NT3/NT3 alleles. The degree of PV immunoreactivity was dependent on BDNF and TrkB recruitment of the adaptor proteins Shc/Frs2. In contrast, PLCgamma signaling from the TrkB receptor was sufficient for dendrite growth in vivo and consistently, blocking PLCgamma prevented BDNF-dependent dendrite development in vitro. Collectively, our results provide genetic evidence that BDNF and TrkB signaling selectively regulate PV expression and dendrite growth in a subset of neurochemically-defined GABAergic interneurons via activation of the PLCgamma pathway.     

TrkB/BDNF signaling regulates photoreceptor progenitor cell fate decisions

Neurotrophins, via activation of Trk receptor tyrosine kinases, serve as mitogens, survival factors and regulators of arborization during retinal development. Brain-derived neurotrophic factor (BDNF) and TrkB regulate neuronal arborization and survival in late retinal development. However, TrkB is expressed during early retinal development where its functions are unclear. To assess TrkB/BDNF actions in the early chick retina, replication-incompetent retroviruses were utilized to over-express a dominant negative truncated form of TrkB (trunc TrkB), or BDNF and effects were assessed at E15. Clones expressing trunc TrkB were smaller than controls, and proliferation and apoptosis assays suggest that decreased clone size correlated with increased cell death when BDNF/TrkB signaling was impaired. Analysis of clonal composition revealed that trunc TrkB over-expression decreased photoreceptor numbers (41%) and increased cell numbers in the middle third of the inner nuclear layer (INL) (23%). Conversely, BDNF over-expression increased photoreceptor numbers (25%) and decreased INL numbers (17%). Photoreceptors over-expressing trunc TrkB demonstrated no increase in apoptosis nor abnormalities in lamination suggesting that TrkB activation is not required for photoreceptor cell survival or migration. These studies suggest that TrkB signaling regulates commitment to and/or differentiation of photoreceptor cells from retinal progenitor cells, identifying a novel role for TrkB/BDNF in regulating cell fate decisions.     

Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system.

Activated receptor tyrosine kinases induce a large number of tyrosine phosphorylation-dependent protein-protein interactions through which they mediate their various ligand-exerted functions including regulation of proliferation, differentiation and survival. TrkB receptor tyrosine kinase activated by binding of brain-derived neurotrophic factor (BDNF) also stimulates various protein interactions in a tyrosine phosphorylation-dependent manner in neuronal cells. To examine tyrosine phosphorylation-dependent interactions stimulated by active TrkB, we developed a modified yeast two-hybrid system, which we call the yeast two-and-a-half-hybrid system. In this system, yeast was engineered to express a tyrosine kinase domain of TrkB as an effector, in addition to two fusion proteins with GAL4 DNA-binding and GAL4 activation domains as bait and prey proteins, respectively. Using this system with Shp2 as the bait, we demonstrated that Shp2 interacts directly with BIT/SHPS-1 (also called SIRP) and Grb2 depending on tyrosine phosphorylation mediated by TrkB. Furthermore, we screened an adult human brain cDNA library with the yeast two-and-a-half-hybrid system in order to identify other Shp2-binding proteins in TrkB-stimulated tyrosine phosphorylation signaling. We found that fibroblast growth factor receptor substrate 2beta (FRS2beta), also called SNT2, interacts with Shp2 dependently on TrkB-mediated tyrosine phosphorylation of FRS2beta/SNT2. Therefore, we show that the two-and-a-half-hybrid system is a powerful tool for studying tyrosine phosphorylation-dependent protein-protein interactions in intracellular signaling pathways stimulated by TrkB receptor tyrosine kinase.     

Control of cortical interneuron migration by neurotrophins and PI3-kinase signaling

During telencephalic development, cells from the medial ganglionic eminence (MGE) are thought to migrate to the neocortex to give rise to a majority of cortical GABAergic interneurons. By combining time-lapse video-microscopy, immunofluorescence and pharmacological perturbations in a new in vitro migration assay, we find that MGE-derived cells migrate through the entire extent of the cortex and into the CA fields of the hippocampus, but avoid the dentate gyrus. Migrating neurons initially travel within the marginal zone and intermediate zone, and can enter the cortical plate from either location. Tangential migration is strongly stimulated by BDNF and NT4 and attenuated by the Trk-family inhibitor, K252a, suggesting that migration is regulated by TrkB signaling. Furthermore, TrkB-null mice show a significant decrease in the number of calbindin-positive neurons migrating tangentially in the embryonic cortex. BDNF and NT4 cause rapid activation of PI3-kinase in MGE cells, and inhibition of PI3-kinase (but not of MAP kinase or PLCgamma) dramatically attenuates tangential migration. These observations suggest that TrkB signaling, via PI3-kinase activation, plays an important role in controlling interneuron migration in the developing cerebral cortex.     

BDNF heightens the sensitivity of motor neurons to excitotoxic insults through activation of TrkB

The survival promoting and neuroprotective actions of brain-derived neurotrophic factor (BDNF) are well known but under certain circumstances this growth factor can also exacerbate excitotoxic insults to neurons. Prior exploration of the receptor through which BDNF exerts this action on motor neurons deflects attention away from p75. Here we investigated the possibility that BDNF acts through the receptor tyrosine kinase, TrkB, to confer on motor neurons sensitivity to excitotoxic challenge. We blocked BDNF activation of TrkB using a dominant negative TrkB mutant or a TrkB function blocking antibody, and found that this protected motor neurons against excitotoxic insult in cultures of mixed spinal cord neurons. Addition of a function blocking antibody to BDNF to mixed spinal cord neuron cultures is also neuroprotective indicating that endogenously produced BDNF participates in vulnerability to excitotoxicity. We next examined the intracellular signaling cascades that are engaged upon TrkB activation. Previously we found that inhibition of the phosphatidylinositide-3'-kinase (PI3'K) pathway blocks BDNF-induced excitotoxic sensitivity. Here we show that expression of a constitutively active catalytic subunit of PI3'K, p110, confers excitotoxic sensitivity (ES) upon motor neurons not incubated with BDNF. Parallel studies with purified motor neurons confirm that these events are likely to be occuring specifically within motor neurons. The abrogation of BDNF's capacity to accentuate excitotoxic insults may make it a more attractive neuroprotective agent.     

Generation of TrkA/TrkB Chimeric Receptor Constructs Reveals Molecular Mechanisms Underlying BDNF-Induced Dendritic Outgrowth in Hippocampal Neurons

Neurotrophins (NTs) regulate neuronal survival, differentiation, and synaptic plasticity through tropomyosin receptor kinases (Trks). The molecular mechanisms underlying these functions, however, have remained incompletely understood. In the present study, we first showed that brain-derived neurotrophic factor (BDNF) increased both the number of primary dendrites and dendritic complexity in cultured hippocampal neurons. Since hippocampal neurons predominantly express the BDNF receptor TrkB, but not the nerve growth factor (NGF) receptor Trk, we generated DNA constructs encoding the extracellular domain of TrkA fused with the transmembrane and intracellular domain of TrkB and introduced these constructs into cultured hippocampal neurons. To visualize the dendrites, the TrkA/TrkB fusion proteins were bicistronically expressed with green fluorescence protein (GFP). Interestingly, the GFP-labeled neurons grew dendrites and activated the TrkA/TrkB receptors in response to NGF, but not BDNF. We next generated a series of TrkA/TrkB receptors with mutations at tyrosine residues in the TrkB kinase domain, and sought to identify the signaling pathway required for NT-induced dendrite outgrowth. Sholl analyses demonstrated that TrkB signaling through Shc, but not through PLC-γ, plays a crucial role in NT-elicited dendritic outgrowth in hippocampal neurons.     

BDNF-induced recruitment of TrkB receptor into neuronal lipid rafts: roles in synaptic modulation

Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity but the underlying signaling mechanisms remain unknown. Here, we show that BDNF rapidly recruits full-length TrkB (TrkB-FL) receptor into cholesterol-rich lipid rafts from nonraft regions of neuronal plasma membranes. Translocation of TrkB-FL was blocked by Trk inhibitors, suggesting a role of TrkB tyrosine kinase in the translocation. Disruption of lipid rafts by depleting cholesterol from cell surface blocked the ligand-induced translocation. Moreover, disruption of lipid rafts prevented potentiating effects of BDNF on transmitter release in cultured neurons and synaptic response to tetanus in hippocampal slices. In contrast, lipid rafts are not required for BDNF regulation of neuronal survival. Thus, ligand-induced TrkB translocation into lipid rafts may represent a signaling mechanism selective for synaptic modulation by BDNF in the central nervous system.