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

Suzuki S, Numakawa T, Shimazu K, Koshimizu H, Hara T, Hatanaka H, Mei L, Lu B, Kojima M - J. Cell Biol. (2004)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Cell Engineering, National Institute for Advanced Industrial Science and Technology, Ikeda, Osaka, Japan.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Attenuation of BDNF enhancement of synaptic exocytosis by lipid raft disruption. Recycling synaptic vesicles in cultured hippocampal or cortical neurons were labeled by FM1-43, and exocytosis was induced by a perfusion of 50 mM KCl containing KRH buffer (high K+ solution). (A) Pseudo-colored images showing high K+ solution-dependent reduction in FM1-43 intensity. FM 1-43 images were captured at the indicated times (s) and baseline intensity was captured at 5 s before depolarization. The monochromic image shows the neuron 5 min before stimulation. Arrowheads indicate representative spots with a gradual reduction of FM dye labeling after depolarization. Bar, 10 μm. (B) Representative recordings of FM 1-43 destaining. Neurons were incubated with or without BDNF for 30 min, followed by a 1-min exposure to high K+ solution to load FM dye. After three washes for 5 min, FM dye was destained with high K+ solution (black arrow). FM 1-43 intensities at 4 s before (gray arrow) and at 15 s after (white arrow) depolarization are defined as Fb and Fa, respectively. ΔF (%) = (Fb−Fa)/Fb × 100. (C–E) Summary of FM dye destaining experiments using hippocampal neurons (C) and cortical neurons (D and E). Cells were pretreated with MCD, MCD–cholesterol complex (MCD-Chol), or filipin for 10 min, followed by BDNF incubation for 30 min and stimulation with high K+ solution. Data were collected at 4 s before and at 15 s after stimulation with high K+ solution. The number associated with each column represents the number of spots recorded for each condition. A 1-min incubation with 5 mM KCl did not elicit any significant change in the intensity of FM 1-43 (not depicted). In all experiments similar results were obtained from at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172613&req=5

fig6: Attenuation of BDNF enhancement of synaptic exocytosis by lipid raft disruption. Recycling synaptic vesicles in cultured hippocampal or cortical neurons were labeled by FM1-43, and exocytosis was induced by a perfusion of 50 mM KCl containing KRH buffer (high K+ solution). (A) Pseudo-colored images showing high K+ solution-dependent reduction in FM1-43 intensity. FM 1-43 images were captured at the indicated times (s) and baseline intensity was captured at 5 s before depolarization. The monochromic image shows the neuron 5 min before stimulation. Arrowheads indicate representative spots with a gradual reduction of FM dye labeling after depolarization. Bar, 10 μm. (B) Representative recordings of FM 1-43 destaining. Neurons were incubated with or without BDNF for 30 min, followed by a 1-min exposure to high K+ solution to load FM dye. After three washes for 5 min, FM dye was destained with high K+ solution (black arrow). FM 1-43 intensities at 4 s before (gray arrow) and at 15 s after (white arrow) depolarization are defined as Fb and Fa, respectively. ΔF (%) = (Fb−Fa)/Fb × 100. (C–E) Summary of FM dye destaining experiments using hippocampal neurons (C) and cortical neurons (D and E). Cells were pretreated with MCD, MCD–cholesterol complex (MCD-Chol), or filipin for 10 min, followed by BDNF incubation for 30 min and stimulation with high K+ solution. Data were collected at 4 s before and at 15 s after stimulation with high K+ solution. The number associated with each column represents the number of spots recorded for each condition. A 1-min incubation with 5 mM KCl did not elicit any significant change in the intensity of FM 1-43 (not depicted). In all experiments similar results were obtained from at least two independent experiments.

Mentions: The biochemical assay described above measures both synaptic and nonsynaptic glutamate release. To determine whether lipid rafts are important for BDNF modulation of neurotransmitter release at synapses, we measured synaptic exocytosis in cultured hippocampal neurons using a style membrane dye FM1-43 (Ryan et al., 1993). A second depolarization of these FM dye-loaded neurons by high K+ solution (see Materials and methods) resulted in a rapid destaining of the FM dye-labeled spots, reflecting transmitter release at the synapses (Fig. 6, A and B). Pre-treatment with BDNF for 30 min enhanced depolarization-induced FM1-43 destaining (Fig. 6 B). A 10-min treatment with MCD (2 mM) before BDNF application prevented the enhancement effect of BDNF (Fig. 6 C). The neurons pretreated with MCD alone exhibited FM1-43 destaining similar to that in control neurons (Fig. 6 C), indicating that neither uptake nor destaining of FM 1-43 dye was affected by MCD pretreatment.


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

Suzuki S, Numakawa T, Shimazu K, Koshimizu H, Hara T, Hatanaka H, Mei L, Lu B, Kojima M - J. Cell Biol. (2004)

Attenuation of BDNF enhancement of synaptic exocytosis by lipid raft disruption. Recycling synaptic vesicles in cultured hippocampal or cortical neurons were labeled by FM1-43, and exocytosis was induced by a perfusion of 50 mM KCl containing KRH buffer (high K+ solution). (A) Pseudo-colored images showing high K+ solution-dependent reduction in FM1-43 intensity. FM 1-43 images were captured at the indicated times (s) and baseline intensity was captured at 5 s before depolarization. The monochromic image shows the neuron 5 min before stimulation. Arrowheads indicate representative spots with a gradual reduction of FM dye labeling after depolarization. Bar, 10 μm. (B) Representative recordings of FM 1-43 destaining. Neurons were incubated with or without BDNF for 30 min, followed by a 1-min exposure to high K+ solution to load FM dye. After three washes for 5 min, FM dye was destained with high K+ solution (black arrow). FM 1-43 intensities at 4 s before (gray arrow) and at 15 s after (white arrow) depolarization are defined as Fb and Fa, respectively. ΔF (%) = (Fb−Fa)/Fb × 100. (C–E) Summary of FM dye destaining experiments using hippocampal neurons (C) and cortical neurons (D and E). Cells were pretreated with MCD, MCD–cholesterol complex (MCD-Chol), or filipin for 10 min, followed by BDNF incubation for 30 min and stimulation with high K+ solution. Data were collected at 4 s before and at 15 s after stimulation with high K+ solution. The number associated with each column represents the number of spots recorded for each condition. A 1-min incubation with 5 mM KCl did not elicit any significant change in the intensity of FM 1-43 (not depicted). In all experiments similar results were obtained from at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172613&req=5

fig6: Attenuation of BDNF enhancement of synaptic exocytosis by lipid raft disruption. Recycling synaptic vesicles in cultured hippocampal or cortical neurons were labeled by FM1-43, and exocytosis was induced by a perfusion of 50 mM KCl containing KRH buffer (high K+ solution). (A) Pseudo-colored images showing high K+ solution-dependent reduction in FM1-43 intensity. FM 1-43 images were captured at the indicated times (s) and baseline intensity was captured at 5 s before depolarization. The monochromic image shows the neuron 5 min before stimulation. Arrowheads indicate representative spots with a gradual reduction of FM dye labeling after depolarization. Bar, 10 μm. (B) Representative recordings of FM 1-43 destaining. Neurons were incubated with or without BDNF for 30 min, followed by a 1-min exposure to high K+ solution to load FM dye. After three washes for 5 min, FM dye was destained with high K+ solution (black arrow). FM 1-43 intensities at 4 s before (gray arrow) and at 15 s after (white arrow) depolarization are defined as Fb and Fa, respectively. ΔF (%) = (Fb−Fa)/Fb × 100. (C–E) Summary of FM dye destaining experiments using hippocampal neurons (C) and cortical neurons (D and E). Cells were pretreated with MCD, MCD–cholesterol complex (MCD-Chol), or filipin for 10 min, followed by BDNF incubation for 30 min and stimulation with high K+ solution. Data were collected at 4 s before and at 15 s after stimulation with high K+ solution. The number associated with each column represents the number of spots recorded for each condition. A 1-min incubation with 5 mM KCl did not elicit any significant change in the intensity of FM 1-43 (not depicted). In all experiments similar results were obtained from at least two independent experiments.
Mentions: The biochemical assay described above measures both synaptic and nonsynaptic glutamate release. To determine whether lipid rafts are important for BDNF modulation of neurotransmitter release at synapses, we measured synaptic exocytosis in cultured hippocampal neurons using a style membrane dye FM1-43 (Ryan et al., 1993). A second depolarization of these FM dye-loaded neurons by high K+ solution (see Materials and methods) resulted in a rapid destaining of the FM dye-labeled spots, reflecting transmitter release at the synapses (Fig. 6, A and B). Pre-treatment with BDNF for 30 min enhanced depolarization-induced FM1-43 destaining (Fig. 6 B). A 10-min treatment with MCD (2 mM) before BDNF application prevented the enhancement effect of BDNF (Fig. 6 C). The neurons pretreated with MCD alone exhibited FM1-43 destaining similar to that in control neurons (Fig. 6 C), indicating that neither uptake nor destaining of FM 1-43 dye was affected by MCD pretreatment.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Cell Engineering, National Institute for Advanced Industrial Science and Technology, Ikeda, Osaka, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus