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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.

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Inhibition of BDNF-induced TrkB-FL translocation and evoked glutamate release by lipid raft disruption. (A) Effect of MCD on BDNF-induced TrkB translocation. Cortical neurons were treated with or without MCD (2 mM) for 10 min. (Left) Raft (fraction 2) and nonraft fractions (fraction 6) were immunoblotted for the indicated proteins. Note that MCD blocked BDNF-induced TrkB phosphorylation in rafts and translocation to rafts. (Middle) TrkB-FLfraction 2/TrkB-FLfraction 6 was quantified and shown as relative to “−BDNF” in control cultures (n = 4 preparations from three independent experiments). *Indicates significantly different from “−BDNF” in control cultures; t test; P < 0.01. (Right) Treatment with 2 mM MCD for 10 min did not influence the amount of caveolin-2 and Fyn in lipid raft fraction. (B) Effect of MCD on BDNF enhancement of 4-AP–evoked glutamate release from cortical neurons. Neurons were pretreated with or without 2 mM MCD for 10 min, followed by incubation with or without BDNF for 30 min. After washing four times, neurons were stimulated with 1 mM 4-AP. *Indicates significantly higher than all other 4-AP–treated groups; ANOVA test; P < 0.05; n = 4 cultures. There was no statistically significant difference among all white bars (ANOVA test, P > 0.48). Similar results were obtained from three independent experiments. (C) Effect of short-term treatment with MCD on the number and distribution of synapses. (Left) Immunocytochemistry for the indicated proteins was done after a 30-min incubation with or without 2 mM MCD. (Right) The number of synapses (doubly labeled by anti–PSD-95 and anti-synaptophysin antibodies) was determined and shown as relative to that of “Control” (n = 90 dendritic segments with 50-μm length from three independent coverslips and experiments). Note that there is no significant difference in distribution and number of the immunopositive synapses between control and MCD-treated cultures. Bar, 5 μm.
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fig5: Inhibition of BDNF-induced TrkB-FL translocation and evoked glutamate release by lipid raft disruption. (A) Effect of MCD on BDNF-induced TrkB translocation. Cortical neurons were treated with or without MCD (2 mM) for 10 min. (Left) Raft (fraction 2) and nonraft fractions (fraction 6) were immunoblotted for the indicated proteins. Note that MCD blocked BDNF-induced TrkB phosphorylation in rafts and translocation to rafts. (Middle) TrkB-FLfraction 2/TrkB-FLfraction 6 was quantified and shown as relative to “−BDNF” in control cultures (n = 4 preparations from three independent experiments). *Indicates significantly different from “−BDNF” in control cultures; t test; P < 0.01. (Right) Treatment with 2 mM MCD for 10 min did not influence the amount of caveolin-2 and Fyn in lipid raft fraction. (B) Effect of MCD on BDNF enhancement of 4-AP–evoked glutamate release from cortical neurons. Neurons were pretreated with or without 2 mM MCD for 10 min, followed by incubation with or without BDNF for 30 min. After washing four times, neurons were stimulated with 1 mM 4-AP. *Indicates significantly higher than all other 4-AP–treated groups; ANOVA test; P < 0.05; n = 4 cultures. There was no statistically significant difference among all white bars (ANOVA test, P > 0.48). Similar results were obtained from three independent experiments. (C) Effect of short-term treatment with MCD on the number and distribution of synapses. (Left) Immunocytochemistry for the indicated proteins was done after a 30-min incubation with or without 2 mM MCD. (Right) The number of synapses (doubly labeled by anti–PSD-95 and anti-synaptophysin antibodies) was determined and shown as relative to that of “Control” (n = 90 dendritic segments with 50-μm length from three independent coverslips and experiments). Note that there is no significant difference in distribution and number of the immunopositive synapses between control and MCD-treated cultures. Bar, 5 μm.

Mentions: What is the functional role of BDNF-induced TrkB translocation into lipid rafts? Because the translocation was observed 1 min after BDNF application (Fig. S2), we reasoned that this process may be involved in the short-term actions of BDNF (Lu, 2003). BDNF has been shown to rapidly enhance transmitter release in cultured cortical neurons (Matsumoto et al., 2001). Therefore, we tested whether disruption of lipid rafts would interfere with BDNF modulation of depolarization-evoked transmitter release at CNS synapses. Methyl-β-cyclodextrin (MCD) binds and depletes cholesterol from the plasma membrane, and thereby disrupts lipid rafts (Simons and Toomre, 2000). A 10-min treatment with MCD (2 mM) removed membrane cholesterol by 33 ± 2% (91 ± 2 ng/well in control cultures, 61.2 ± 2 ng/well in MCD-treated cultures, n = 4 independent experiments), but did not affect the amount of caveolin-2 and Fyn in lipid rafts (Fig. 5 A, right). Treatment of the cultured cortical neurons with 2 mM MCD significantly attenuated the BDNF-induced recruitment of TrkB-FL into the rafts (Fig. 5 A, left and middle). Consequently, the amount of phosphorylated TrkB was also reduced in the rafts. Importantly, TrkB phosphorylation in the nonraft fraction was not affected. Thus, cholesterol may play a role in TrkB translocation into lipid rafts.


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)

Inhibition of BDNF-induced TrkB-FL translocation and evoked glutamate release by lipid raft disruption. (A) Effect of MCD on BDNF-induced TrkB translocation. Cortical neurons were treated with or without MCD (2 mM) for 10 min. (Left) Raft (fraction 2) and nonraft fractions (fraction 6) were immunoblotted for the indicated proteins. Note that MCD blocked BDNF-induced TrkB phosphorylation in rafts and translocation to rafts. (Middle) TrkB-FLfraction 2/TrkB-FLfraction 6 was quantified and shown as relative to “−BDNF” in control cultures (n = 4 preparations from three independent experiments). *Indicates significantly different from “−BDNF” in control cultures; t test; P < 0.01. (Right) Treatment with 2 mM MCD for 10 min did not influence the amount of caveolin-2 and Fyn in lipid raft fraction. (B) Effect of MCD on BDNF enhancement of 4-AP–evoked glutamate release from cortical neurons. Neurons were pretreated with or without 2 mM MCD for 10 min, followed by incubation with or without BDNF for 30 min. After washing four times, neurons were stimulated with 1 mM 4-AP. *Indicates significantly higher than all other 4-AP–treated groups; ANOVA test; P < 0.05; n = 4 cultures. There was no statistically significant difference among all white bars (ANOVA test, P > 0.48). Similar results were obtained from three independent experiments. (C) Effect of short-term treatment with MCD on the number and distribution of synapses. (Left) Immunocytochemistry for the indicated proteins was done after a 30-min incubation with or without 2 mM MCD. (Right) The number of synapses (doubly labeled by anti–PSD-95 and anti-synaptophysin antibodies) was determined and shown as relative to that of “Control” (n = 90 dendritic segments with 50-μm length from three independent coverslips and experiments). Note that there is no significant difference in distribution and number of the immunopositive synapses between control and MCD-treated cultures. Bar, 5 μm.
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fig5: Inhibition of BDNF-induced TrkB-FL translocation and evoked glutamate release by lipid raft disruption. (A) Effect of MCD on BDNF-induced TrkB translocation. Cortical neurons were treated with or without MCD (2 mM) for 10 min. (Left) Raft (fraction 2) and nonraft fractions (fraction 6) were immunoblotted for the indicated proteins. Note that MCD blocked BDNF-induced TrkB phosphorylation in rafts and translocation to rafts. (Middle) TrkB-FLfraction 2/TrkB-FLfraction 6 was quantified and shown as relative to “−BDNF” in control cultures (n = 4 preparations from three independent experiments). *Indicates significantly different from “−BDNF” in control cultures; t test; P < 0.01. (Right) Treatment with 2 mM MCD for 10 min did not influence the amount of caveolin-2 and Fyn in lipid raft fraction. (B) Effect of MCD on BDNF enhancement of 4-AP–evoked glutamate release from cortical neurons. Neurons were pretreated with or without 2 mM MCD for 10 min, followed by incubation with or without BDNF for 30 min. After washing four times, neurons were stimulated with 1 mM 4-AP. *Indicates significantly higher than all other 4-AP–treated groups; ANOVA test; P < 0.05; n = 4 cultures. There was no statistically significant difference among all white bars (ANOVA test, P > 0.48). Similar results were obtained from three independent experiments. (C) Effect of short-term treatment with MCD on the number and distribution of synapses. (Left) Immunocytochemistry for the indicated proteins was done after a 30-min incubation with or without 2 mM MCD. (Right) The number of synapses (doubly labeled by anti–PSD-95 and anti-synaptophysin antibodies) was determined and shown as relative to that of “Control” (n = 90 dendritic segments with 50-μm length from three independent coverslips and experiments). Note that there is no significant difference in distribution and number of the immunopositive synapses between control and MCD-treated cultures. Bar, 5 μm.
Mentions: What is the functional role of BDNF-induced TrkB translocation into lipid rafts? Because the translocation was observed 1 min after BDNF application (Fig. S2), we reasoned that this process may be involved in the short-term actions of BDNF (Lu, 2003). BDNF has been shown to rapidly enhance transmitter release in cultured cortical neurons (Matsumoto et al., 2001). Therefore, we tested whether disruption of lipid rafts would interfere with BDNF modulation of depolarization-evoked transmitter release at CNS synapses. Methyl-β-cyclodextrin (MCD) binds and depletes cholesterol from the plasma membrane, and thereby disrupts lipid rafts (Simons and Toomre, 2000). A 10-min treatment with MCD (2 mM) removed membrane cholesterol by 33 ± 2% (91 ± 2 ng/well in control cultures, 61.2 ± 2 ng/well in MCD-treated cultures, n = 4 independent experiments), but did not affect the amount of caveolin-2 and Fyn in lipid rafts (Fig. 5 A, right). Treatment of the cultured cortical neurons with 2 mM MCD significantly attenuated the BDNF-induced recruitment of TrkB-FL into the rafts (Fig. 5 A, left and middle). Consequently, the amount of phosphorylated TrkB was also reduced in the rafts. Importantly, TrkB phosphorylation in the nonraft fraction was not affected. Thus, cholesterol may play a role in TrkB translocation into lipid rafts.

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