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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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Requirement of TrkB activation for BDNF-induced TrkB translocation into lipid rafts. Immunoblots were performed using anti-TrkB and anti–phospho-Trk antibodies. (A) TrkB-FL recruited into rafts is tyrosine phosphorylated. Cortical neurons were treated with (200 ng/ml) or without BDNF for 30 min. (Top) Sample blots. (Bottom) Quantification. pTrkB-FL/total TrkB-FL was quantified in fractions 2 and 6, and shown as relative to that of “+BDNF” in fraction 6. n = 5 preparations from five independent experiments. (B) Inhibition of TrkB-FL activation prevents TrkB-FL translocation into lipid rafts. Neurons were treated with 100 nM K252a or 10 μM AG879 for 3 h, followed by a 30-min stimulation with 200 ng/ml BDNF. (C) Effect of a brief exposure to BDNF on TrkB-FL translocation. Cortical neurons were treated with 200 ng/ml BDNF for only 1 min followed by incubating with medium containing no BDNF for the indicated times. Note that once activated, TrkB-FL continued to be partitioned into rafts in the absence of BDNF.
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fig2: Requirement of TrkB activation for BDNF-induced TrkB translocation into lipid rafts. Immunoblots were performed using anti-TrkB and anti–phospho-Trk antibodies. (A) TrkB-FL recruited into rafts is tyrosine phosphorylated. Cortical neurons were treated with (200 ng/ml) or without BDNF for 30 min. (Top) Sample blots. (Bottom) Quantification. pTrkB-FL/total TrkB-FL was quantified in fractions 2 and 6, and shown as relative to that of “+BDNF” in fraction 6. n = 5 preparations from five independent experiments. (B) Inhibition of TrkB-FL activation prevents TrkB-FL translocation into lipid rafts. Neurons were treated with 100 nM K252a or 10 μM AG879 for 3 h, followed by a 30-min stimulation with 200 ng/ml BDNF. (C) Effect of a brief exposure to BDNF on TrkB-FL translocation. Cortical neurons were treated with 200 ng/ml BDNF for only 1 min followed by incubating with medium containing no BDNF for the indicated times. Note that once activated, TrkB-FL continued to be partitioned into rafts in the absence of BDNF.

Mentions: Binding of BDNF to TrkB-FL induces the autophosphorylation of its tyrosine residues on the intracellular kinase domain, leading to its activation (Kaplan and Miller, 2000). To test whether TrkB recruited into rafts is activated, we performed Western blot analysis using anti–phospho-Trk antibody (pY490; Binder et al., 1999). In neurons treated with BDNF for 30 min, a substantial amount of TrkB-FL recruited into rafts was tyrosine phosphorylated (Fig. 2 A, top). In BDNF-treated cultures, TrkB-FL tyrosine phosphorylation relative to total TrkB-FL protein was not significantly different between in rafts and in nonrafts (Fig. 2 A, bottom; P > 0.64). To investigate whether activation of TrkB tyrosine kinase was required for the translocation of TrkB-FL into lipid rafts, we performed the following experiments. First, we treated cortical neurons with the Trk kinase inhibitors, K252a or AG879 for 3 h before BDNF stimulation (Fig. 2 B). K252a (100 nM), which reduced BDNF-dependent TrkB tyrosine phosphorylation by 68.8 ± 12.2% in rafts and by 49.4 ± 7.0% in the nonrafts (n = 3 independent experiments), inhibited BDNF-induced TrkB-FL translocation (Fig. 2 B). BDNF-dependent recruitment of TrkB-FL into rafts was also blocked by another Trk kinase inhibitor AG879 (10 μM). Second, because TrkB activation could be induced by a 1-min exposure to BDNF (Takei et al., 1998), we tested whether this short-term stimulation would allow recruitment and partition of TrkB-FL in rafts. Cultured cortical neurons were stimulated with BDNF for 1 min, followed by incubation with medium containing no BDNF for 5–360 min. As shown in Fig. 2 C, a 1-min stimulation with BDNF was sufficient to recruit TrkB-FL into rafts. The amount of TrkB-FL continued to increase after BDNF was washed out, suggesting that once activated TrkB-FL can move into lipid rafts. Third, TrkB-T1, a truncated form of the TrkB receptor lacking tyrosine kinase domain, did not appear to be partitioned into rafts by BDNF (Fig. S3A, available at http://www.jcb.org/cgi/content/full/jcb.200404106/DC1). Finally, NT-4 (200 ng/ml, 30 min), another ligand that activates TrkB tyrosine kinase, recruited TrkB-FL into rafts (Fig. S3 B). Together, these results suggest that BDNF-induced translocation of TrkB into rafts requires its tyrosine kinase activity. Because cultured astrocytes expressed TrkB-T1, but not TrkB-FL (Fig. S3 C), the TrkB-FL translocation is likely to occur in neurons only.


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)

Requirement of TrkB activation for BDNF-induced TrkB translocation into lipid rafts. Immunoblots were performed using anti-TrkB and anti–phospho-Trk antibodies. (A) TrkB-FL recruited into rafts is tyrosine phosphorylated. Cortical neurons were treated with (200 ng/ml) or without BDNF for 30 min. (Top) Sample blots. (Bottom) Quantification. pTrkB-FL/total TrkB-FL was quantified in fractions 2 and 6, and shown as relative to that of “+BDNF” in fraction 6. n = 5 preparations from five independent experiments. (B) Inhibition of TrkB-FL activation prevents TrkB-FL translocation into lipid rafts. Neurons were treated with 100 nM K252a or 10 μM AG879 for 3 h, followed by a 30-min stimulation with 200 ng/ml BDNF. (C) Effect of a brief exposure to BDNF on TrkB-FL translocation. Cortical neurons were treated with 200 ng/ml BDNF for only 1 min followed by incubating with medium containing no BDNF for the indicated times. Note that once activated, TrkB-FL continued to be partitioned into rafts in the absence of BDNF.
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fig2: Requirement of TrkB activation for BDNF-induced TrkB translocation into lipid rafts. Immunoblots were performed using anti-TrkB and anti–phospho-Trk antibodies. (A) TrkB-FL recruited into rafts is tyrosine phosphorylated. Cortical neurons were treated with (200 ng/ml) or without BDNF for 30 min. (Top) Sample blots. (Bottom) Quantification. pTrkB-FL/total TrkB-FL was quantified in fractions 2 and 6, and shown as relative to that of “+BDNF” in fraction 6. n = 5 preparations from five independent experiments. (B) Inhibition of TrkB-FL activation prevents TrkB-FL translocation into lipid rafts. Neurons were treated with 100 nM K252a or 10 μM AG879 for 3 h, followed by a 30-min stimulation with 200 ng/ml BDNF. (C) Effect of a brief exposure to BDNF on TrkB-FL translocation. Cortical neurons were treated with 200 ng/ml BDNF for only 1 min followed by incubating with medium containing no BDNF for the indicated times. Note that once activated, TrkB-FL continued to be partitioned into rafts in the absence of BDNF.
Mentions: Binding of BDNF to TrkB-FL induces the autophosphorylation of its tyrosine residues on the intracellular kinase domain, leading to its activation (Kaplan and Miller, 2000). To test whether TrkB recruited into rafts is activated, we performed Western blot analysis using anti–phospho-Trk antibody (pY490; Binder et al., 1999). In neurons treated with BDNF for 30 min, a substantial amount of TrkB-FL recruited into rafts was tyrosine phosphorylated (Fig. 2 A, top). In BDNF-treated cultures, TrkB-FL tyrosine phosphorylation relative to total TrkB-FL protein was not significantly different between in rafts and in nonrafts (Fig. 2 A, bottom; P > 0.64). To investigate whether activation of TrkB tyrosine kinase was required for the translocation of TrkB-FL into lipid rafts, we performed the following experiments. First, we treated cortical neurons with the Trk kinase inhibitors, K252a or AG879 for 3 h before BDNF stimulation (Fig. 2 B). K252a (100 nM), which reduced BDNF-dependent TrkB tyrosine phosphorylation by 68.8 ± 12.2% in rafts and by 49.4 ± 7.0% in the nonrafts (n = 3 independent experiments), inhibited BDNF-induced TrkB-FL translocation (Fig. 2 B). BDNF-dependent recruitment of TrkB-FL into rafts was also blocked by another Trk kinase inhibitor AG879 (10 μM). Second, because TrkB activation could be induced by a 1-min exposure to BDNF (Takei et al., 1998), we tested whether this short-term stimulation would allow recruitment and partition of TrkB-FL in rafts. Cultured cortical neurons were stimulated with BDNF for 1 min, followed by incubation with medium containing no BDNF for 5–360 min. As shown in Fig. 2 C, a 1-min stimulation with BDNF was sufficient to recruit TrkB-FL into rafts. The amount of TrkB-FL continued to increase after BDNF was washed out, suggesting that once activated TrkB-FL can move into lipid rafts. Third, TrkB-T1, a truncated form of the TrkB receptor lacking tyrosine kinase domain, did not appear to be partitioned into rafts by BDNF (Fig. S3A, available at http://www.jcb.org/cgi/content/full/jcb.200404106/DC1). Finally, NT-4 (200 ng/ml, 30 min), another ligand that activates TrkB tyrosine kinase, recruited TrkB-FL into rafts (Fig. S3 B). Together, these results suggest that BDNF-induced translocation of TrkB into rafts requires its tyrosine kinase activity. Because cultured astrocytes expressed TrkB-T1, but not TrkB-FL (Fig. S3 C), the TrkB-FL translocation is likely to occur in neurons only.

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