|
|
|
|
|
|
|
||
Article |
BDNF-induced recruitment of TrkB receptor into neuronal lipid rafts
: roles in synaptic modulation
2 Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan
3 Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
4 National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, 187-8502, Japan
5 Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912
6 Section on Neural Development and Plasticity, NICHD, National Institutes of Health, Bethesda, MD 20892
Correspondence to Masami Kojima: m-kojima{at}aist.go.jp; or Bai Lu: bailu{at}mail.NIH.gov
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
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.
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Despite rapid progress in this area, the molecular mechanisms remain ill defined (Lu, 2003). All the functions of BDNF are mediated by TrkB, a receptor tyrosine kinase (RTK; Kaplan and Miller, 2000). Binding of BDNF rapidly activates its tyrosine kinase, which in turn triggers multiple intracellular signaling pathways. Downstream pathways include MAPK, phosphatidylinositol 3-kinase (PI3-K) and PLC
. A critical yet poorly understood issue is how signals from this receptor are transduced to mediate diverse biological functions in CNS neurons.
One idea for specific signal-function coupling is that different signaling pathways may be transduced in different subcellular compartments. More specifically, it has been proposed that cholesterol/sphingolipid-rich microdomains called lipid rafts make a specialized signaling platform in the plasma membrane, and therefore can transduce signals different from those in the nonraft membrane (Simons and Toomre, 2000; Anderson and Jacobson, 2002). Because both lipid components are resistant to solubilization with nonionic detergents, lipid rafts can be biochemically isolated as detergent-resistant membrane fractions. Raft fractions prepared from brain tissues are enriched in proteins that carry lipid modifications such as glycosylphosphatidylinositol (GPI)-anchored proteins, as well as palmitylated or myristoylated proteins such as Src-family kinases and trimeric or small G proteins, suggesting a crucial role of lipid rafts in signal transduction in the CNS (Paratcha and Ibanez, 2002). Recently, lipid rafts have been shown to serve as organizing platforms for chemotrophic guidance of nerve growth cones (Guirland et al., 2004). Transmembrane RTKs, including EGF receptor (Mineo et al., 1999) and FGF receptor (Citores et al., 1999) are associated with rafts. The localization of certain signaling molecules in the rafts allows them to interact with each other more efficiently, and prevents them from interacting with the proteins outside rafts (Simons and Toomre, 2000). Thus, entering and exiting lipid rafts of RTKs represent a unique mechanism that transduces differential signals at the subcellular levels. In the present study, we used brain tissues, slices and dissociated cultures to examine whether TrkB receptor is localized in lipid rafts of the plasma membrane, and if so, how the localization is regulated and what the functional roles are. Our results reveal a BDNF-induced TrkB translocation into the lipid rafts, and such translocation is important for BDNF-induced synaptic modulation in CNS neurons.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
To directly test whether BDNF recruits TrkB into lipid rafts, we prepared the rafts in cultured cortical neurons treated with BDNF. In the cortical cultures used here, 93.3 ± 2.4% and 3.8 ± 0.7% are NSE-positive neurons and GFAP-positive astrocytes, respectively (n = 6 independent experiments). As shown in Fig. 1 A, application of BDNF induced an increase in TrkB-FL in the raft fraction. There was a low amount of TrkB-FL in lipid rafts in naïve neurons, suggesting that in naive cells TrkB-FL may be associated with rafts with a low affinity. In cultures stimulated with 200 ng/ml BDNF for 30 min, the amount of TrkB-FL was markedly increased in rafts (TrkB-FLBDNF-treated/TrkB-FLcontrol: 3.4 ± 0.8-fold, P < 0.03) but decreased slightly in nonrafts (23.1 ± 6.4%). Total proteins in both regions were not changed by the BDNF treatment (Fig. 1 A). When expressed as TrkB-FL/total protein, BDNF increased TrkB-FL in rafts by 3.63 ± 0.73-fold, indicating that BDNF selectively increases the amount of TrkB-FL, but not protein concentration, in rafts.
|
TrkB translocation: dependence on its tyrosine kinase activity
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.
|
|
, leading to activation of MAPK (Erks), PLC, and PI3-K pathways (Kaplan and Miller, 2000). We tested whether TrkB-FL, upon BDNF stimulation, carries these signaling molecules into lipid rafts during its translocation. In naïve neurons, Erks extended throughout the gradient (fractions 2–5), whereas Shc, Grb2, p85 subunit of PI3-K, Akt, and PLC were primarily localized in the bottom, nonraft fraction (Fig. 4 A, left). BDNF application did not increase the amount of any of these proteins in fraction 2 (Fig. 4 A, right), suggesting that TrkB-FL does not carry its associated signaling molecules into lipid rafts during its translocation. When a milder detergent Triton X-165 was used to prepare rafts (Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200404106/DC1), many of them (Shc, Grb2, Erks, and PLC) were extended throughout the gradient whereas p85 subunit of PI3-K and Akt appeared to be still in nonraft fraction. It was notable that BDNF did not stimulate the translocation of these signaling molecules into rafts (Fig. S4 A). Parallel to these, BDNF-induced translocation of TrkB-FL (Fig. 4 B, left) was accompanied by a significant increase in the phosphorylation of Erks (Figs. 4 B, middle and Fig. 4 D), but not that of Akt (Fig. 4 B, right and Fig. 4 D), in lipid rafts. In nonraft regions, however, both Erks and Akt were activated by BDNF application (Fig. 4, C and D). Thus, although TrkB-FL does not move with its associated proteins during translocation, the translocation of TrkB-FL itself into rafts may be a key event in forming TrkB signaling complex, including TrkB-FL and its associated proteins, in rafts, leading to preferential activation of Erks over Akt in neuronal lipid rafts.
|
|
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.
|
One of the major functions of BDNF in the intact hippocampal synaptic circuits is to attenuate synaptic fatigue induced by a train of high frequency stimulation (HFS; or tetanus, 100 Hz, 1 s; Figurov et al., 1996). We next examined the role of lipid rafts in this form of synaptic modulation. In neonatal hippocampal slices (P12-13) in which the level of endogenous BDNF is low, application of tetanus resulted in pronounced synaptic fatigue at Schaffer collateral-CA1 synapses (Fig. 7 A). Consistent with our previous reports (Figurov et al., 1996), treatment with exogenous BDNF (2 nM) for 1–2 h significantly attenuated the synaptic fatigue (Fig. 7 B). However, pretreatment with MCD for 30 min completely abolished the attenuating effect of BDNF on HFS-induced synaptic fatigue. Quantitative analysis indicated that treatment with BDNF markedly increased the rate constant (
) for synaptic fatigue and disruption of lipid rafts with MCD completely prevented such an increase (Fig. 7 C). It is important to note that treatment with MCD for 3 h had no effect on synaptic responses to HFS (Fig. 7, B and C), nor did MCD affect basal synaptic transmission or tetanus induced LTP (Ma et al., 2003). These results suggest that short-term exposure to MCD per se does not affect the number of readily releasable vesicles in the presynaptic terminals or the number or properties of AMPA or NMDA receptors on the postsynaptic density.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
BDNF-induced translocation of activated TrkB into lipid rafts
One of the key findings in the present study is that TrkB translocation to rafts depends on its tyrosine kinase activity. This notion is supported by a number of observations: (a) inhibition of the Trk kinase activity by K252a or AG879 significantly inhibited BDNF-induced translocation of TrkB-FL; (b) NT-4, which activates the TrkB RTK, also induced TrkB-FL translocation; (c) 1-min exposure of BDNF, which activates TrkB, was sufficient to partition TrkB-FL into rafts. Thus, TrkB activation has to occur before the translocation; and (d) TrkB-T1, the truncated form of TrkB lacking the tyrosine kinase domain, was not recruited into rafts by BDNF. This last result, however, has to be interpreted with caution. It is also possible that the intracellular domain of the TrkB receptor may contain motifs that interact with other proteins important for the delivery of TrkB to the lipid rafts.
Another important observation is that removing of cholesterol, a major component in the lipid rafts, with 2 mM MCD inhibited TrkB signaling, leading to impairments in synaptic transmission and plasticity. The most straightforward interpretation is that MCD disrupted BDNF-induced translocation of TrkB-FL into rafts, and consequently impaired TrkB signaling in rafts. Alternatively, TrkB may need to interact with cholesterol to become fully functional, and therefore depletion of cholesterol directly affects TrkB signaling, rather than TrkB translocation. Consistent with this idea, BDNF failed to induce TrkB phosphorylation on the cell surface in cultured striatal neurons from Niemann-Pick type C mice (NPC–/–), which have abnormal cholesterol metabolism (Henderson et al., 2000). The fact that there was a small amount of TrkB in lipid rafts in naïve cells (Fig. 1 A) also supports the idea that TrkB interacts with rafts, perhaps with a low affinity. However, several observations argue against this idea. First, in cultured striatal neurons from NPC–/–, the content of free cholesterol was normal, suggesting that the cholesterol-rich microdomains on cell surface are probably normal. Thus, the dysfunction of TrkB on cell surface in NPC–/– is probably due to reasons other than lack of cholesterol in the plasma membrane. Second, we demonstrated that treatment with 2 mM MCD, which removed membrane cholesterol by 33%, interfered with TrkB recruitment into rafts, but not TrkB activation outside rafts (Fig. 5 A). The basal level of TrkB localization in rafts in resting cells was normal as well. Finally, treatment with 10 mM MCD, which removed >95% cholesterol, did not diminish TrkB kinase activity (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200404106/DC1). These data suggest that cholesterol is involved in the recruitment of TrkB into lipid rafts, but not directly in TrkB function.
A third possible mechanism for TrkB translocation is that BDNF signaling involves an interaction between TrkB and protein X, which could be a protein embedded in cholesterol-rich membranes. Cholesterol depletion could in principle lead to structural changes in the protein X (Simons and Toomre, 2000; Munro, 2003; Glebov and Nichols, 2004), and therefore disturb its interaction with TrkB. Although the nature of protein X remains unknown, we tested whether p75NTR, which is primarily associates with rafts (Higuchi et al., 2003) and is capable of binding to Trk receptors (Hempstead, 2002), facilitates BDNF-dependent translocation of TrkB into rafts. Interestingly, expression of exogenous p75NTR in cortical neurons inhibited TrkB translocation into lipid rafts, as well as the level of TrkB phosphorylation in the rafts, but not the basal association of TrkB with rafts or TrkB phosphorylation outside rafts (Fig. 3). These data are opposite to the above prediction and suggest that p75NTR has an inhibitory function in TrkB translocation and lipid raft-mediated BDNF/TrkB signaling. Thus, although it is unlikely that BDNF-induced TrkB translocation into lipid rafts is mediated by p75NTR through its interaction with TrkB, our experiments could not rule out the possibility that a protein X is an intermediate for BDNF-induced TrkB translocation into rafts.
The dynamic behavior of TrkB into lipid rafts is most analogous to that of two tyrosine kinase receptors, c-Ret for glial cell line–derived neurotrophic factor (GDNF; Tansey et al., 2000; Paratcha et al., 2001) and ErbB4 for neuregulin (Ma et al., 2003). c-Ret is recruited into lipid rafts upon GDNF stimulation by two distinct mechanisms. In cells expressing c-Ret and GPI-anchored protein GFR
1 (GDNF family receptor 1), which is primarily associated with lipid rafts, the binding of GDNF to GFR1 results in a transient recruitment of c-Ret into the compartment. Unlike TrkB translocation, this movement is independent of the kinase activity. In cells lacking GPI-anchored GFR1, however, coapplication of GDNF and soluble GFR1 allows stabilization of c-Ret in rafts, and this effect is tyrosine kinase-activity dependent. These mechanisms lead to the association of c-Ret with different adaptor proteins inside rafts and elicit neuronal survival and differentiation. Similar to TrkB-FL, ErbB4 is recruited into lipid rafts by neuregulin. However, it is unclear whether ErbB4 translocation depends on its kinase activity. Although neuregulin translocates the whole receptor complex, including ErbB4 and its adaptor proteins, such as Grb2 and Shc, into rafts, BDNF recruited TrkB alone into lipid rafts, without carrying its associated proteins Shc, Grb2, and PLC (Fig. 4 A and Fig. S4 A). However, we cannot completely rule out the possibility that the movement of these associated proteins into rafts was under the detection limit of our immunoblotting analysis. Given that Shc, Grb2, Erks, and PLC were present in lipid rafts in an experiment using a milder detergent Triton X-165 (Fig. S4 A), it is possible that the movement of TrkB-FL into rafts allows TrkB to interact with signaling molecules inside the rafts, a step crucial in transmitting lipid raft-mediated BDNF/TrkB signal transduction.
The raft localization of detergent insoluble proteins including RTKs depends on cell type and detergent stringency (Pike, 2003; Schuck et al., 2003). For example, when a less stringent detergent (Triton X-165) was used, a larger amount of TrkB-FL was associated with rafts in naïve cells, and BDNF-induced movement of TrkB-FL from nonrafts to rafts became less apparent (Fig. S4 B). In PC12 cells, both TrkA and p75NTR are constitutively localized in caveolae/lipid rafts (Bilderback et al., 1999; Huang et al., 1999) and NGF binding does not alter the partition of TrkA and p75NTR in lipid rafts (Huang et al., 1999). This may be due to the specific cell types they are expressed or their association with lipid raft-anchored proteins.
Role of BDNF-dependent TrkB translocation into lipid raft in synaptic modulation
The present study has revealed the importance of TrkB translocation into rafts in short-term modulation of synaptic transmission and plasticity by BDNF. In contrast, lipid rafts are not required for BDNF regulation of cortical neuron survival. In neurons, lipid rafts are preferentially distributed in synaptic membranes (Ma et al., 2003). In cerebral cortex in vivo, TrkB-FL is present in lipid rafts (Fig. S1 B). Moreover, the endogenous BDNF appears to be enriched in lipid raft fractions in the adult cortex (Fig. S1 B), stored in vesicles in synaptosomes (Fawcett et al., 1997), and secreted at synapses in an activity-dependent manner (Hartmann et al., 2001; Kojima et al., 2001). Thus, the translocation of TrkB into lipid rafts may imply a BDNF-induced recruitment of TrkB into the synapses.
Because BDNF elicits the synaptic effects through presynaptic mechanisms (Gottschalk et al., 1998; Lessmann, 1998), activated TrkB may need to be translocated to the rafts in the presynaptic terminals to interact with its target molecules. The specific target molecules mediating the presynaptic effects of BDNF, however, remain to be fully identified. In purified nerve terminals (synaptosomes) from cerebral cortices, application of BDNF facilitates evoked glutamate release and increases the MAPK-dependent phosphorylation of synapsin I, a synaptic vesicle-associated protein implicated in the control of transmitter release (Jovanovic et al., 2000), suggesting a critical role in the TrkB/MAPK/synapsin signaling cascade. BDNF also rapidly induces MAPK phosphorylation in these slices and inhibition of MAPK signaling blocks BDNF modulation of HFS response (Gottschalk et al., 1999). In the present study, when TrkB was translocated into lipid rafts, Erks appeared to be activated in the rafts (Fig. 4 B). Assuming that TrkB-FL is rapidly translocated into the rafts in synaptic membranes, activation of Erks in rafts may lead to the phosphorylation of presynaptic molecules (e.g., synapsin, synaptobrevin, or synaptophysin) necessary to mediate BDNF effects at CNS synapses.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Preparation of lipid rafts and Western blot analysis
Lipid rafts were prepared according to the method of Kawabuchi et al. (2000). Cultured neurons (3.5 x105 cells/cm2) were rinsed with PBS and quickly lysed in 0.5 ml ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.15 M NaCl, 10 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 1 mM PMSF, 10 mM Na2P2O7, 100 µM phenylarsine oxide) and incubated at 4°C for 30 min. For a separate experiment (Fig. S4), 1% Triton X-165 was used. The lysates were mixed with an equal volume of 100% (wt/vol) sucrose in buffer A (50 mM Tris-HCl, pH 7.4, 5 mM NaCl, 1 mM Na3VO4, 1 mM PMSF, 100 µM phenylarsine oxide). The mixture was transferred to a centrifuge tube, and 8 ml of 35% (wt/vol) sucrose in buffer A and 3.5 ml of 5% (wt/vol) sucrose in buffer A were overlaid sequentially. After centrifugation at 2 x 105 g for 13 h at 4°C, six fractions were collected from the top of the gradient (the first fraction, 2.5 ml; other fractions, 2.0 ml). To measure cholesterol, 50 µl of each fraction was analyzed with the cholesterol assay kit (Wako). To isolate lipid rafts from cortex, rats of different ages were decapitated and cortexes were removed quickly. This procedure was strictly in accord with the protocols approved by the Institutional Animal Care and Use Committee of AIST. Western blot analysis was performed as described previously (Guirland et al., 2004). To determine the concentration of protein, we used BCA protein assay kit (Pierce Chemical Co.).
Recombinant adenovirus
The rat p75NTR cDNA was supplied by E. Shooter (Stanford University, Stanford, CA). The adenovirus vector pAxCAwt was kindly provided by I. Saito (University of Tokyo, Tokyo, Japan). For adenovirus generation, see Online supplemental material. The recombinant adenovirus was used at a multiplicity of infection of 5. After 6 d in culture, cells were infected with adenovirus for 24 h followed by a 24-h incubation in serum-free DME to assay.
Glutamate release
For this assay, cultured neurons (6 x105 cells/cm2) were prepared as described previously (Matsumoto et al., 2001). After 5 h incubation in serum-free medium, the neurons were washed four times with Krebs'-Ringer's-Henseleit (KRH) buffer containing 130 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose, and 25 mM Hepes, pH 7.4, and pretreated with or without MCD for 10 min followed by BDNF incubation for 30 min. To induce glutamate release, 1 mM 4-AP was applied (Matsumoto et al., 2001). The glutamate released was measured using Amplex red glutamic acid oxidase kit (Molecular Probes).
Exocytosis assay using FM dye destaining
Dissociated neurons (5 x104 cells/cm2) were cultured on polyethylenimine-coated glasses (Matsunami; Numakawa et al., 2002). After washing three times with KRH buffer, neurons were pretreated with or without MCD, MCD–cholesterol complex or filipin for 10 min followed by BDNF incubation for 30 min. FM dye (2 mM; Molecular Probes) was loaded by incubating the cells with the high K+ solution (KRH buffer containing 56 mM KCl and 79 mM NaCl) for 1 min at 37°C. After washing the cells with KRH buffer, fluorescence images of FM-labeled spots were taken every second using fluorescence microscope (model IX70; Olympus) equipped with a Cool SNAP HQ CCD camera (Roper Scientific) and a 20 x 0.8 NA objective (Olympus). The baseline intensity was at 4 s before the stimulation with high K+ solution. To induce FM dye destaining, neurons were depolarized with high K+ solution. FM-labeled spots/50-µm dendrite were analyzed using a quantification menu of the MetaMorph software (Universal Imaging Co.).
Slice preparation and electrophysiology
Recording of hippocampal slices, prepared from neonatal rat (P12-13), was described previously (Figurov et al., 1996). Field EPSPs were evoked in CA1 stratum radiatum by stimulating Schaeffer-commissurals and were recorded with ACSF-filled glass pipettes (<5 M
). Only slices exhibiting EPSPs of 2–3mV in amplitude without superimposed population spikes were used. Stimulus intensity was adjusted to evoked EPSPs of 
1.3 mV. A train of HFS (100 Hz, 1 s) was used to induce synaptic depression. All experimental data were collected at least 10 min after stable EPSPs were achieved. Slices were treated with MCD (2 mM) or BDNF (2 nM) or them together for 2 h before synaptic responses to HFS were tested.
Assays of cell survival and dendritic growth
Cortical neurons (5 x104 cells/cm2) were cultured in Neurobasal medium containing B27 supplement (GIBCO BRL), 0.5 mM glutamine, for 3 d, and treated with indicated agents in Neurobasal medium containing no B27 supplement for 3 d. Cell survival was quantitated by WST-1 assay, which determines cell viability based on the cleavage activity of a soluble tetrazolium salt WST-1 by mitochondrial dehydrogenases. In brief, cultured cortical neurons were incubated with a tetrazolium salt WST-1 (Roche) for 30 min and viable cells were then determined by a microplate reader at 450 nm with 650 nm as a reference wavelength. Alternatively, the number of MAP2-positive neurons was counted (Yamada et al., 2001). To identify primary dendrites from each neuron, cells were stained with anti-MAP2 antibody, followed by DAPI staining. After taking images of MAP2-positive neurons with cell body of diameter (16.9 ± 0.5 µm diam, n = 32 cells from four independent coverslips), the number of the primary dendrites was counted.
Online supplemental material
Fig. S1 shows localization of TrkB and BDNF in lipid rafts during cortical development. Fig. S2 shows association of TrkB-FL with lipid rafts 1 min after BDNF stimulation. Fig. S3 shows TrkB-T1 expression in rafts and nonrafts and NT-4–induced recruitment of TrkB-FL into lipid rafts. Fig. S4 shows association of signaling molecules and TrkB with lipid rafts. Fig. S5 shows that treatment with 10 mM MCD lead to a partial decrease in BDNF-induced activation of TrkB-FL. Further comments on the data reported can be found in the legends. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200404106/DC1.
|
Acknowledgments |
|---|
Submitted: 26 April 2004
Accepted: 19 October 2004
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Anderson, R.G., and K. Jacobson. 2002. A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science. 296:1821–1825.
Bibel, M., and Y.A. Barde. 2000. Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system. Genes Dev. 14:2919–2937.
Bilderback, T.R., V.R. Gazula, M.P. Lisanti, and R.T. Dobrowsky. 1999. Caveolin interacts with Trk A and p75 (NTR) and regulates neurotrophin signaling pathways. J. Biol. Chem. 274:257–263.
Binder, D.K., M.J. Routbort, and J.O. McNamara. 1999. Immunohistochemical evidence of seizure-induced activation of trk receptors in the mossy fiber pathway of adult rat hippocampus. J. Neurosci. 19:4616–4626.
Citores, L., J. Wesche, E. Kolpakova, and S. Olsnes. 1999. Uptake and intracellular transport of acidic fibroblast growth factor: evidence for free and cytoskeleton-anchored fibroblast growth factor receptors. Mol. Biol. Cell. 10:3835–3848.
Fawcett, J.P., R. Aloyz, J.H. McLean, S. Pareek, F.D. Miller, P.S. McPherson, and R.A. Murphy. 1997. Detection of brain-derived neurotrophic factor in a vesicular fraction of brain synaptosomes. J. Biol. Chem. 272:8837–8840.
Figurov, A., L.D. Pozzo-Miller, P. Olafsson, T. Wang, and B. Lu. 1996. Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature. 381:706–709.[CrossRef][Medline]
Ghosh, A., J. Carnahan, and M.E. Greenberg. 1994. Requirement for BDNF in activity-dependent survival of cortical neurons. Science. 263:1618–1623.
Glebov, O.O., and B.J. Nichols. 2004. Lipid raft proteins have a random distribution during localized activation of the T-cell receptor. Nat. Cell Biol. 6:238–243.[Medline]
Gottschalk, W.A., L.D. Pozzo-Miller, A. Figurov, and B. Lu. 1998. Presynaptic modulation of synaptic transmission and plasticity by brain-derived neurotrophic factor in the developing hippocampus. J. Neurosci. 18:6830–6839.
Gottschalk, W.A., H. Jiang, N. Tartaglia, L. Feng, A. Figurov, and B. Lu. 1999. Signaling mechanisms mediating BDNF modulation of synaptic plasticity in the hippocampus. Learn. Mem. 6:243–256.
Guirland, C., S. Suzuki, M. Kojima, B. Lu, and J.Q. Zheng. 2004. Lipid rafts mediate chemotropic guidance of nerve growth cones. Neuron. 42:51–62.[CrossRef][Medline]
Hartmann, M., R. Heumann, and V. Lessmann. 2001. Synaptic secretion of BDNF after high-frequency stimulation of glutamatergic synapses. EMBO J. 20:5887–5897.[CrossRef][Medline]
Henderson, L.P., L. Lin, A. Prasad, C.A. Paul, T.Y. Chang, and R.A. Maue. 2000. Embryonic striatal neurons from niemann-pick type C mice exhibit defects in cholesterol metabolism and neurotrophin responsiveness. J. Biol. Chem. 275:20179–20187.
Hering, H., C.C. Lin, and M. Sheng. 2003. Lipid rafts in the maintenance of synapses, dendritic spines, and surface AMPA receptor stability. J. Neurosci. 23:3262–3271.
Hempstead, B.L. 2002. The many faces of p75NTR. Curr. Opin. Neurobiol. 12:260–267.[CrossRef][Medline]
Higuchi, H., T. Yamashita, H. Yoshikawa, and M. Tohyama. 2003. PKA phosphorylates the p75 receptor and regulates its localization to lipid rafts. EMBO J. 22:1790–1800.[CrossRef][Medline]
Huang, C.S., J. Zhou, A.K. Feng, C.C. Lynch, J. Klumperman, S.J. DeArmond, and W.C. Mobley. 1999. Nerve growth factor signaling in caveolae-like domains at the plasma membrane. J. Biol. Chem. 274:36707–36714.
Jovanovic, J.N., A.J. Czernik, A.A. Fienberg, P. Greengard, and T.S. Sihra. 2000. Synapsins as mediators of BDNF-enhanced neurotransmitter release. Nat. Neurosci. 3:323–329.[CrossRef][Medline]
Kaplan, D.R., and F.D. Miller. 2000. Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol. 10:381–391.[CrossRef][Medline]
Kawabuchi, M., Y. Satomi, T. Takao, Y. Shimonishi, S. Nada, K. Nagai, A. Tarakhovsky, and M. Okada. 2000. Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases. Nature. 404:999–1003.[CrossRef][Medline]
Kojima, M., N. Takei, T. Numakawa, Y. Ishikawa, S. Suzuki, T. Matsumoto, R. Katoh-Semba, H. Nawa, and H. Hatanaka. 2001. Biological characterization and optical imaging of brain-derived neurotrophic factor-green fluorescent protein suggest an activity-dependent local release of brain-derived neurotrophic factor in neurites of cultured hippocampal neurons. J. Neurosci. Res. 64:1–10.[CrossRef][Medline]
Lessmann, V. 1998. Neurotrophin-dependent modulation of glutamatergic synaptic transmission in the mammalian CNS. Gen. Pharmacol. 31:667–674.[Medline]
Lu, B. 2003. BDNF and activity-dependent synaptic modulation. Learn. Mem. 10:86–98.
Ma, L., Y.Z. Huang, G.M. Pitcher, J.G. Valtschanoff, Y.H. Ma, L.Y. Feng, B. Lu, W.C. Xiong, M.W. Salter, R.J. Weinberg, and L. Mei. 2003. Ligand-dependent recruitment of the ErbB4 signaling complex into neuronal lipid rafts. J. Neurosci. 23:3164–3175.
Matsumoto, T., T. Numakawa, N. Adachi, D. Yokomaku, S. Yamagishi, N. Takei, and H. Hatanaka. 2001. Brain-derived neurotrophic factor enhances depolarization-evoked glutamate release in cultured cortical neurons. J. Neurochem. 79:522–530.[CrossRef][Medline]
McAllister, A.K., D.C. Lo, and L.C. Katz. 1995. Neurotrophins regulate dendritic growth in developing visual cortex. Neuron. 15:791–803.[CrossRef][Medline]
Mineo, C., G.N. Gill, and R.G. Anderson. 1999. Regulated migration of epidermal growth factor receptor from caveolae. J. Biol. Chem. 274:30636–30643.
Munro, S. 2003. Lipid rafts: elusive or illusive? Cell. 115:377–388.[CrossRef][Medline]
Murphy, D.D., and M. Segal. 1996. Regulation of dendritic spine density in cultured rat hippocampal neurons by steroid hormones. J. Neurosci. 16:4059–4068.
Numakawa, T., D. Yokomaku, K. Kiyosue, N. Adachi, T. Matsumoto, Y. Numakawa, T. Taguchi, H. Hatanaka, and M. Yamada. 2002. Basic fibroblast growth factor evokes a rapid glutamate release through activation of the MAPK pathway in cultured cortical neurons. J. Biol. Chem. 277:28861–28869.
Paratcha, G., and C.F. Ibanez. 2002. Lipid rafts and the control of neurotrophic factor signaling in the nervous system: variations on a theme. Curr. Opin. Neurobiol. 12:542–549.[CrossRef][Medline]
Paratcha, G., F. Ledda, L. Baars, M. Coulpier, V. Besset, J. Anders, R. Scott, and C.F. Ibanez. 2001. Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts. Neuron. 29:171–184.[CrossRef][Medline]
Patterson, S.L., T. Abel, T.A. Deuel, K.C. Martin, J.C. Rose, and E.R. Kandel. 1996. Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron. 16:1137–1145.[CrossRef][Medline]
Pike, L.J. 2003. Lipid rafts: bringing order to chaos. J. Lipid Res. 44:655–667.
Ryan, T.A., H. Reuter, B. Wendland, F.E. Schweizer, R.W. Tsien, and S.J. Smith. 1993. The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron. 11:713–724.[CrossRef][Medline]
Schuck, S., M. Honsho, K. Ekroos, A. Shevchenko, and K. Simons. 2003. Resistance of cell membranes to different detergents. Proc. Natl. Acad. Sci. USA. 100:5795–5800.
Simons, K., and D. Toomre. 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1:31–39.[CrossRef][Medline]
Takei, N., T. Numakawa, S. Kozaki, N. Sakai, Y. Endo, M. Takahashi, and H. Hatanaka. 1998. Brain-derived neurotrophic factor induces rapid and transient release of glutamate through the non-exocytotic pathway from cortical neurons. J. Biol. Chem. 273:27620–27624.
Tansey, M.G., R.H. Baloh, J. Milbrandt, and E.M. Johnson Jr. 2000. GFRalpha-mediated localization of RET to lipid rafts is required for effective downstream signaling, differentiation, and neuronal survival. Neuron. 25:611–623.[CrossRef][Medline]
Thiele, C., M.J. Hannah, F. Fahrenholz, and W.B. Huttner. 2000. Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles. Nat. Cell Biol. 2:42–49.[CrossRef][Medline]
Yamada, M., K. Tanabe, K. Wada, K. Shimoke, Y. Ishikawa, T. Ikeuchi, S. Koizumi, and H. Hatanaka. 2001. Differences in survival-promoting effects and intracellular signaling properties of BDNF and IGF-1 in cultured cerebral cortical neurons. J. Neurochem. 78:940–951.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
F (%) = (Fb–Fa)/Fb x 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.
