Regulation of TrkB receptor translocation to lipid rafts by adenosine A(2A) receptors and its functional implications for BDNF-induced regulation of synaptic plasticity

Abstract

Brain-derived neurotrophic factor (BDNF) signalling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signalling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A(2A) receptor activation, we hypothesized that activation of A(2A) receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A(2A) receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansyl cadaverine (100 μM) did not modify the effects of the A(2A) receptor agonists, but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A(2A) receptor activation in TrkB localization was mimicked by 5 μM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors RpcAMPs and PKI-(14-22) and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF upon hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts, induced by the activation of adenosine A(2A) receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.

Fig. 1

Activation of adenosine receptors enhances the levels of TrkB-FL and potentiates BDNF-induced pTrkB localization in lipid rafts. DIV 7-11 cortical neurons were starved for 4 h and incubated with/without 20 nM CGS 21680 or 50 nM ZM 241385 for 30 min prior to 5 min-incubation with 20 ng/ml BDNF, as indicated. Lysates were prepared in 0.5 % Triton X-100-containing buffer and fractioned in a discontinuous Optiprep gradient, as described in “Materials and methods”. a Equal volumes of each gradient fraction were probed for TrkB (1:200) and Fyn (1:400). Note that Fyn, a lipid raft marker, was only detected in fraction #2, which therefore was considered the lipid raft-containing fraction. b Quantification of cholesterol content in each gradient fraction. Fraction #2 was highly enriched in cholesterol, containing approximately 22 % of total cholesterol. c Representative Western blot analysis of the lipid raft fraction (#2) obtained from the Optiprep density gradients. Antibodies used were TrkB (1:200), pTrk (pY490, 1:750) and Fyn (1:400). d Quantitative analysis of TrkB staining in fraction #2, normalized by Fyn staining in this fraction; 100 % represents staining in the absence of any drug. e Quantification analysis of BDNF-induced changes in #2 pTrk, normalized by #2 Fyn (left) or by total TrkB (right), in cells pre-incubated with CGS 21680 or ZM 241385 as indicated below each bar. One hundred percent corresponds to the staining obtained in the presence of BDNF alone. f Fraction #2 obtained from the density gradients of cells incubated with CGS 21680 for the indicated times were probed for TrkB and Fyn, which was used as a loading control. g Densitometry analysis of #2 TrkB/#2 Fyn. h Analysis of TrkB and pTrk staining in total lysates of cells treated with/without CGS 21680, ZM 241385 or 20 ng/ml BDNF, as indicated. TrkB, pTrkB, Fyn or β-actin band corresponds to approximately 150, 150, 60 and 42 kDa proteins. Results are expressed as mean ± SEM of three to seven independent experiments. *p < 0.05, compared to 100 %

Fig. 2

CGS 21680 treatment increases co-patching between TrkB receptors and cholera toxin subunit B (Chol. Tox. B). a Alexa Fluor 594-coupled cholera toxin subunit B (2 μg/ml) and an anti-TrkB receptor antibody (1:500) raised against the extracellular domain of TrkB receptors were used in the co-patching experiments of cultured cortical neurons. TrkB receptor patches were labelled with an Alexa Fluor 488-coupled goat anti-rabbit antibody (1:300). Scale bar, 5 μm. b Quantification of the percentage of TrkB receptors co-patched with cholera toxin subunit B. Results are expressed as mean ± SEM of five independent experiments. *p < 0.05, compared to control

Fig. 3

Time course of TrkB receptor phosphorylation in lipid rafts by BDNF. a Lipid raft (fraction #2) analysis of pTrk and TrkB staining in lipid rafts from cells incubated with BDNF for 1–40 min, in the absence or presence of the A_2A receptor agonist CGS 21680 (20 nM), as indicated. Fraction #2 was probed for pTrk, TrkB and Fyn. b, c Densitometry analysis of #2 pTrk staining, normalized by total #2 TrkB (b) and #2 Fyn (c). d Densitometry analysis of #2 TrkB staining, normalized by #2 Fyn. Results are expressed as mean ± SEM of two to three (1 and 20 min) or seven to ten (5 and 40 min) experiments. *p < 0.05, compared to BDNF alone

Fig. 4

A_2A receptors are not required for maximal BDNF-induced TrkB translocation to lipid rafts. DIV 7-11 cortical neurons were starved for 4 h prior to treatment with/without 20 nM CGS 21680, 50 nM ZM 241385 and/or 20 ng/ml BDNF (40 min), as indicated. a Equal volumes of each density gradient fraction were immunoblotted for TrkB and the lipid raft marker Fyn. b Staining of lipid raft fraction #2. Membranes were probed for TrkB, pTrk and Fyn. c Densitometry analysis of TrkB staining in lipid rafts (fraction #2), normalized by #2 Fyn. d Densitometric analysis of the pTrk staining in fraction #2 normalized by Fyn and total TrkB. One hundred percent corresponds to pTrk staining in the presence of BDNF alone. e Total lysates were treated as described in “Materials and methods”, lysed and probed for TrkB, pTrk and β-actin. Results are expressed as mean ± SEM of three to six independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001, compared to 100 %, unless otherwise indicated

Fig. 5

Influence of clathrin-dependent endocytosis on CGS 21680-induced and BDNF-induced TrkB recruitment and phosphorylation to lipid rafts. DIV 7-11 cortical neurons were treated with 20 nM CGS 21680 for 30 min or 20 ng/ml BDNF for 40 min in the presence or absence of the clathrin-dependent endocytosis inhibitor monodansyl cadaverine (100 μM), where indicated. a Density gradient fraction #2 was immunoblotted and probed for TrkB, pTrk and Fyn. b Quantification of #2 TrkB/#2 Fyn. c Quantification of #2 pTrk/#2 TrkB after BDNF treatment, in the presence/absence of MDC, as indicated below each bar. Data are expressed as mean ± SEM of five to seven independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001, compared to 100 %, unless otherwise indicated. Note that the clathrin-dependent endocytosis inhibitor, MDC, attenuated BDNF-induced TrkB translocation to lipid rafts, but did not influence CGS 21680-induced TrkB recruitment to these membrane domains

Fig. 6

Signalling pathways involved in CGS 21680-induced TrkB translocation to lipid rafts. Cultured cortical neurons were incubated with/without 20 nM CGS 21680 for 30 min in the presence of the adenylate cyclase activator forskolin (FSK, 5 μM), the PKA inhibitor H-89 (1 μM), the cAMP antagonist Rp-cAMPs (100 μM), the PKA inhibitor PKI-(14-22) (1 μM), the phospholipase C inhibitor U73122 (4 μM) or the Src-family kinase inhibitor PP2 (500 nM), as indicated. Cells were lysed and processed for lipid raft isolation. a–c Fraction #2 obtained from the density gradients of cells under different conditions were probed for total TrkB and Fyn, which was used as a loading control. d Densitometry analysis of TrkB/Fyn staining obtained in a–c. Data are expressed as mean ± SEM of four to nine independent experiments. *p < 0.05; **p < 0.01; NS, no statistical difference (p > 0.05), compared to 100 %, except when otherwise indicated

Fig. 7

Effects of cholesterol depletion and loading on the partition of TrkB receptors to lipid rafts and on adenosine A_2A receptor binding properties. Cultured cortical neurons were treated with/without 20 nM CGS 21680 for 30 min after incubation with 3 mM MβCD or MβCD–cholesterol complexes (water-soluble cholesterol, wsCLT, 50 μg/ml cholesterol). a, b Lipid raft (fraction #2) analysis of the effect of MβCD on TrkB sublocalization after CGS 21680 treatment. MβCD–cholesterol complexes were used to load cholesterol to membranes. c Cholesterol content in lipid raft (#2) and non-raft fractions (#8) [56] in control conditions and after 20 nM CGS 21680 incubation for 30 min. d Saturation curves for the specific binding of the A_2A receptor antagonist [³H] ZM 241385 in cortical membranes from cells in control conditions (open circles), after incubation with MβCD (closed squares) or with wsCLT (closed triangles) treatment. Data are expressed as mean ± SEM of three to five independent experiments. *p < 0.05, compared to 100 %

Fig. 8

BDNF increases glutamate release in an adenosine- and lipid raft-dependent manner. a Averaged time course of [³H] glutamate release from cortical synaptosomes. Synaptosomes were labelled with [³H] glutamate and stimulation of neurotransmitter release was induced twice, at 5–7 min (S₁) and 29–31 min (S₂), as described in “Materials and methods”. Samples were collected every 2 min. BDNF (20 ng/ml) was added at 9 min and remained in the perfusion solution until the end of the experiments (closed circles). Control curves in the absence of any drug, performed in parallel with the same synaptosomal batch, are represented by the open circles. b, c, S₂/S₁ ratios, calculated in each experiment from the time courses curves, as described in “Materials and methods”. BDNF (20 ng/ml) was tested in the presence/absence of 1 U/ml ADA, 1 mM MβCD or 2 U/ml cholesterol oxidase (Chol.Oxi.), as indicated below each bar. In each experiment, the S₂/S₁ ratio obtained while BDNF was present during S₂ was normalized taking as 100 % the S₂/S₁ ratio obtained in parallel chambers under the same drug conditions but absence of BDNF. Data are represented as mean ± SEM of three to six independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001, compared to 100 %, except when otherwise indicated

Fig. 9

High-frequency stimulation of hippocampal slices induces TrkB translocation and increases pTrk staining in lipid rafts in an adenosine-dependent manner. Hippocampal slices were superfused with 1 U/ml ADA, where indicated, 30 min prior to the high-frequency stimulation (HFS). HFS was applied for 1 min as described in “Materials and methods”, and after 30 min, slices were homogenized and lipid rafts were isolated by discontinuous Optiprep gradients. a Fraction #2, containing lipid raft membranes, was immunoblotted for TrkB, pTrk and Fyn, which was used as a loading control. b Quantifications of #2 TrkB/#2 Fyn. c Quantifications of #2 pTrk/#2 Fyn. Data are represented as mean ± SEM of five to seven independent experiments. *p < 0.05, **p < 0.01, compared to 100 %

Fig. 10

BDNF enhances long-term potentiation in a lipid raft-dependent manner. a Schematic representation of a transverse hippocampal slice with the electrode configuration used to record fEPSPs in the CA1 apical dendritic layer (stratum radiatum) evoked by electric stimulation of two independent pathways of the Schaffer fibres, S₀ and S₁. In b–e, the averaged time course changes in the fEPSP slope are shown. The small inhibition of fEPSP caused by 1 mM MβCD is illustrated in b. c–e Changes in the fEPSP slope induced by the θ-burst stimulation of slices (see “Materials and methods”), as indicated by the arrow in each panel. Zero percent corresponds to the averaged slopes recorded for 10 min before MβCD (b −0.55 ± 0.02 mV/ms, n = 7) or θ-burst stimulation (c white circle, −0.49 ± 0.02 mV/ms; black circle, −0.49 ± 0.02 mV/ms, n = 3; d, white circle, −0.52 ± 0.01 mV/ms, black circle, −0.49 ± 0.04 mV/ms, n = 6; e white circle −0.49 ± 0.01 mV/ms, black circle, −0.47 ± 0.01 mV/ms, n = 5). f, g Recordings from representative experiments, where each trace represents the average of eight consecutive responses obtained before and after LTP induction, in the absence (f, left) or presence of BDNF (f, right), or in the presence of MβCD (g, left) or MβCD + BDNF (g, right). Recordings under same conditions, but before and 60 min after LTP induction are superimposed. All recordings in f were obtained from a single slice at approximately the points indicated in d. All recordings in g were obtained from a single slice at approximately the points indicated in e. Each recording is composed by the stimulus artifact, followed by the presynaptic volley and the fEPSP. h Comparison of the effect of BDNF upon LTP in the absence or presence of MβCD, as indicated. *p < 0.05, compared to the first column