Brain-derived neurotrophic factor-tropomyosin-related kinase B signaling contributes to activity-dependent changes in synaptic proteins

Abstract

The ability of synapses to undergo changes in structure and function in response to alterations of neuronal activity is an essential property of neural circuits. One way that this is achieved is through global changes in the molecular composition of the synapse; however, it is not clear how these changes are coupled to the dynamics of neuronal activity. Here we found that, in cultured rat cortical neurons, bidirectional changes of neuronal activity led to corresponding alterations in the expression of brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor tropomyosin-related kinase B (TrkB), as well as in the level of synaptic proteins. Exogenous BDNF reversed changes in synaptic proteins induced by chronic activity blockade, while inhibiting Trk kinase activity or depleting endogenous BDNF abolished the concentration changes induced by chronic activity elevation. Both tetrodotoxin and bicuculline had significant, but opposite, effects on synaptic protein ubiquitination in a time-dependent manner. Furthermore, exogenous BDNF was sufficient to increase ubiquitination of synaptic proteins, whereas scavenging endogenous BDNF or inhibiting Trk kinase activity prevented the ubiquitination of synaptic proteins induced by chronic elevation of neuronal activity. Inhibiting the proteasome or blocking protein polyubiquitination mimicked the effect of tetrodotoxin on the levels of synaptic proteins and canceled the effects of BDNF. Our study indicates that BDNF-TrkB signaling acts upstream of the ubiquitin proteasome system, linking neuronal activity to protein turnover at the synapse.

Figures

FIGURE 1.

Activity-dependent BDNF mRNA expression. A, semiquantitative RT-PCR analysis of BDNF and GAPDH mRNA in cultured cortical neurons (DIV12) treated with TTX (2 μm), bicuculline (Bic) (40 μm), or standard medium (untreated control, Ctrl) for 8 h. Inset, representative RT-PCR. Quantification of BDNF mRNA band intensities normalized to GAPDH mRNA, presented as the mean ± S.E. relative to control (n = 4). B, time course of BDNF mRNA expression in response to chronic changes in neuronal activity. Cultured cortical neurons treated with TTX or bicuculline for 1, 4, 8, 12, 24, or 48 h. Top, representative RT-PCR. Bottom, mean BDNF levels calculated as in A (*, p < 0.05; **, p < 0.001 compared with control, n = 3-5 for each treatment and time point). C, neuronal activity regulates TORC1 nuclear translocation. Top, TORC1 (green) in cultured hippocampal neurons counterstained with Hoechst (blue) after treatment with TTX or bicuculline for 8 h. Bottom, quantification of the ratio of nuclear TORC1 (N) to cytoplasmic TORC1 (C) (*, p < 0.05; **, p < 0.01, n = 31/group). D, neuronal activity modulates TORC1 phosphorylation. Western analysis of TORC1 in cultured neurons treated with TTX or bicuculline for 8 h. E, calcineurin phosphatase is required for activity-dependent BDNF expression. Cortical neuron cultures were treated for 10 min with cyclosporine A and FK506 (CsA, 10 μm; FK, 10 μm) before and during 8 h of treatment with bicuculline. BDNF mRNA was measured as in A. F, TORC1 is required for neuronal activity-induced BDNF expression. DIV10 cortical neurons were infected with pSFV(pd) EGFP or pSFV (pd)-EGFP-DN-TORC1 2 days before 8 h of incubation with bicuculline. BDNF mRNA was measured as in A (*, p < 0.05; **, p < 0.01, n = 4).

FIGURE 2.

Activity-dependent Trk phosphorylation. A, top, Western blot analysis of Trk phosphorylation and TrkB expression. Lysates were obtained from cultured cortical neurons treated with TTX or bicuculline (Bic) for 12 h and probed with anti-phosphorylated Trk (pTrk), and the blots were stripped and reprobed with anti-TrkB antibody. Bottom, quantification of pTrk normalized to TrkB expression, presented as the mean ± S.E. (*, p < 0.05; **, p < 0.001, n = 3/group). B, time course of Trk phosphorylation in response to chronic changes in neuronal activity. Cultured cortical neurons treated with TTX or bicuculline for 1, 4, 8, 12, 24, or 48 h. Top, representative immunoblot of cortical neuron lysate blotted with anti-pTrk; the blots were stripped and reprobed with anti-TrkB antibody. Bottom, mean level of pTrk normalized to TrkB (± S.E., *, p < 0.05; **, p < 0.001,#, p < 0.05; n = 5/group). C, activity-dependent Trk phosphorylation requires BDNF. Cortical neurons were pretreated with TrkB-IgG (10-20 μg/ml) for 6 h or K252a (100 nm) for 0.5 h prior to 24 h of incubation with bicuculline and TrkB-IgG or K252a. Left, representative anti-pTrk and anti-TrkB Western blot. Right, mean level of phosphorylated Trk, normalized to TrkB (± S.E., **, p < 0.001 compared with control, n = 6/group).

FIGURE 3.

Activity-dependent changes in synaptic protein concentrations are mediated by BDNF-TrkB signaling. A, Western blot analysis of 17 proteins present in synaptosomal fractions isolated from cortical neurons treated for 48 h with TTX, BDNF (25 ng/ml), TTX and BDNF (TTX+BDNF), bicuculline (Bic), TrkB-IgG, bicuculline and K252a (Bic+K252a), or bicuculline and TrkB-IgG (Bic+TrkB-IgG). B, quantification of immunoblot band intensities normalized to levels in untreated controls, presented as means ± S.E. (n = 3-8 for each protein and treatment). *, p < 0.05.

FIGURE 4.

BDNF-TrkB signaling regulates ubiquitination of synaptic proteins. A, time course of global ubiquitination in the synaptosomal fraction in response to modulation of neuronal activity with TTX (left) or bicuculline (Bic) (right) for 1, 4, 8, 12, 24, or 48 h. Synaptosomal fraction lysate from cortical neurons was blotted with anti-polyubiquitin (Ub), stripped, and reprobed with anti-actin. B, the mean total anti-Ub signal normalized to actin, relative to untreated control (± S.E., *, p < 0.05; **, p < 0.001, n = 5/group). C, top, Western blot analysis of ubiquitin levels in the synaptosomal fraction of cortical neurons treated for 48 h with TTX, TTX and BDNF, bicuculline, bicuculline and K252a, or bicuculline and TrkB-IgG. Bottom, quantification of ubiquitination levels measured as in A (**, p < 0.001, n = 6/group). D, BDNF-TrkB signaling regulates ubiquitination of AKAP 79/150. Lysates from cortical neurons treated for 24 h were immunoprecipitated with anti-polyubiquitin (Ub) or control rabbit IgG (control IgG) and blotted with anti-AKAP 79/150 antibody.

FIGURE 5.

BDNF is sufficient to increase synaptic protein ubiquitination but does not affect proteasome activity. A, BDNF increases ubiquitination of synaptic proteins. Ubiquitination levels in lysates from cortical neurons treated with BDNF, BDNF and K252a, or TrkB-IgG for 2 h were analyzed by anti-Ub Western blotting. B, quantification of ubiquitination after treatment as in A; mean levels are presented relative to control (± S.E., *, p < 0.05; **, p < 0.01, n = 6/group). C, ubiquitination of AKAP 79/150 in cultured cortical neurons treated with BDNF for 2 h. Cell lysates were immunoprecipitated with anti-polyubiquitin and blotted with anti-AKAP 79/150, (n = 4 from separate culture preparation). D, ubiquitination of AKAP 79/150 in cultured cortical neurons treated for 2 h with BDNF (100 ng/ml) in the presence of MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor PD98059 (20 μm) or U0126 (20 μm) or control solution. Cell lysates were immunoprecipitated with anti-Ub and blotted with 7 anti-AKAP 79/150. E and F, BDNF has no effect on proteasome activity. E, GFP fluorescence in cortical neurons infected with pSFV(pd)-GFPu (see “Experimental Procedures”) and treated with MG132 (10 μm) for 6 h or BDNF (100 ng/ml) for 2 h (scale bar, 50 μm). F, representative anti-GFP immunoblots of cell lysates from cortical neurons infected with pSFV(pd)-EGFP or pSFV(pd)-GFPu and treated as in E. Note that the molecular mass of GFPu is slightly higher than EGFP due to the addition of the ubiquitination signal sequence at the C terminus. G, quantification of GFP and GFPu expression in Western blot. Data represent mean ± S.E. relative to untreated controls (*, p < 0.05, n = 2 blots/group).

FIGURE 6.

BDNF-TrkB signaling acts upstream of the ubiquitin-proteasome system. A, Western blot analysis of proteins present in synaptosomal fractions isolated from cortical neurons treated for 24 h with BDNF (25 ng/ml), MG132 (5 μm), or both or isolated from neurons infected with pSFV(pd)-GFP (GFP) or pSFV(pd)-DN-Ubquitin (DN-Ub). B, quantification of immunoblot band intensities of synaptosomal proteins isolated from neurons treated with bicuculline (Bic) (40 μm, 48 h), TTX (2 μm, 48 h), MG132 (5 μm, 24 h), BDNF (25 ng/ml, 48 h), or MG132+BDNF (24 h) or infected with GFP or DN-Ub. Data represent means ± S.E. normalized to untreated controls and are presented as means ± S.E. (*, p < 0.05, n = 3-6 for each protein and treatment). C, BDNF-TrkB signaling pathway is not affected by proteasome inhibition. Cortical neurons were lysed immediately after stimulation with MG132 (1 h), BDNF (100 ng/ml, 10 min), or MG132 (1 h) + BDNF (10 min) and analyzed by Western blotting with anti-phospho-Trk, anti-phospho-ERK1/2, anti-phospho-Akt, or anti-phospho-phospholipase C-γ. Immunoblots for TrkB, ERK1/2, Akt, and phospholipase C-γ served as loading controls (n = 3).