Research ArticleNeuroscience

BDNF increases synaptic NMDA receptor abundance by enhancing the local translation of Pyk2 in cultured hippocampal neurons

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Science Signaling  18 Jun 2019:
Vol. 12, Issue 586, eaav3577
DOI: 10.1126/scisignal.aav3577
  • Fig. 1 BDNF stimulation up-regulates the synaptic expression of GluN2B-containing NMDAR in a protein synthesis–dependent manner.

    (A) Representative images of hippocampal neurons (DIV 14 and 15) that were preincubated with cycloheximide (CHX) (50 μg/ml) or vehicle (DMSO; 1:1000 dilution for 45 min) and then either maintained under the same conditions or stimulated with BDNF (50 ng/ml for 30 min), as indicated. Neurons were then live-immunostained for GluN2B using an antibody against an extracellular epitope in the GluN2B N terminus, fixed, and then further immunostained for PSD-95, vGlut1, and MAP2. Arrowheads indicate surface GluN2B–PSD-95–vGlut1–colocalized puncta. Scale bar, 5 μm. (B to G) Images described in (A) were analyzed for the total number (B) and intensity (C) of surface GluN2B puncta per dendritic length and GluN2B immunoreactivity per puncta (D). Synaptic (PSD-95– and vGlut1-colocalized) surface GluN2B number (E) and intensity (F) of puncta per density of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per dendrite length), as well as the immunoreactivity per synaptic puncta (G), were analyzed. Data are relative to the DMSO control and are the means ± SEM for the indicated number of neurons (n) in at least three independent experiments performed in different preparations. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way analysis of variance (ANOVA) with Bonferroni test.

  • Fig. 2 BDNF treatment increases the abundance of functional synaptic GluN2B-containing NMDARs in cultured hippocampal neurons.

    (A to D) NMDAR mEPSCs were recorded in cultured rat hippocampal neurons after incubation in culture medium under control conditions or in the presence of BDNF (50 ng/ml) for 30 to 40 min. mEPSCs were recorded in a Na+ salt solution, and where indicated, the medium was supplemented with conantokin G (Con G; 3 μM). The average mEPSC traces recorded are shown in (A), and representative traces are shown in (C). Data (B and D) are mean ± SEM mEPSC amplitude and frequency, respectively, for the indicated number of neurons (n) from at least three independent preparations. **P < 0.01, ****P < 0.0001 by one-way ANOVA with Bonferroni test.

  • Fig. 3 GluN2B-NMDAR surface dynamics is altered by BDNF.

    (A) Representative trajectories of extrasynaptic (blue lines) and synaptic (red lines) GluN2B-NMDARs in hippocampal neurons transfected with PSD95-FingR-GFP (green fluorescent protein) at DIV 10 and 4 days later incubated with or without BDNF (50 ng/ml for 30 min). The cells were then incubated for 3 min in a solution containing QD particles coupled to a primary antibody against GluN2B-NMDAR subunits. Imaging captured 200 frames with a 50-ms acquisition frequency. Scale bar, 1 μm. (B) Percentage of extrasynaptic and synaptic immobile GluN2B-NMDAR trajectories in neurons described in (A). ns, not significant. (C and D) MSD over time of the extrasynaptic (C) and synaptic (D) surface GluN2B-NMDAR trajectories in hippocampal neurons described in (A). (E and F) Comparison of GluN2B-NMDAR instantaneous diffusion coefficient (μm2 s−1) within the extrasynaptic (E) and PSD area (F) in neurons described in (A). Data are means ± SEM from five independent experiments performed in distinct preparations. *P < 0.05, ****P < 0.0001 by unpaired Student’s t test.

  • Fig. 4 BDNF-induced increase in the synaptic surface expression of NMDAR-containing GluN2B subunits requires Pyk2.

    (A) Representative images of rat hippocampal neurons transfected with sh1-Scramble (sh1-Scrbl) or one of two Pyk2-targeted (shA2-Pyk2 or shA4-Pyk2) shRNA at DIV 12. At DIV 15, cultures were then either maintained under the same conditions or stimulated with BDNF (50 ng/ml for 30 min) as indicated. Neurons were live-immunostained for GluN2B using an antibody against an extracellular epitope located in the GluN2B N terminus, fixed and permeabilized, and then further immunostained for vGLUT1, GFP, and MAP2. Scale bar, 5 μm. (B to H) Images represented in (A) were analyzed for the number (B) and intensity (C) of surface GluN2B puncta per dendritic length, as well as GluN2B immunoreactivity per puncta (D), quantified relative to those in the sh1-Scramble control condition. The number (E) and intensity (F) of synaptic (vGlut1-colocalized) surface GluN2B puncta per density of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per dendrite length), the immunoreactivity per synaptic puncta (G), and the percentage of synapses containing surface GluN2B (number of vGluT1 puncta colocalized with surface GluN2B/total vGluT1 number of puncta) (H) were also analyzed. Data are means ± SEM for the indicated number of neurons (n) from at least four independent experiments, performed in independent preparations. ***P < 0.001, ****P < 0.0001 between indicated conditions; ##P < 0.01, ####P < 0.0001 versus scramble control by one-way ANOVA with Bonferroni test.

  • Fig. 5 The up-regulation of NMDAR-mediated mEPSCs by BDNF is mediated by Pyk2.

    (A to D) Rat hippocampal neurons were either untransfected (Ctrl) or transfected with sh1-Scramble (Sh1-Scrbl) or Sh4-Pyk2 at DIV 12 and, at DIV 15, were then either maintained under control conditions or stimulated with BDNF (50 ng/ml) for 30 to 40 min. Average NMDAR-mediated mEPSC traces recorded at −60 mV are shown in (A), and representative traces are shown in (C). Mean ± SEM amplitude (B) and frequency (D) of NMDAR-mediated mEPSCs were quantified. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Bonferroni test.

  • Fig. 6 BDNF-induced increase in synaptic expression of GluN2B-containing NMDAR is mediated by activation of Pyk2.

    (A to C) Representative Western blots (A) and analysis (B and C) of synaptic, phosphorylated (Tyr402) Pyk2 abundance in hippocampal synaptoneurosomes that were first warmed for 5 min at 30°C and then either unperturbed or stimulated with BDNF (50 ng/ml) for 10, 20, or 30 min. (D to F) As in (A) to (C) in cultured hippocampal neurons (high-density cultures; DIV 14 and 15) either maintained under control conditions or stimulated with BDNF (50 ng/ml) for 20 min. Data are means ± SEM for the number of independent experiments indicated (n). *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test. (G) Representative images of hippocampal neurons that were transfected with wild-type (WT) or phospho-mutant, kinase-deficient Pyk2 (Y402F) at DIV 12, and at DIV 15 were maintained under control conditions or stimulated with BDNF (50 ng/ml) for 30 min, live-immunostained for GluN2B (using an antibody against an extracellular epitope in the GluN2B N terminus), fixed and permeabilized, and further immunostained for Flag (transfection marker) and MAP2. Scale bar, 5 μm. (H to J) Analysis of total number (H) and intensity (I) of surface GluN2B puncta per dendritic length, as well as for GluN2B immunoreactivity per puncta (J), in neurons described in (G). Data were quantified relative to the empty vector control (Flag-Empty) and are means ± SEM for the indicated number of neurons (n) from at least three independent experiments. *P < 0.05, ***P < 0.001 between the indicated conditions; #P < 0.05, ##P < 0.01, ###P < 0.0001 versus empty control by one-way ANOVA with Bonferroni test.

  • Fig. 7 BDNF increases the synaptic synthesis of Pyk2.

    (A) Representative images of hippocampal neurons (DIV 14 and 15) that were incubated with azidohomoalanine (AHA) (4 mM) in the presence or absence of BDNF (50 ng/ml) for 30 min, fixed, and then subjected to the click chemistry technique (FUNCAT) to biotinylate AHA. Antibodies against biotin and Pyk2 were used to detect close proximity between Pyk2 and newly synthetized proteins. The FUNCAT-PLA signal (green) was obtained using PLAminus and PLAplus oligonucleotides coupled to secondary antibodies, together with detection reagents for ligation and amplification. PLA signal was amplified through binding of fluorescent detection probes. Scale bar, 5 μm. Arrowheads indicate PLA–PSD-95–colocalized puncta. (B to G) PLA neurons described in (A) were immunostained for PSD-95 and MAP2 and analyzed for the total number (B) and intensity (C) of PLA puncta per dendritic length, as well as for the immunoreactivity per puncta (D). The number (F) and intensity (G) of synaptic (PSD-95–colocalized) PLA puncta per dendrite length and the immunoreactivity per synaptic puncta (G) were also analyzed. Data were quantified relative to control and are means ± SEM for the indicated number of neurons (n) in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test.

  • Fig. 8 BDNF treatment increases local protein translation and up-regulates Pyk2 protein levels in hippocampal synaptoneurosomes.

    (A) Timeline of the protocol used to test the effect of BDNF on Pyk2 protein abundance in hippocampal synaptoneurosomes. (B and C) Representative Western blot (B) and analysis (C) of Pyk2 abundance in hippocampal synaptoneurosomes that underwent the protocol described in (A). Data are means ± SEM from four or five independent experiments as indicated (n). Densitometry of Pyk2 bands was normalized to that of β-tubulin. Statistical significance was calculated using the one-way ANOVA (P < 0.0001) with Bonferroni’s multiple comparison test: ***P < 0.001, ****P < 0.0001. (D and E) As described for (B) and (C) in cultured hippocampal neurons. Statistical analysis was performed using Student’s t test.

  • Fig. 9 BDNF up-regulates synaptic Pyk2 protein abundance and the percentage of synapses containing Pyk2 in a protein synthesis–dependent manner.

    (A) Representative images of rat hippocampal neurons maintained under control conditions (Ctrl) or incubated with BDNF (50 ng/ml) for 30 or 60 min in the presence or absence of cycloheximide (50 μg/ml), as indicated. Neurons were live-immunostained for GluN2B using an antibody against an extracellular epitope located in the GluN2B N terminus, fixed and permeabilized, and then further immunostained for Pyk2, MAP2, and PSD-95. Hippocampal neurons were preincubated with cycloheximide (50 μg/ml) or vehicle (DMSO, 1:1000 dilution) for 45 min before stimulation with BDNF, as indicated. Arrowheads indicate Pyk2–PSD-95–colocalized puncta. Scale bars, 5 μm. (B to I) Images represented in (A) were analyzed for the number (B) and intensity (C) of Pyk2 puncta per dendritic length, as well as for Pyk2 immunoreactivity per puncta (D), quantified as percentage of the respective DMSO control (at 30 or 60 min). The number (E) and intensity (F) of synaptic (PSD-95–colocalized) Pyk2 as well as Pyk2 immunoreactivity per puncta (G) were also analyzed. The percentage of Pyk2 showing a synaptic distribution (number of Pyk2 puncta colocalized with PSD-95/total Pyk2 number of puncta) and the percentage of synapses containing Pyk2 (number of PSD-95 puncta colocalized with Pyk2/total PSD-95 number of puncta) are shown in (H) and (I), respectively. Data are average ± SEM for the indicated number of neurons (n) from at least three independent experiments performed in independent preparations. **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Bonferroni test.

  • Fig. 10 hnRNP K mediates the effects of BDNF on dendritic Pyk2 protein abundance.

    (A) Immunoprecipitation (IP) of and Western blotting (WB) for hnRNP K from cultured hippocampal neuron lysates. Blot is representative of three experiments performed in independent preparations. (B) Amount of Pyk2, GluA1, and NPAS4 mRNA that coimmunoprecipitated with hnRNP K in extracts from cultured hippocampal neurons (DIV 15), stimulated with BDNF (50 ng/ml for 20 min) relative to controls, assessed by qPCR. Data are means ± SEM from three or four independent experiments performed in distinct preparations as indicated (n). *P < 0.05 versus control by Student’s t test. (C to F) Effect of knockdown of hnRNP K on the BDNF-induced increase in dendritic Pyk2 protein abundance in hippocampal neurons. Cultures were infected at DIV 10 with control [sh1-Scramble (sh1-Scrbl)] or hnRNP K–targeted (sh6–hnRNP K) shRNA and, at DIV 14, were either unperturbed or stimulated with BDNF (50 ng/ml for 30 min) and then fixed and immunostained for Pyk2, GFP, and MAP2. Representative images are shown in (C). Images [represented in (C); scale bar, 5 μm] were analyzed for the number (D) and intensity (E) of Pyk2 puncta per dendritic length and for the Pyk2 immunoreactivity per puncta (F). Results are expressed as percentage to sh1-Scramble control. **P < 0.01, ****P < 0.0001 by one-way ANOVA with Bonferroni test. (G to J) As in (C) to (F) assessing the effect of overexpression of hnRNP K compared with treatment with BDNF on dendritic Pyk2 protein abundance in hippocampal neurons transfected at DIV 11 and 12. Data were quantified relative to the GFP-Empty control. Scale bar, 5 μm. (K) Assessment of the total number of GFP–hnRNP K puncta per dendritic length under the same conditions as described in (G) to (J), as a control. Data are means ± SEM from the indicated number of neurons (n) in at least three independent experiments. #P < 0.05, ##P < 0.01, ####P < 0.0001 versus GFP-Empty control by one-way ANOVA followed by the Bonferroni test.

Supplementary Materials

  • This PDF file includes:

    • Fig. S1. Evaluation of Pyk2 knockdown efficiency by shRNA.
    • Fig. S2. hnRNP K overexpression increases the synaptic abundance of Pyk2.
    • Table S1. shRNA sequences.

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