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Neurogranin Expression Is Regulated by Synaptic Activity and Promotes Synaptogenesis in Cultured Hippocampal Neurons

  • Alberto Garrido-García
  • Raquel de Andrés
  • Amanda Jiménez-Pompa
  • Patricia Soriano
  • Diego Sanz-Fuentes
  • Elena Martínez-Blanco
  • F. Javier Díez-GuerraEmail author
Article

Abstract

Neurogranin (Ng) is a calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC) and is highly enriched in the dendrites and spines of telencephalic neurons. It is proposed to be involved in regulating CaM availability in the post-synaptic environment to modulate the efficiency of excitatory synaptic transmission. There is a close relationship between Ng and cognitive performance; its expression peaks in the forebrain coinciding with maximum synaptogenic activity, and it is reduced in several conditions of impaired cognition. We studied the expression of Ng in cultured hippocampal neurons and found that both protein and mRNA levels were about 10% of that found in the adult hippocampus. Long-term blockade of NMDA receptors substantially decreased Ng expression. On the other hand, treatments that enhanced synaptic activity such as long-term bicuculline treatment or co-culture with glial cells or cholesterol increased Ng expression. Chemical long-term potentiation (cLTP) induced an initial drop of Ng, with a minimum after 15 min followed by a slow recovery during the next 2–4 h. This effect was most evident in the synaptosome-enriched fraction, thus suggesting local synthesis in dendrites. Lentiviral expression of Ng led to increased density of both excitatory and inhibitory synapses in the second and third weeks of culture. These results indicate that Ng expression is regulated by synaptic activity and that Ng promotes the synaptogenesis process. Given its relationship with cognitive function, we propose targeting of Ng expression as a promising strategy to prevent or alleviate the cognitive deficits associated with aging and neuropathological conditions.

Keywords

Neurogranin Synaptic plasticity Glutamate receptors Synaptogenesis Hippocampal neurons 

Notes

Acknowledgments

We thank Dr. FG Scholl (IBiS, Sevilla, Spain) and P. Scheifelle (Biozentrum, Basel, Switzerland) for providing the lentivector pLOX-Syn-DsRed-Syn-GFP. We would like to thank the Advanced Light Microscopy Core Facility, from Centro de Biología Molecular Severo Ochoa (CSIC-UAM), for assistance with the imaging studies.

Author Contributions

AG-G, RA, AJ-P, PS, DS-F, and EM-B carried out the experiments and analyzed the data. FJD-G conceived the study and wrote the manuscript. All the authors have read and approved the final version of the manuscript submitted.

Funding Information

This work was supported by the Spanish Ministry of Science and Innovation and MINECO (grants BFU2010-18297 and SAF2014- 55686-R). We also thank the “Fundación Ramón Areces” for providing institutional support to CBMSO.

Compliance with Ethical Standards

All procedures were carried out in accordance with the Spanish Royal Decree 1201/2005 for the protection of animals used in scientific research, and the European Union Directive 2010/63/EU regarding the protection of animals used for scientific purposes. The procedures were approved by local Ethical Committees.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary Figure 1

Maturation profiles of several proteins in cultured hippocampal neurons. Hippocampal neurons were harvested at several times of culture and processed for western blot; equal amounts of total protein were loaded from samples collected at each day in vitro (DIV). Antibodies used are listed in Supplementary Table 1. (PNG 926 kb)

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High resolution image (TIF 3030 kb)
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Supplementary Figure 2

Distribution of excitatory and inhibitory neurons in hippocampal neurons in culture. DIV16 hippocampal neurons were fixed and processed for immunofluorescence. 10 x 10 images were acquired with a 25X oil objective for each channel (DAPI, MAP 2, GluN1, GAD6) and stitched using Metamorph software. Upper panel shows complete tilescans of the total number of cells (DAPI), total number of neurons (MAP 2), excitatory neurons (GluN1), and inhibitory neurons (GAD6). White square shows the ROI detailed in the lower panel. Only a small proportion (11,41% ±3,43 SD n = 2) of neurons in culture expressed the inhibitory marker GAD6 when quantified, while the vast majority (82,55% ±1,41 SD n = 2) were excitatory neurons and expressed GluN1 marker. (PNG 3355 kb)

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Supplementary Figure 3

Example of the dense network of excitatory buttons in hippocampal neurons in culture. Immunofluorescence showing DIV16 hippocampal neurons labeled with DAPI, NeuN and vGluT1 antibodies. Note the dense network of excitatory synapses labeled with anti-vGluT1 antibody. (PNG 3010 kb)

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High resolution image (TIF 7426 kb)
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Supplementary Figure 4

Ng is expressed in a reduced fraction of hippocampal neurons in culture. DIV16 hippocampal neurons were fixed and processed for immunofluorescence. 10 x 10 images were acquired with a 25X oil objective for each channel (DAPI, MAP 2, Ng, GFAP) and stitched using Metamorph software. Upper panel shows complete tilescans of the total number of cells (DAPI), total number of neurons (MAP 2), Ng expressing neurons (Ng), and astrocytes (GFAP). White square shows the ROI detailed in the lower panel. (PNG 2999 kb)

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Supplementary Figure 5

Ng localizes to the dendritic spines of cultured hippocampal neurons. (Upper panel) Immunofluorescence showing a hippocampal neuron after 18 days in vitro (DIV) labeled with anti-Ng antibody and several other cells labeled only with DAPI. (Lower panel) Detail of Ng-labeled dendrites and dendritic spines (red arrowheads). (PNG 1551 kb)

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Supplementary Figure 6.

Analysis of Ng-expressing cells in cultured hippocampal neurons. Hippocampal neurons cultured at different cell densities were processed for immunofluorescence with anti-NeuN, anti-Ng antibodies and DAPI. 15 x 15 images covering a central rectangle (7.2 x 5.7 mm) of each coverslip were acquired for each channel and stitched using Metamorph software. Then, for each coverslip, cell density and time of culture (see Fig. 2c), total number of cells (DAPI), total number of neurons (NeuN/DAPI) and total number of Ng-expressing neurons (Ng/NeuN) were measured. The table below gives the total number of cells analyzed at each cell density and the percentage of those that were identified as neurons. (PNG 1016 kb)

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Supplementary Figure 7.

Long-term manipulation of endogenous synaptic activity modulates Ng expression. Examples of the western blots quantified for Fig. 4b. Hippocampal neurons were treated as in stated in fig. 4a for the indicated periods, starting at different times and all them collected at DIV18 for western blot analysis. (PNG 802 kb)

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Supplementary Figure 8.

Long-term manipulation of endogenous activity alters postsynaptic composition. Hippocampal neurons were kept in control medium or treated with tetrodotoxin (0′5 μM) or bicuculline (25 μM) for 48 h and extracted at DIV18. PSD fractions were purified as described in Methods. 4 μg of total protein of post-synaptic membrane-enriched fractions (Triton X-100 resistant pellet, TxP) were separated by SDS-PAGE and analyzed by western blot with anti-GluN1 (NR1, Millipore AB9864R), anti-GluN2B (NR2B, Millipore MAB5220), anti-PKCɛ (Millipore 06-991) and anti-β-actin (Sigma clone AC-15). (PNG 214 kb)

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Supplementary Figure 9.

Recovery of Ng expression after long-term treatment with AP5, TTX and NBQX. Hippocampal neurons were treated with 50 μM AP5, 1 μM TTX, AP5 + TTX or 10 μM NBQX for 1, 2 or 3 days and then harvested after 18 days in vitro (DIV18) for western blot analysis of their Ng and β3-tubulin content. Additionally, hippocampal neurons that were previously treated (Rec.) or not (Cont.) for 3 days with the above drugs were cultured for 3 additional days in normal growth medium (without drugs) and then collected to analyze recovery of Ng expression. Results are expressed as the ratio of Ng:β3-tubulin normalized to the ratio obtained for controls at DIV18. Results are means ± SEM, n = 5. Statistical comparisons were performed between each treatment and the corresponding controls at DIV18. *: p < 0.05, **: p < 0.01, ***: p < 0.001. (PNG 238 kb)

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Supplementary Figure 10.

Effect of several inhibitors on Ng levels after cLTP and cLTD induction. Cultured hippocampal neurons after 17 or 18 days in vitro (DIV) were processed for induction of (a) chemical long-term potentiation (cLTP) or (b) chemical long-term depression (cLTD). Lysates were obtained at the indicated times of the recovery period to analyze their Ng content by western blot. Inhibitors (MG132, 10 μM; anisomycin, 40 μM; calpeptin, 10 μM; cycloheximide [CHX], 25 μg/ml; AP5, 100 μM; NBQX, 20 μM; and MCPG, 125 μM) were added 15 min before cLTP induction and maintained thereafter (means ± SEM, n = 3). Statistical comparisons were performed between each treatment and the corresponding controls at DIV18. *: p < 0.05, **: p < 0.01, ***: p < 0.001. (PNG 285 kb)

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Supplementary Figure 11

Flowchart of the image analysis procedure used to quantify synaptogenesis in hippocampal neurons in culture. (PNG 1020 kb)

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Supplementary Figure 12

Typical captures for analyzing excitatory synapses. Hippocampal neurons were fixed and processed for immunofluorescence at DIV18. Upper panel shows typical example images labeling of MAP 2, vGluT1 and PSD-95, used for the synaptogenesis analysis in Fig. 6. White squares in the merge image show the ROIs detailed in the lower panel. (PNG 2561 kb)

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Supplementary Table 1 (DOCX 17 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Alberto Garrido-García
    • 1
    • 2
  • Raquel de Andrés
    • 1
  • Amanda Jiménez-Pompa
    • 1
    • 3
  • Patricia Soriano
    • 1
  • Diego Sanz-Fuentes
    • 1
  • Elena Martínez-Blanco
    • 1
  • F. Javier Díez-Guerra
    • 1
    • 4
    Email author
  1. 1.Departamento de Biología Molecular and Centro de Biología Molecular “Severo Ochoa” (UAM-CSIC)Universidad Autónoma de MadridMadridSpain
  2. 2.Instituto Cajal (CSIC)MadridSpain
  3. 3.Departamento de Farmacología y Terapéutica, Facultad de MedicinaUniversidad Autónoma de MadridMadridSpain
  4. 4.Laboratory of Neuronal Plasticity, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM)Universidad Autónoma de MadridMadridSpain

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