Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Apr 22;291(17):9105-18.
doi: 10.1074/jbc.M115.691717. Epub 2016 Feb 16.

Subunit-selective N-Methyl-d-aspartate (NMDA) Receptor Signaling through Brefeldin A-resistant Arf Guanine Nucleotide Exchange Factors BRAG1 and BRAG2 during Synapse Maturation

Mohammad Nael Elagabani  1 , Dušica Briševac  1 , Michael Kintscher  2 , Jörg Pohle  3 , Georg Köhr  3 , Dietmar Schmitz  2 , Hans-Christian Kornau  4
Affiliations

Subunit-selective N-Methyl-d-aspartate (NMDA) Receptor Signaling through Brefeldin A-resistant Arf Guanine Nucleotide Exchange Factors BRAG1 and BRAG2 during Synapse Maturation

Mohammad Nael Elagabani et al. J Biol Chem. .

Abstract

The maturation of glutamatergic synapses in the CNS is regulated by NMDA receptors (NMDARs) that gradually change from a GluN2B- to a GluN2A-dominated subunit composition during postnatal development. Here we show that NMDARs control the activity of the small GTPase ADP-ribosylation factor 6 (Arf6) by consecutively recruiting two related brefeldin A-resistant Arf guanine nucleotide exchange factors, BRAG1 and BRAG2, in a GluN2 subunit-dependent manner. In young cortical cultures, GluN2B and BRAG1 tonically activated Arf6. In mature cultures, Arf6 was activated through GluN2A and BRAG2 upon NMDA treatment, whereas the tonic Arf6 activation was not detectable any longer. This shift in Arf6 regulation and the associated drop in Arf6 activity were reversed by a knockdown of BRAG2. Given their sequential recruitment during development, we examined whether BRAG1 and BRAG2 influence synaptic currents in hippocampal CA1 pyramidal neurons using patch clamp recordings in acute slices from mice at different ages. The number of AMPA receptor (AMPAR) miniature events was reduced by depletion of BRAG1 but not by depletion of BRAG2 during the first 2 weeks after birth. In contrast, depletion of BRAG2 during postnatal weeks 4 and 5 reduced the number of AMPAR miniature events and compromised the quantal sizes of both AMPAR and NMDAR currents evoked at Schaffer collateral synapses. We conclude that both Arf6 activation through GluN2B-BRAG1 during early development and the transition from BRAG1- to BRAG2-dependent Arf6 signaling induced by the GluN2 subunit switch are critical for the development of mature glutamatergic synapses.

Keywords: ADP ribosylation factor (ARF); BRAG; GTPase; N-methyl-D-aspartate receptor (NMDA receptor, NMDAR); X chromosome-linked intellectual disability (XLID); guanine nucleotide exchange factor (GEF); neurodevelopment; postsynaptic density (PSD); subunit switch; synapse.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Ligand binding to NMDARs stimulates BRAG-mediated Arf6 activation in a subtype-selective and Ca2+-dependent manner. A, subtype-selective stimulation of BRAG1 and BRAG2 by ligand binding to NMDARs in HEK293 cells. Shown are representative immunoblots of Arf6GTP-specific pulldown assays from HEK-BRAG1 and HEK-BRAG2 cells expressing Arf6-HA, GluN1, and either GluN2A or GluN2B. Bars illustrate Arf6 activation calculated as the Arf6GTP/Arf6total ratio (pd/t) of cells treated for 5 min with 1 mm l-glutamate (glu) normalized to untreated controls (paired t test: HEK-BRAG1: GluN2A, *, p = 0.0099, n = 9; GluN2B, *, p = 0.0033, n = 7; HEK-BRAG2: GluN2A, *, p = 0.043, n = 6; GluN2B, *, p = 0.025, n = 6). B, NMDAR-BRAG signaling requires Ca2+. Shown are representative immunoblots of Arf6GTP-specific pulldown assays from HEK-BRAG1 cells expressing Arf6-HA, GluN1, and GluN2B and from HEK-BRAG2 cells expressing Arf6-HA, GluN1, and GluN2A. Cells were stimulated by application of an extracellular solution containing l-glutamate (glu) with or without 1.3 mm Ca2+. Bars illustrate Arf6 activation calculated as the Arf6GTP/Arf6total ratio (pd/t) of cells treated with glutamate in the presence or absence of extracellular Ca2+ normalized to untreated controls (paired t test: HEK-BRAG1: without (w/o) Ca2+, p = 0.63, n = 11; with Ca2+, *, p = 0.0009, n = 6; HEK-BRAG2: without Ca2+, p = 0.88, n = 8; with Ca2+, *, p = 0.010, n = 6). Error bars indicate S.E. C, subcellular distribution and expression levels of NMDARs, BRAGs, and Arf6 in transfected HEK-BRAG1 and -BRAG2 cells. NMDARs with HA-tagged GluN2 subunits and untagged Arf6 were expressed to compare the localization of GluN2B and BRAG1 or GluN2A and BRAG2 (left column). NMDARs with untagged GluN2 subunits and Arf6-HA (right column, as in A and B) were expressed to compare the localization of BRAG1 or BRAG2 and Arf6. The distribution of the stained proteins was similar upon expression of GluN2A in HEK-BRAG1 and GluN2B in HEK-BRAG2 cells (not shown).Western blots show the expression levels of GluN2 subunits, BRAGs, and Arf6-HA in homogenates of HEK-BRAG1 and HEK-BRAG2 cells transfected as in A and B. Scale bar, 10 μm.
FIGURE 2.
FIGURE 2.
Physical interaction between NMDARs and BRAG proteins. A, the central part of the intracellular domain of GluN2A binds to BRAG2. An immunoblot of recombinant FLAG (F)-tagged BRAG2 pulled down from a HEK293 cell extract by GST fused to aa 838–1037 (GluN2A-CT200a), aa 1038–1237 (GluN2A-CT200b), and aa 1238–1464 (GluN2A-CT227) of GluN2A in the presence of 2 mm EDTA is shown. GluN2A-CT200a was recovered from inclusion bodies (50). Input, 5% of cell extract. B, Ca2+ promotes selective interactions between GluN2 fragments and BRAGs. Immunoblots of recombinant FLAG-tagged BRAG2 or BRAG1 recovered from HEK293 cell extracts by GluN2A-CT200b and by GST fused to aa 1036–1243 of GluN2B (GluN2B-CT208) in the presence of 2 mm EDTA or 100 μm Ca2+ are shown. Input, 5% of cell extract. C, BRAGs interact with short regions in GluN2 subunits. Immunoblots of recombinant FLAG-tagged BRAG proteins recovered from HEK293 cell extracts by GST pulldown with 40- or 100-amino acid fragments of GluN2A (GluN2A-CT100a, aa 1038–1137; GluN2A-CT100b, aa 1138–1237; GluN2A-CT40a, aa 1058–1097; GluN2A-CT40b, aa 1078–1117; GluN2A-CT40c, aa 1098–1137) and GluN2B (GluN2B-CT40a, aa 1115–1154; GluN2B-CT40b, aa 1135–1174; GluN2B-CT40c, aa 1155–1194) in the presence of 2 mm Ca2+. Input, 5% of cell extract. D, interaction sites for BRAG2 and BRAG1 within GluN2 intracellular C-terminal tails. The scheme indicates the location of the protein fragments used for the interaction analyses within the C-terminal regions of GluN2A and GluN2B. NMDAR fragments shown to interact with BRAG1 or BRAG2 are depicted in green. Sequences of the shortest interacting fragments in single letter code of amino acids are shown below with conserved motifs in red and blue. Importance of the KTK/RTK motif (bold) was tested in Fig. 3.
FIGURE 3.
FIGURE 3.
NMDAR-BRAG signaling requires physical interaction. A and B, BRAG recruitment upon ligand binding to NMDARs depends on a physical interaction. Shown are results of Arf6GTP-specific pulldown assays from HEK-BRAG1 and HEK-BRAG2 cells expressing Arf6-HA, GluN1, and GluN2A (A) or GluN2B (B) with or without the indicated deletions or mutations. ΔB2BD, deletion of BRAG2-binding domain in GluN2A (aa 1078–1117; GluN2A-CT40b in Fig. 2C); KTK-AAA, mutation K1081A/T1082A/K1083A in GluN2A; ΔB1BD, deletion of BRAG1-binding domain in GluN2B (aa 1115–1154; GluN2B-CT40a in Fig. 2C); RTK-AAA, mutation R1138A/T1139A/K1140A in GluN2B. Western blots on the bottom indicate that the mutations did not affect the expression levels of GluN2A or GluN2B. Bars illustrate Arf6 activation calculated as the Arf6GTP/Arf6total ratio (pd/t) of cells treated with 1 mm glutamate for 5 min normalized to untreated controls (paired t test: A, HEK-BRAG2: GluN2A, *, p = 0.0012, n = 14; ΔB2BD, *, p = 0.030, n = 7; Δ260, *, p = 0.017, n = 6; K1081A/T1082A/K1083A, p = 0.075, n = 9; B, HEK-BRAG1: GluN2B, *, p = 0.0007, n = 14; ΔB1BD, p = 0.14, n = 8; R1138A/T1139A/K1140, p = 0.14, n = 10). Error bars indicate S.E. Left, representative immunoblots.
FIGURE 4.
FIGURE 4.
Tonic Arf6 activation through GluN2B-BRAG1 signaling in young neurons. A, differences in Arf6 activity between immature and mature cortical neuron cultures. Shown are results of Arf6GTP-specific pulldown assays from cultured cortical neurons at DIV7 and DIV21 (±1 day for each) treated with or without 100 μm d-AP5 (AP5) for 1 h (paired t test: DIV7, *, p < 0.0001, n = 8; DIV21, p = 0.057, n = 6; unpaired t test: basal DIV7 versus DIV21, *, p = 0.038, n = 6–8). Levels of Arf6 activity are shown as Arf6GTP/Arf6total ratios (pd/t). B, tonic Arf6 activation in immature neuronal cultures depends on GluN2B. Shown are results of Arf6GTP-specific pulldown assays from cultured cortical neurons at DIV7 treated for 1 h with 100 μm MK-801 (MK), 3 μm ifenprodil (ifen), or 300 nm Zn2+. Changes in Arf6 activity by drugs were calculated as the Arf6GTP/Arf6total ratio (pd/t) normalized to an untreated control (paired t test, MK-801, *, p = 0.0054, n = 7; ifenprodil, *, p = 0.0005, n = 8; Zn2+, p = 0.83, n = 6). C, tonic Arf6 activation through GluN2B in young cortical cultures is mediated by BRAG1. Cultured cortical neurons (DIV7) infected with lentiviral vectors delivering shRNAs to BRAG1 (B1-KD), BRAG2 (B2-KD), or a scrambled control hairpin (ctrl) at DIV2 were treated with or without 3 μm ifenprodil for 1 h. Changes in Arf6 activity were calculated as the Arf6GTP/Arf6total ratio (pd/t) normalized to the respective control without ifenprodil (paired t test: ctrl, *, p = 0.021, n = 9; BRAG1-KD, *, p = 0.031, n = 11; BRAG2-KD, *, p = 0.007, n = 10). Top, timeline of neurons in culture indicating times of infection and experiment (black arrows). Error bars indicate S.E. Left, representative immunoblots. D, the distribution and expression level of GluN2B were not affected by BRAG1 depletion in young neuronal cultures. Left, at DIV8, BRAG1 co-localized with the synaptic marker VGLUT 1 in mouse hippocampal neurons. A subset of GluN2B co-localized with VGLUT 1 in neurons infected with a control virus as well as in neurons infected for BRAG1 RNAi at DIV2. Right, immunoblots of cell homogenates indicated that the expression levels of GluN2B, GluN2A, and Arf6 were not altered by depletion of BRAG1 or BRAG2 in young rat cortical cultures. Scale bars, 5 μm.
FIGURE 5.
FIGURE 5.
NMDA-triggered Arf6 activation through GluN2A-BRAG2 signaling in mature neurons. A, NMDA-triggered Arf6 activity in mature cortical cultures depends on GluN2A. Shown are results of Arf6GTP-specific pulldown assays from cultured cortical neurons at DIV21 stimulated by 100 μm NMDA for 5 min in the presence or absence of 100 μm d-AP5 (AP5), 100 μm MK-801 (MK), 3 μm ifenprodil (ifen), or 300 nm Zn2+. Arf6 activation by NMDA was calculated as the Arf6GTP/Arf6total ratio (pd/t) normalized to the respective control without NMDA (paired t test: −, *, p = 0.0007, n = 15; d-AP5, p = 0.059, n = 9; MK-801, p = 0.058, n = 10; ifenprodil, *, p = 0.0016, n = 10; Zn2+, *, p = 0.0049, n = 10). The NMDA-triggered Arf6 activation was not altered by ifenprodil (unpaired t test: p = 0.24, n = 10–15). ns, not significant. B, NMDA-triggered Arf6 activation in mature cortical cultures is mediated by BRAG2. Cultured cortical neurons (DIV21) infected at DIV15 with lentiviral vectors delivering shRNAs to BRAG1 (B1-KD), BRAG2 (B2-KD), or a control hairpin (ctrl) were treated with or without 100 μm NMDA for 5 min. Arf6 activation was calculated as the Arf6GTP/Arf6total ratio (pd/t) normalized to the respective control without NMDA (paired t test: ctrl, *, p = 0.0021, n = 17; BRAG1-KD, *, p = 0.012, n = 8; BRAG2-KD, *, p = 0.044, n = 12; BRAG1/2-KD, p = 0.35, n = 9). Top, timeline of neurons in culture indicating times of infection and experiment (black arrows). Error bars indicate S.E. Left, representative immunoblots. C, synaptic localization and expression levels of the proteins involved in NMDAR-Arf6 signaling. Left, BRAG1, BRAG2, GluN2A, and GluN2B partly co-localized with the synaptic marker PSD-95 in mouse hippocampal neuron cultures at DIV21. Arf6 was broadly distributed in dendritic shafts, and a subset co-localized with Homer1. These patterns were not changed by the knockdown of BRAG2 (B2-KD). Right, immunoblots of cell homogenates indicated that the levels of the GluN2 subunits and Arf6 were not altered by knockdown of BRAG1 (B1-KD), BRAG2 (B2-KD), or both (B1/2-KD) in mature rat cortical cultures. Scale bar, 5 μm.
FIGURE 6.
FIGURE 6.
Upon BRAG2 knockdown or GluN2A blockade, neurons revert to tonic Arf6 activation through GluN2B-BRAG1 signaling. A, tonic Arf6 activation by GluN2B and BRAG1 in mature cortical cultures upon depletion of BRAG2. Cultured cortical neurons (DIV21) infected at DIV15 with lentiviral vectors delivering shRNAs to BRAG2 (B2-KD), to both BRAG1 and BRAG2 (B1/2-KD), or a scrambled control hairpin (ctrl) were treated with or without 3 μm ifenprodil (ifen) for 1 h (paired t test: ctrl, p = 0.20, n = 10; BRAG2-KD, *, p < 0.0001, n = 12; BRAG1/2-KD, p = 0.89, n = 6; unpaired t test: BRAG2-KD − versus ctrl −, *, p < 0.0001, n = 10–13; BRAG1/2-KD − versus ctrl −, p = 0.86, n = 6–10). Levels of Arf6 activity are shown as Arf6GTP/Arf6total ratios (pd/t). B and C, tonic Arf6 activation by GluN2B and BRAG1 in mature cortical cultures upon Zn2+ treatment. B, cultured cortical neurons (DIV21) were treated with or without 300 nm Zn2+ for 1 h and subsequently with or without 3 μm ifenprodil for an additional hour (unpaired t test: Zn2+ effect, *, p = 0.016, n = 10; paired t test: ifenprodil effect without prior Zn2+ treatment, p = 0.63, n = 6; ifenprodil effect after Zn2+ treatment, *, p < 0.0001, n = 10). C, mature neuronal cultures (DIV21) infected at DIV15 with lentiviral vectors delivering an shRNA to BRAG1 (B1-KD) or a scrambled control hairpin (ctrl) were treated with or without 300 nm Zn2+ for 1 h (paired t test: ctrl, *, p = 0.0015, n = 9; BRAG1-KD, p = 0.56, n = 8; unpaired t test: knock-down effect, p = 0.49, n = 8–9). Levels of Arf6 activity are shown as Arf6GTP/Arf6total ratios (pd/t). Error bars indicate S.E. Left, representative immunoblots.
FIGURE 7.
FIGURE 7.
Effects of the knockdown of BRAG1 and BRAG2 on the number and size of glutamatergic synapses. VGLUT 1 and PSD-95 were stained in mature rat hippocampal neurons (DIV21–28) infected at DIV15 with lentiviral vectors delivering shRNAs to BRAG1 (B1-KD), BRAG2 (B2-KD), or a control hairpin (ctrl). Bars illustrate the number and size of overlapping PSD-95 and VGLUT 1 puncta in dendritic segments normalized to the average values of control-infected neurons in each of five independent experiments (Mann-Whitney test (*, p < 0.05): number: ctrl, n = 75 dendritic segments; BRAG1-KD, n = 56, *, p = 0.0067; BRAG2-KD, n = 60, *, p = 0.0096; size: ctrl, n = 75; BRAG1-KD, n = 56, p = 0.96; BRAG2-KD, n = 60, *, p = 0.0072). Error bars indicate S.E. Scale bars, 10 μm.
FIGURE 8.
FIGURE 8.
NMDA-triggered AMPAR internalization in mature neuronal cultures lacking BRAG1 or BRAG2. The endocytosis rate of the AMPAR subunit GluA2 with or without NMDA treatment was evaluated using a fluorescence internalization assay in rat hippocampal neurons (DIV24/25) infected at DIV15 with lentiviral vectors delivering shRNAs to BRAG1 (B1-KD), BRAG2 (B2-KD), or a control hairpin (ctrl). GluA2 internalization was calculated as the ratio between internalized (int) and surface-remaining (surf) GluA2 fluorescence signals in individual dendritic segments. Top, representative pictures of fluorescence signals in neurons and dendritic segments (indicated by rectangles). Bottom, bars showing average GluA2 internalization from four independent experiments (Mann-Whitney test (*, p < 0.05): ctrl − NMDA, n = 66 dendritic segments versus ctrl + NMDA, n = 93, *, p < 0.0001; BRAG1-KD −, n = 90 versus BRAG1-KD +, n = 92, *p = 0.023; BRAG2-KD −, n = 84 versus BRAG2-KD +, n = 97, p = 0.17; BRAG1-KD − versus ctrl −, p = 0.79; BRAG2-KD − versus ctrl −, *, p = 0.019). Error bars indicate S.E. Scale bar, 10 μm.
FIGURE 9.
FIGURE 9.
Loss of BRAG1 or BRAG2 alters AMPAR mEPSC frequency and amplitude distribution in CA1 pyramidal neurons. A–C, the impact of BRAG1 or BRAG2 signaling on synaptic transmission during development was assessed by recording of spontaneous AMPAR mEPSCs at a holding potential of −70 mV. Recordings were performed around P16 for P0-injected mice using viral constructs for shRNA to BRAG1 (A) or shRNA to BRAG2 (B) or after P35 for P21-injected mice using shRNA to BRAG2 (C). Example traces for uninfected (ctrl) and infected cells are shown below the experimental timelines indicating times of infection and experiment (black arrows). Data are plotted for mEPSC frequency and amplitude and reported as mean ± S.E. (error bars); *, p < 0.05 using Student's unpaired t test. Relative frequency distribution plots for 5-pA bins are shown on the bottom (amplitude values indicate the center of each bin). These plots contain data sets of an equal number of events per cell and condition to prevent overrepresentation of single cells. D, expression levels of NMDAR subunits, BRAGs, and Arf6 in postnatal mouse hippocampi and in rat cortical neuron cultures (top) as well as during mouse forebrain development (bottom). Shown are Western blots of nucleus-free tissue extracts and of neuronal culture homogenates adjusted to obtain comparable βIII-tubulin immunoreactivities.
FIGURE 10.
FIGURE 10.
Effects of BRAG2 depletion on evoked AMPAR and NMDAR currents in mature CA1 pyramidal neurons. A, representative current traces to compare NMDAR decay (averages of 15 sweeps) recorded at −70 mV (AMPAR current) and following AMPAR blockade at +40 mV (NMDAR current) from uninfected (ctrl) and Cre-infected green CA1 pyramidal neurons (ΔBRAG2) in acute slices from mice homozygous for a floxed BRAG2 allele (Iqsec1fl/fl) upon Schaffer collateral stimulation. B, rise and decay of NMDAR currents is unaffected and shown for individual neurons as marks and as mean ± S.E. (error bars) (15 repetitions per cell). C, AMPA/NMDA ratio is enhanced in infected neurons (15 repetitions per cell for AMPAR and NMDAR, respectively). D, CV of evoked NMDAR currents is smaller than CV of evoked AMPAR currents in uninfected but not in infected neurons (15 repetitions per cell for AMPAR and NMDAR, respectively). E, paired pulse ratio of evoked AMPAR currents (30 repetitions per cell). F, AMPAR (left) and NMDAR (right) average EPSC per cell versus corresponding CV−2 as a measure for number of stimulated synapses because release probability is unchanged (see E). Linear regressions through the origin indicate reduced AMPAR as well as NMDAR quantal sizes in infected neurons. The upper x axes illustrate (for release probability 0.3) that a larger number of actually releasing synapses needs to be activated upon BRAG2 deletion to evoke AMPAR and NMDAR currents with comparable amplitudes (15–30 repetitions per cell for AMPAR and 15 repetitions per cell for NMDAR). *, p < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hanse E., Seth H., and Riebe I. (2013) AMPA-silent synapses in brain development and pathology. Nat. Rev. Neurosci. 14, 839–850 - PubMed
    1. Paoletti P., Bellone C., and Zhou Q. (2013) NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat. Rev. Neurosci. 14, 383–400 - PubMed
    1. Carmignoto G., and Vicini S. (1992) Activity-dependent decrease in NMDA receptor responses during development of the visual cortex. Science 258, 1007–1011 - PubMed
    1. Monyer H., Burnashev N., Laurie D. J., Sakmann B., and Seeburg P. H. (1994) Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12, 529–540 - PubMed
    1. Sheng M., Cummings J., Roldan L. A., Jan Y. N., and Jan L. Y. (1994) Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368, 144–147 - PubMed

Publication types

MeSH terms

Substances