Arf6-GEF BRAG1 regulates JNK-mediated synaptic removal of GluA1-containing AMPA receptors: a new mechanism for nonsyndromic X-linked mental disorder

Abstract

Activity-dependent modifications of excitatory synapses contribute to synaptic maturation and plasticity, and are critical for learning and memory. Consequently, impairments in synapse formation or synaptic transmission are thought to be responsible for several types of mental disabilities. BRAG1 is a guanine nucleotide exchange factor for the small GTP-binding protein Arf6 that localizes to the postsynaptic density of excitatory synapses. Mutations in BRAG1 have been identified in families with X-linked intellectual disability (XLID). These mutations mapped to either the catalytic domain or an IQ-like motif; however, the pathophysiological basis of these mutations remains unknown. Here, we show that the BRAG1 IQ motif binds apo-calmodulin (CaM), and that calcium-induced CaM release triggers a reversible conformational change in human BRAG1. We demonstrate that BRAG1 activity, stimulated by activation of NMDA-sensitive glutamate receptors, depresses AMPA receptor (AMPA-R)-mediated transmission via JNK-mediated synaptic removal of GluA1-containing AMPA-Rs in rat hippocampal neurons. Importantly, a BRAG1 mutant that fails to activate Arf6 also fails to depress AMPA-R signaling, indicating that Arf6 activity is necessary for this process. Conversely, a mutation in the BRAG1 IQ-like motif that impairs CaM binding results in hyperactivation of Arf6 signaling and constitutive depression of AMPA transmission. Our findings reveal a role for BRAG1 in response to neuronal activity with possible clinical relevance to nonsyndromic XLID.

Figure 1.

BRAG1 binds Ca²⁺-free calmodulin. A, Domain organization of the three BRAG family members. B, Consensus sequence for IQ motifs, compared with the IQ-like motif in wild-type BRAG1. The bottom sequence shows mutations introduced to generate BRAG1-IQ. C, CaM binding to wild-type and mutant BRAG1. HeLa cells were transiently transfected with myc-tagged BRAG1-WT, BRAG1-EK, BRAG1-IQ, or BRAG1-ΔN and lysed in the presence of either 2 mm EGTA or 2 mm CaCl₂. Cell lysates were then incubated with CaM-Sepharose, and BRAG1 binding was analyzed by anti-myc immunoblotting. Binding was quantified by densitometry. The signal for BRAG1-WT was normalized to a value of 1 and relative binding values are shown. The blot shown is representative of three independent experiments.

Figure 2.

BRAG1 mutants localize to excitatory synapses in hippocampal neurons. A, Dissociated rat hippocampal neurons were fixed at DIV 21, and coimmunostained with rabbit polyclonal anti-BRAG1 and mouse anti-PSD-95 antibodies. B–F, Hippocampal neurons were transfected (DIV 6) with the indicated mCherry-tagged constructs and fixed (DIV 21) for epifluorescence imaging. Cells expressing mCherry (B), mCherry-BRAG1-WT (C), mCherry-BRAG1-EK (D), mCherry-BRAG1-IQ (E), and mCherry-BRAG1-ΔN (F) were costained with PSD-95 antibody to detect excitatory synapses. Scale bars: 10 μm.

Figure 3.

Ca²⁺ influx triggers a reversible conformational change in BRAG1. A, B, Formation of cytoplasmic puncta in response to calcium influx. HeLa cells expressing mCherry-BRAG1-WT (A) or mCherry-BRAG1-IQ (B) were stimulated with 5 μm ionomycin for 3 min. Single frames from time-lapse movies beginning at 30 s before ionomycin treatment (−30) are shown. Note that BRAG1-IQ is punctate in the absence of ionomycin and does not change its distribution after ionomycin addition. C, Calcium-induced condensation of BRAG1 is reversible. HeLa cells were either left untreated (left), were treated with 5 μm ionomycin for 3 min (center), or were treated with 5 μm ionomycin for 3 min followed by incubation with 5 mm BAPTA-AM for 10 min (right). D, BRAG1-ΔN does not form puncta in response to ionomycin stimulation. Cells expressing mCherry-BRAG1-ΔN were treated with ionomycin for 3 min and imaged as described above. E, Quantification of calcium-induced puncta formation. Cells expressing BRAG1-WT, BRAG1-IQ or BRAG1-ΔN were treated with either vehicle (DMSO) or ionomycin for 3 min, then fixed and imaged. Puncta were quantified as described previously (see Materials and Methods) from at least 18 cells from three independent experiments. Mean number of BRAG1 puncta per 100 μm² pre- and post-ionomycin stimulation (WT-pre: 4.6 ± 0.9 puncta, n = 19; WT-post: 66.6 ± 13.5 puncta; n = 19, p < 0.001; IQ-pre: 67.5 ± 7.4 puncta, n = 17; IQ-post: 76.6 ± 19.5 puncta; n = 17, p = 0.25; ΔN-pre: 0.0 ± 0. puncta, n = 18; ΔN-post: 13.8 ± 3.8 puncta; n = 19, p < 0.005). *p ≤ 0.005 (Student's t test). F, The N terminus of BRAG1 is necessary for self-association. Myc-BRAG1-WT or myc-BRAG1-ΔN were coexpressed in HeLa cells with GFP-BRAG1 WT. Cell lysates were then immunoprecipitated with anti-GFP, and the presence of bound myc-BRAG1 detected by immunoblotting with anti-myc. Scale bars: 10 μm.

Figure 4.

Stimulation of NMDA-Rs in hippocampal neurons triggers a conformational change in BRAG1. A–C, Frames from time-lapse movies of DIV 12 hippocampal neurons expressing mCherry-tagged BRAG1 constructs and treated with 30 μm NMDA for the times shown. A–C, Shows BRAG1-WT (A), BRAG1-IQ (B), and BRAG1-ΔN (C). Arrowheads indicate dendritic spines where single BRAG1 foci resolve into distinct puncta. Arrows indicate formation of new puncta in the dendritic shaft. D, Quantitation of puncta formation. Mean number of puncta per 100 μm² pre- and post-NMDA stimulation (WT-pre: 17.5 ± 1.4 puncta; WT-post: 46.1 ± 6.6 puncta; n = 3, p < 0.05; IQ-pre: 20.0 ± 3.4 puncta; IQ-post: 20.5 ± 2.4 puncta; n = 4, p = 0.5; ΔN-pre: 6.0 ± 4.1 puncta; ΔN-post: 7.0 ± 3.9 puncta; n = 3, p = 0.5). *p ≤ 0.05 (Wilcoxon). Scale bars: 10 μm.

Figure 5.

The IQ motif and N terminus are dispensable for BRAG1 catalytic activity in heterologous cells. A, Pulldown assay for Arf6 activity. Active GTP-bound Arf6 was precipitated with GST-GGA3 from lysates of HeLa cell coexpressing Arf6-HA and the indicated myc-tagged BRAG1 constructs. B, Quantitation of Arf6 activation (Ctrl: 100.0 ± 55.6%; WT: 362.0 ± 99.4%; n = 3; p < 0.05; EK: 117.3 ± 78.9%; n = 3; p = 0.5; IQ: 376.8 ± 86.0%; n = 3; p < 0.05; ΔN: 340.6 ± 131.3%; n = 3; p < 0.05). The relative values and SEs were normalized to average amounts of Arf6-GTP from control cells expressing Arf6-HA alone. **p < 0.05 (Wilcoxon test).

Figure 6.

Expression of BRAG1 does not alter the basic membrane properties of CA1 neurons. A, Two-photon fluorescence imaging indicates that mCherry-BRAG1 is expressed in the dendrites and spines (inset) of CA1 pyramidal neurons. B, Evoked responses to step depolarizing and hyperpolarizing pulses recorded from neighboring nonexpressing (Ctrl) and BRAG1-expressing cells. C, Resting membrane potentials (Ctrl: −58.1 ± 1.1 mV; BRAG1: −57.2 ± 0.9 mV; n = 20; p = 0.25), input resistances (Ctrl: 189.8 ± 8.2 MΩ; BRAG1: 190.2 ± 13.2 MΩ; n = 20; p = 0.50), and time constants (Ctrl: 27.3 ± 1.7 ms; BRAG1: 28.5 ± 2.1 ms; n = 20; p = 0.97) in mCherry-BRAG1-expressing cells are plotted against those obtained from control nonexpressing cells. Blue dots indicate the average points.

Figure 7.

BRAG1-WT and BRAG1 mutants differentially affect synaptic transmission. A, Evoked AMPA-R-mediated (−60 mV) and NMDA-R-mediated (+40 mV) responses recorded from neighboring nonexpressing (Ctrl) and mCherry-tagged BRAG1-WT- or BRAG1-ΔN-expressing neurons cultured in normal media or media containing 100 μm dl-APV. B, Values for AMPA responses in neurons expressing BRAG1-WT and BRAG1-ΔN cultured in normal media (Ctrl: −31.6 ± 2.5 pA; WT: −22.9 ± 2.4 pA; n = 38; p < 0.05; Ctrl: −14.2 ± 2.1 pA; ΔN: −20.1 ± 3.2 pA; n = 16; p < 0.05), in media containing high Mg²⁺ (Ctrl: −26.8 ± 2.6 pA; WT: −28.8 ± 3.1 pA; n = 22; p = 0.94; Ctrl: −20.3 ± 1.7 pA; ΔN: −21.4 ± 2.3 pA; n = 23; p = 0.93), or in media containing dl-APV (Ctrl: −31.7 ± 2.3 pA; WT: −30.1 ± 2.4 pA; n = 19; p = 0.51; Ctrl: −22.3 ± 3.8 pA; ΔN: −23.8 ± 2.5 pA; n = 19; p = 0.69), and for NMDA responses in neurons expressing Brag1-WT and BRAG1-ΔN cultured in normal media (Ctrl: 79.3 ± 6.5 pA; WT: 75.9 ± 6.8 pA; n = 38; p = 0.60; Ctrl: 46.6 ± 9.7 pA; ΔN: 41.3 ± 8.5 pA; n = 16; p = 0.33), in media containing high Mg²⁺ (Ctrl: 66.4 ± 11.6 pA; WT: 61.6 ± 9.4 pA; n = 22; p = 0.78; Ctrl: 53.8 ± 5.3 pA; ΔN: 54.3 ± 7.4 pA; n = 23; p = 0.72), or in media containing dl-APV (Ctrl: 59.2 ± 7.3 pA; WT: 59.3 ± 6.7 pA; n = 19; p = 0.94; Ctrl: 47.4 ± 6.8 pA; ΔN: 44.4 ± 5.5 pA; n = 19; p = 0.57) related to control nonexpressing neurons. C, Evoked AMPA-R-mediated (−60 mV) and NMDA-R-mediated (+40 mV) responses recorded from neighboring nonexpressing (Ctrl) with either mCherry-tagged BRAG1-EK or BRAG1-IQ expressing neurons cultured in normal media or media containing 100 μm dl-APV. D, Values for AMPA responses in neurons expressing BRAG1-EK and BRAG1-IQ cultured in normal media (Ctrl: −24.1 ± 2.8 pA; EK: −26.5 ± 2.5 pA; n = 21; p = 0.52; Ctrl: −25.4 ± 2.6 pA; IQ: −19.4 ± 1.9 pA; n = 25; p < 0.05), in neurons expressing BRAG1-IQ cultured in media containing high Mg²⁺ (Ctrl: −23.0 ± 2.3 pA; IQ: −15.4 ± 1.3 pA; n = 25; p < 0.005), or dl-APV (Ctrl: −26.7 ± 2.8 pA; IQ: −19.9 ± 1.7 pA; n = 24; p < 0.01), and for NMDA responses in neurons expressing BRAG1-EK or BRAG1-IQ cultured in normal media (Ctrl: 77.1 ± 9.1 pA; EK: 74.1 ± 8.0 pA; n = 21; p = 0.54; Ctrl: 52.5 ± 5.2 pA; IQ: 50.8 ± 6.6 pA; n = 25; p = 0.14), in neurons expressing BRAG1-IQ cultured in media containing high Mg²⁺ (Ctrl: 57.6 ± 5.7 pA; IQ: 52.9 ± 5.7 pA; n = 25; p = 0.20), or dl-APV (Ctrl: 68.9 ± 6.8 pA; IQ: 64.4 ± 5.8 pA; n = 24; p = 0.67) related to control nonexpressing neurons. The relative values and SEs were normalized to average amounts of AMPA and NMDA responses from control CA1 cells. *p < 0.05 (Wilcoxon test).

Figure 8.

BRAG1 regulates endogenous Arf6 signaling. A, Relative levels of GTP-bound Arf6 and total Arf6 in control hippocampal CA1 regions, and hippocampal CA1 regions expressing BRAG1-IQ or BRAG1-ΔN cultured in normal media. For each set of hippocampal CA1 cell lysate, 35 μg protein was used to precipitate and blot GTP-bound Arf6 and 7.5 μg protein was used to directly blot total Arf6. B, Relative amounts of Arf6-GTP (Ctrl: 100.0 ± 6.5%; IQ: 120.9 ± 6.3%; n = 22; p < 0.005; ΔN: 87.1 ± 7.7%; n = 22; p < 0.05) and total Arf6 (Ctrl: 100.0 ± 7.1%; IQ: 90.7 ± 7.5%; n = 22; p = 0.21; ΔN: 100.6 ± 9.6%; n = 22; p = 0.86) in control hippocampal CA1 regions, and hippocampal CA1 regions expressing BRAG1-IQ or BRAG1-ΔN. The relative values and SEs were normalized to average amounts of Arf6-GTP or total Arf6 from control untreated cells. *p < 0.05 (Wilcoxon test).

Figure 9.

BRAG1 signals synaptic depression via JNK. A, Evoked AMPA-R-mediated (−60 mV) and NMDA-R-mediated (+40 mV) responses recorded from neighboring nonexpressing (Ctrl) and mCherry-tagged BRAG1-IQ or BRAG1-ΔN expressing neurons cultured in media containing 5 μm SP600125 or 2 μm SB203580. B, Values for AMPA responses in neurons expressing BRAG1-IQ and BRAG1-ΔN cultured in media containing SP600125 (Ctrl: −23.0 ± 2.1 pA; IQ: minus]23.5 ± 2.8 pA; n = 26, p = 0.81; Ctrl: −18.4 ± 1.6 pA; ΔN: −19.7 ± 2.0 pA; n = 23, p = 0.48) or SB203580 (Ctrl: −26.0 ± 2.2 pA; IQ: −18.2 ± 1.4 pA; n = 28, p < 0.005; Ctrl: −17.9 ± 1.6 pA; ΔN: −25.5 ± 1.9 pA; n = 23, p < 0.01), and for NMDA responses in neurons expressing BRAG1-IQ and BRAG1-ΔN cultured in media containing SP600125 (Ctrl: 55.4 ± 4.7 pA; IQ: 55.4 ± 6.0 pA; n = 26, p = 0.53; Ctrl: 60.7 ± 10.6 pA; ΔN: 58.3 ± 12.1 pA; n = 23, p = 0.38) or SB203580 (Ctrl: 51.3 ± 6.7 pA; IQ: 54.0 ± 6.2 pA; n = 28, p = 0.93; Ctrl: 60.6 ± 6.3 pA; IQ: 56.4 ± 7.7 pA; n = 23, p = 0.43) related to control nonexpressing neurons. C, Western blots of phospho-p46/54 JNK or p38 MAPK in control hippocampal CA1 regions, hippocampal CA1 regions expressing BRAG1-IQ, and hippocampal CA1 regions expressing BRAG1-ΔN. Each lane was loaded with the same amount of protein (45 μg). D, Left, Relative amounts of phospho-p46 (Ctrl: 100.0 ± 5.3%; IQ: 118.9 ± 7.5%; n = 12; p < 0.005; ΔN: 89.6 ± 5.8%; n = 12; p < 0.01) and phospho-p54 (Ctrl: 100.0 ± 11.0%; IQ: 117.4 ± 11.6%; n = 12; p < 0.005; ΔN: 81.6 ± 9.9%; n = 12; p < 0.005) JNK in hippocampal CA1 regions expressing BRAG1-IQ and BRAG1-ΔN. Relative amounts of total p46 (Ctrl: 100.0 ± 7.7%; IQ: 101.2 ± 8.2%; n = 12; p = 0.64; ΔN: 98.3 ± 8.1%; n = 12; p = 0.43) and total p54 (Ctrl: 100.0 ± 10.2%; IQ: 107.2 ± 11.0%; n = 12; p = 0.14; ΔN: 101.3 ± 10.2%; n = 12; p = 0.24) JNK in hippocampal CA1 regions expressing BRAG1-IQ and BRAG1-ΔN. Right, Relative amounts of phospho-p38 MAPK (Ctrl: 100.0 ± 9.1%; IQ: 105.1 ± 9.5%; n = 12; p = 0.31; ΔN: 107.9 ± 8.7%; n = 12; p = 0.11) in hippocampal CA1 regions expressing BRAG1-IQ and BRAG1-ΔN. Relative amounts of total p38 MAPK (Ctrl: 100.0 ± 9.9%; IQ: 103.0 ± 10.8%; n = 12; p = 0.64; ΔN: 106.0 ± 10.7%; n = 12; p = 0.94) in hippocampal CA1 regions expressing BRAG1-IQ and BRAG1-ΔN. The relative values and SEs were normalized to average amounts of AMPA and NMDA responses from control CA1 cells, phospho-JNK, phospho-p38 MAPK, total JNK, or total p38 MAPK from control hippocampal CA1 regions. *p < 0.05 (Wilcoxon test).

Figure 10.

BRAG1 signaling regulates synaptic trafficking of GluA1-containing AMPA-Rs. A, Evoked AMPA-R-mediated (−60 mV) and NMDA-R-mediated (+40 mV) responses recorded from neighboring nonexpressing (Ctrl) and mCherry-tagged BRAG1-IQ or BRAG1-ΔN expressing neurons prepared from GluA1 or GluA2 knock-out mice. B, Values for AMPA responses in BRAG1-IQ and BRAG1-ΔN expressing neurons prepared from GluA1 (Ctrl: −23.5 ± 1.8 pA; IQ: −24.1 ± 2.0 pA; n = 20, p = 0.88; Ctrl: −26.2 ± 2.5 pA; ΔN: −26.2 ± 1.5 pA; n = 20, p = 0.82) or GluA2 (Ctrl: −30.9 ± 2.1 pA; IQ: −20.9 ± 1.6 pA; n = 20, p < 0.005; Ctrl: −24.5 ± 1.7 pA; ΔN: −31.5 ± 2.9 pA; n = 24, p < 0.005) knock-out mice, and for NMDA responses in BRAG1-IQ and BRAG1-ΔN expressing neurons prepared from GluA1 (Ctrl: 94.9 ± 7.6 pA; IQ: 91.9 ± 8.4 pA; n = 20, p = 0.91; Ctrl: 113.6 ± 8.7 pA; ΔN: 111.9 ± 9.8 pA; n = 20, p = 0.97) or GluA2 (Ctrl: 54.5 ± 5.0 pA; IQ: 54.8 ± 5.2 pA; n = 20, p = 0.46; Ctrl: 60.1 ± 5.2 pA; ΔN: 61.8 ± 4.4 pA; n = 24, p = 0.63) knock-out mice. The relative values and SEs were normalized to average amounts of AMPA and NMDA responses from control CA1 cells. *p < 0.05 (Wilcoxon test).

Figure 11.

Model for BRAG1-mediated synaptic trafficking of GluA1-containing AMPA-Rs. BRAG1 is anchored at PSDs via its interaction with PSD-95, which also interacts with NMDA receptors. Calcium influx triggered by NMDAR activation causes CaM release from BRAG1, which causes a conformational change leading to enhanced catalytic activity. BRAG1 activates Arf6 which, through downstream activation of JNK, triggers internalization of GluA1-containing AMPA receptors.