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. 2016 Mar 24:7:11080.
doi: 10.1038/ncomms11080.

Bidirectional regulation of synaptic transmission by BRAG1/IQSEC2 and its requirement in long-term depression

Joshua C Brown  1 , Amber Petersen  1 , Ling Zhong  1 , Miranda L Himelright  2 , Jessica A Murphy  2 , Randall S Walikonis  2 , Nashaat Z Gerges  1
Affiliations

Bidirectional regulation of synaptic transmission by BRAG1/IQSEC2 and its requirement in long-term depression

Joshua C Brown et al. Nat Commun. .

Abstract

Dysfunction of the proteins regulating synaptic function can cause synaptic plasticity imbalance that underlies neurological disorders such as intellectual disability. A study found that four distinct mutations within BRAG1, an Arf-GEF synaptic protein, each led to X-chromosome-linked intellectual disability (XLID). Although the physiological functions of BRAG1 are poorly understood, each of these mutations reduces BRAG1's Arf-GEF activity. Here we show that BRAG1 is required for the activity-dependent removal of AMPA receptors in rat hippocampal pyramidal neurons. Moreover, we show that BRAG1 bidirectionally regulates synaptic transmission. On one hand, BRAG1 is required for the maintenance of synaptic transmission. On the other hand, BRAG1 expression enhances synaptic transmission, independently of BRAG1 Arf-GEF activity or neuronal activity, but dependently on its C-terminus interactions. This study demonstrates a dual role of BRAG1 in synaptic function and highlights the functional relevance of reduced BRAG1 Arf-GEF activity as seen in the XLID-associated human mutations.

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Figures

Figure 1
Figure 1. BRAG1 enhances AMPA receptor-mediated responses independently of its enzymatic activity.
(a–d) Left: sample traces of AMPAR- and NMDAR-mediated synaptic responses recorded at −60 and+40 mV (the amplitude at 60 ms latency, after AMPAR EPSCs are decayed), respectively. Scale bars, 20 pA, 20 ms. Data represent averaged evoked EPSCs recorded for AMPAR (left graphs), NMDAR (middle graphs) and AMPA/NMDA ratio (right graphs) simultaneously from pairs of untransfected (control) CA1 neurons and neurons transfected with BRAG1-Q801P (a; AMPA: n=11, P=0.0050; NMDA: n=8, P=0.17; AMPA/NMDA: n=8, P=0.019), BRAG1-E849K (b; AMPA: n=15, P=0.0079; NMDA: n=11, P=0.55; AMPA/NMDA: n=11, P=0.0063), BRAG1 (c; AMPA: n=18, P=0.00034; NMDA: n=18, P=0.40; AMPA/NMDA: n=14, P=0.0019) or GFP-tagged BRAG1 (d; AMPA: n=10, P=0.010; NMDA: n=9, P=0.92; AMPA/NMDA: n=8, P=0.013). * Indicates significance (P≤0.05). (e) Confocal images of GFP-BRAG1 co-expressed with td-Tomato in hippocampal CA1 neurons (left; scale bars, 10 μm) and their dendritic distribution (right; scale bars, 1 μm) showing preferential localization of BRAG1 at dendritic spines.
Figure 2
Figure 2. BRAG1-mediated enhancement of synaptic transmission depends on its C-terminus interactions.
(a,b) BRAG1's C terminus interacts with PSD-95 in vitro. (a) Protein overlay assay demonstrating the interaction between WT BRAG1 and recombinant PSD-95. Top: HEK293 cell lysates expressing FLAG-WT BRAG1, FLAG-BRAG1 C-terminal deletion mutant (FLAG-BRAG1ΔCt) or no plasmid (untransfected) were run on the gel, and the membrane was overlaid with recombinant GST-PSD-95, and stained with antiserum UCT80 against PSD-95. Middle and bottom: the blot was also probed with anti-FLAG to detect total BRAG1 in the lysates and with anti-tubulin as a loading control. (b) Left: GST-PSD-95 and GST were separated by SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, which was overlaid with a HEK293 cell lysate overexpressing FLAG-WT BRAG1 and stained with antiserum C3 against BRAG1. BRAG1 binds to GST-PSD-95, but not GST (indicated by the *). Right: protein overlay assay with FLAG-BRAG1ΔCt. GST-PSD-95 and GST were run on the gel, and the membrane was overlaid with a HEK293 cell lysate overexpressing FLAG-BRAG1 ΔCt and stained with anti-FLAG to detect BRAG1. WT BRAG1 but not BRAG1ΔCt binds to GST-PSD-95. Molecular weight markers in kDa are shown on the left. (c–e) Insets: sample traces of AMPAR- and NMDAR-mediated synaptic responses recorded at −60 (left) and +40 mV (right), respectively. Scale bars, 20 pA, 20 ms. Data represent averaged evoked EPSCs recorded for AMPA (left graphs), NMDA (centre graphs) and AMPA/NMDA ratios (right graphs) simultaneously from pairs of untransfected (control) CA1 neurons and neurons transfected with BRAG1-ΔCt (c; AMPA: n=18, P=0.24; NMDA: n=18, P=0.24; AMPA/NMDA: n=15, P=0.12) or BRAG1 (d,e). In d and e, shortly after BRAG1 transfection, TAT-BRAG1-Ct peptide (d; AMPA: n=10, P=0.67; NMDA: n=7, P=0.25; AMPA/NMDA: n=7, P=0.97) or TAT-Ct control peptide (e; AMPA: n=7, P=0.031; NMDA: n=8, P=0.42, AMPA/NMDA: n=7, P=0.047) was added to the culture media. * Indicates significance (P≤0.05).
Figure 3
Figure 3. Expression of BRAG1, but not BRAG2, lacking the PDZ-binding sequence interfere with BRAG1-mediated enhancement of synaptic transmission.
(a,b) Insets: sample traces of AMPAR- and NMDAR-mediated responses recorded at −60 and +40 mv, respectively. Scale bars, 20 pA, 20 ms. Data represent averaged evoked EPSCs recorded for AMPA (left graphs), NMDA (centre graphs) and AMPA/NMDA ratios (right graphs) simultaneously from pairs of untransfected (control) CA1 neurons and neurons transfected with BRAG1+BRAG1-ΔCt (a; AMPA: n=11, P=0.65; NMDA: n=11, P=0.34; AMPA/NMDA: n=11, P=0.08) or BRAG1+BRAG2-ΔCt (b; AMPA: n=8, P=0.0063; NMDA: n=8, P=0.28; AMPA/NMDA: n=8, P=0.05). * Indicates significance (P≤0.05).
Figure 4
Figure 4. BRAG1 enhances synaptic strength independently of synaptic activity or GluA1 insertion.
(a,b) Insets: sample traces of AMPAR- and NMDAR-mediated synaptic responses recorded at −60 (left) and +40 mV (right), respectively. Scale bars, 20 pA, 20 ms. Data represent averaged evoked EPSCs recorded for AMPA (left graphs), NMDA (middle graphs) and AMPA/NMDA ratios (right graphs) simultaneously from pairs of nearby untransfected and BRAG1-transfected CA1 neurons treated overnight with TTX (a; AMPA: n=8, P=0.013; NMDA: n=8, P=0.51; AMPA/NMDA: n=7, P=0.020) or APV (b; AMPA: n=9, P=0.023; NMDA: n=6, P=0.64; AMPA/NMDA: n=6, P=0.031). (c) Insets: sample traces of evoked AMPAR-mediated synaptic responses recorded at −60 and +40 mV from control cells or cells co-transfected with BRAG1 and GluA1. Scale bars, 20 pA, 20 ms. Left graph shows AMPAR-mediated responses recorded at −60 mV (n=14, P=0.027). Right graph shows the rectification index, which was calculated as the ratio of the amplitude of AMPAR-mediated responses at −60 mV to that at +40 mV (n=13, P=0.39). * Indicates significance (P≤0.05).
Figure 5
Figure 5. BRAG1 increases the synaptic pool of AMPA receptors that constitutively recycle.
(a) Graph of representative time-course experiment of whole-cell double patch-clamp recordings from a control neuron and a neuron transfected with BRAG1. In these experiments, patch pipettes contain 10 μM pep2m in the internal solution. Insets: traces of AMPAR-mediated responses of the representative experiment at baseline (thin line, left) and at the new steady state following pep2m infusion (thick line, right). Scale bars, 20 pA, 20 ms. (b) Averaged EPSC values from adjacent transfected and control cells. Time zero indicates the time at which neurons were patch clamped at −60 mV. Note that immediately following patch clamping, BRAG1-transfected neurons have higher AMPAR-mediated responses. Pep2m caused rundown in BRAG1-transfected neurons to a higher degree than in control ones. (c) Series resistance values corresponding to EPSC values over time (control versus BRAG1, 0–55 min, n=8, P=0.51). (d) Quantification of average EPSC values of baseline (up to 12 min, n=8, P<0.05) and steady state following peptide infusion (35–55 min, n=8, P=0.56). * Indicates significance (P≤0.05).
Figure 6
Figure 6. BRAG1 increases surface GluA2 but not GluA1.
(a) Left: Data represent the average normalized spine-to-dendrite ratios of SEP-GluA2 in the presence and absence of BRAG1. Right: representative confocal images of SEP-GluA2 and td-Tomato co-expressed with either pCAGGS (left, n=114) or pCAGGS-BRAG1 (right, n=73). (b) Left: data represent the average normalized spine-to-dendrite ratios of SEP-GluA1 in the presence and absence of BRAG1. Right: representative confocal images of SEP-GluA1and td-Tomato co-expressed with either pCAGGS (left, n=102) or pCAGGS-BRAG1 (right, n=102). * Indicates significance (P≤0.05). Scale bars, 1 μm.
Figure 7
Figure 7. BRAG1 is required for the maintenance of synaptic transmission.
(a) Representative confocal images from organotypic hippocampal slices transfected with pSuper plasmid containing BRAG1-siRNA and expressing GFP (to visualize the transfected cells) and immunostained 2 days later for BRAG1 (red signal) under permeabilized conditions. Note, the cell transfected with the BRAG1-siRNA (green cell) has low levels of endogenous BRAG1 (red signal). Scale bars, 10 μm. (b) Representative line plot analysis of BRAG1 (red signal) and GFP (green signal). CB1 is a cell body of non-transfected neuron; CB2 is that of a transfected neuron. (c) Normalized intensity from the line plots for endogenous BRAG1 for the three different RNAi used; RNAi significantly (P≤0.05) decreased BRAG1 levels. RNAi #1: n=14, P=0.0112; RNAi #2: n=7, P=0.0132; RNAi #3: n=10, P=0.0417. (d,e) Simultaneous whole-cell double recordings from nearby pairs of untransfected (control) neurons and those transfected with either BRAG1-siRNA#2 (d) or BRAG1-siRNA#3 (e). Left graph: comparisons of evoked AMPAR-mediated responses. Right graph: simultaneous recordings of evoked NMDAR-mediated responses (P=0.21). (f) RNAi #3 was co-expressed with RNAi-resistant WT BRAG1 to restore BRAG1 levels. Data represent average AMPAR EPSCs (left; n=10, P=0.15), NMDAR EPSCs (middle; n=8, P=0.51) and AMPA/NMDA ratios (right; n=8, P=0.60). * Indicates significance (P≤0.05).
Figure 8
Figure 8. BRAG1–PDZ-binding sequence, but not its Arf-GEF enzymatic activity, is required for the maintenance of synaptic transmission.
(a,b) Left: sample traces of AMPAR- and NMDAR-mediated responses recorded at −60 and +40 mv, respectively. Scale bars, 20 pA, 20 ms. Data represent averaged evoked EPSCs recorded for AMPA (left graphs), NMDA (centre graphs) and AMPA/NMDA ratios (right graphs) simultaneously from pairs of untransfected (control) CA1 neurons and neurons transfected with BRAG1 RNAi+BRAG1-Q801P (a) or BRAG1 RNAi+BRAG1-ΔCt (b). * Indicates significance (P≤0.05).
Figure 9
Figure 9. BRAG1 is not required for LTP.
(a) LTP was induced by pairing 3 Hz presynaptic stimulation (300 pulses) with 0 mV postsynaptic depolarization (indicated by an arrow) in CA1 neurons while expressing BRAG1 (black circles, n=7, P=0.96), BRAG1 RNAi (grey circles, n=5, P=0.86) or untransfected neurons (white circles, n=19). Insets: sample traces of evoked AMPAR-mediate responses recorded at −60 mV before pairing (thin line) and 20 min after pairing (thick line) from control or transfected cells as indicated. Scale bars, 20 pA, 20 ms. (b) Normalized averaged steady-state AMPAR-mediated responses in paired (LTP induction) and control (unpaired pathway).
Figure 10
Figure 10. Arf-GEF activity and PDZ-binding sequence are required for LTD.
LTD was induced by pairing a low-frequency stimulation (500 pulses at 1 Hz) with −40 mV postsynaptic depolarization (indicated by a dark line) in untransfected CA1 neurons or neurons transfected with BRAG1-E849K (a; n=9), BRAG1-Q801P (a; n=8), wild-type BRAG1 (c; n=7), BRAG1 RNAi (e; n=7), BRAG1 RNAi+wild-type BRAG1 (e; n=9) or BRAG1 RNAi+BRAG1-ΔCt (g; n=6). Insets: sample traces of evoked AMPAR-mediated synaptic responses recorded at −60 mV before pairing (thin line) and 20 min after pairing (thick line) from control or transfected cells as indicated. Scale bars, 20 pA, 20 ms. (b,d,f,h) Normalized average steady-state AMPAR-mediated responses in paired (LTD induction) and control (unpaired pathway). * Indicates significance (P≤0.05).
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