A negative feedback loop controls NMDA receptor function in cortical interneurons via neuregulin 2/ErbB4 signalling

Abstract

The neuregulin receptor ErbB4 is an important modulator of GABAergic interneurons and neural network synchronization. However, little is known about the endogenous ligands that engage ErbB4, the neural processes that activate them or their direct downstream targets. Here we demonstrate, in cultured neurons and in acute slices, that the NMDA receptor is both effector and target of neuregulin 2 (NRG2)/ErbB4 signalling in cortical interneurons. Interneurons co-express ErbB4 and NRG2, and pro-NRG2 accumulates on cell bodies atop subsurface cisternae. NMDA receptor activation rapidly triggers shedding of the signalling-competent NRG2 extracellular domain. In turn, NRG2 promotes ErbB4 association with GluN2B-containing NMDA receptors, followed by rapid internalization of surface receptors and potent downregulation of NMDA but not AMPA receptor currents. These effects occur selectively in ErbB4-positive interneurons and not in ErbB4-negative pyramidal neurons. Our findings reveal an intimate reciprocal relationship between ErbB4 and NMDA receptors with possible implications for the modulation of cortical microcircuits associated with cognitive deficits in psychiatric disorders.

Figure 1. Pro-NRG2 protein accumulates atop subsurface cisternae in ErbB4-positive interneurons.

(a) Double-label fluorescence ISH of NRG2 and ErbB4 in the mouse hippocampus, showing overlapping signals in a neuron located in the stratum oriens of area CA1. The pyramidal cell layer is visible in the lower right corner. DAPI was added to label nuclei (blue). Note that ErbB4-negative cells have much lower or no NRG2 signal. Magnified areas on the right outlined by boundary box. (b) Immunogen sites in NRG2 used to raise poly- and monoclonal NRG2 antibodies. Ig-like and EGF-like domains in the ECD, the transmembrane (TM) and ICD are shown. (c) NRG2 immunoreactivity in ErbB4⁺ interneurons in rat CA1 strata pyramidale and radiatum. (d) Somatodendritic NRG2 surface puncta on a DIV 28 cultured ErbB4⁺ hippocampal neuron. (e) Immunogold electron micrograph of a DIV 35 hippocampal neuron with a patch of concentrated label for NRG2 (black particles depict silver-enhanced signals) at the plasma membrane atop intracellular membrane stacks characteristic of SSCs. Asterisks mark the lumen of the open cisterns that are continuous and surround the flattened stacks of the specialized endoplasmic reticulum (ER) membranes (circle) in the centre of the SSC. Astroglial processes (astro, identified by a gap junction (GJ) between two processes) are often closely associated with SSCs. (f) Super-resolution microscopy image of a 2-month old hippocampal neuron double-labelled for NRG2 and Kv2.1. Magnified areas illustrate how NRG2 frequently resides in the centre of doughnut-shaped Kv2.1 clusters. (g) Immunofluorescence signals obtained with ECD antibody 8D11 (surface-labelled) and ICD antibody mAB11 exhibit extensive overlap, suggesting that puncta consist of unprocessed pro-NRG2. (h,i) The ∼110 kDa pro-NRG2 protein is enriched in SMs and partitions with the Triton X-100 (TX-100) insoluble fraction in the rat cortex (Ctx), hippocampus (Hc) and cerebellum (Cb). NRG2 was detected with polyclonal antibody 1349 against the carboxyl terminus. M, molecular mass standards; S1, crude extract; P2, crude membranes; SM, synaptosomal membranes; Sol, detergent-soluble; Ins, detergent-insoluble. Scale bars, 10 μm (a,c), 5 μm (d,f,g and inset in a), 1 μm (inset in f), 0.1 μm (e). Image shown in a is representative of two experiments, micrographs in c–g are representative of at least three replicates.

Figure 2. Activity and glutamate dynamically regulate NRG2 puncta.

(a–c) Effect of chronic and acute burst firing on NRG2 puncta. (a) Representative confocal images of NRG2 puncta on cultured ErbB4⁺ interneurons in untreated controls, and after 12 h of Bic/4AP without or with the NMDAR inhibitor AP5. (b,c) Mean NRG2 puncta intensity (AU) and size (μm²) in ErbB4⁺ neurons. (d–f) Glutamate downregulates NRG2 puncta in an NMDAR-dependent manner. (d) Representative confocal images of NRG2 puncta on ErbB4⁺ interneurons, either untreated or after 10 min of 20 μM glutamate without or with 50 μM AP5. (e,f) Mean NRG2 puncta intensity (AU) and size (μm²) in ErbB4⁺ neurons. In both experiments, n=30 neurons from three independent experiments were analysed for each data point. Data represent the mean±s.e.m. *P<0.05; **P<0.01; ****P<0.0001 (one-way analysis of variance). Scale bars, 5 μm. Ctrl, control; Glu, glutamate.

Figure 3. Analysis of glutamate-mediated NRG2 downregulation by live-cell imaging.

(a) Schematic of the AAV construct to express Venus-NRG2 from the neuron-selective human Synapsin I promoter. (b,d) Representative images of live neurons expressing Venus-NRG2, 3 min before (left) and 6 min after the onset of 20 μM glutamate treatment in the absence (b) or presence of 50 μM AP5 (d). (c,e) Line graphs illustrating the time course of fluorescence intensities for neurons shown on the left. (f) Summary analysis of the effects of glutamate acting via NMDARs on Venus-NRG2 fluorescence. Fluorescence intensities are normalized to baseline (−3 min). N=17 (Ctrl), 18, (Glu) and 17 (Glu+AP5) neurons from three independent experiments. Data represent the mean±s.e.m. ****P<0.0001 (one-way analysis of variance). Scale bar, 5 μm. Ctrl, control; Glu, glutamate.

Figure 4. NMDAR activation triggers NRG2 ectodomain shedding.

(a) Pro-Venus-NRG2 in whole-cell lysates of DIV 21 hippocampal neurons, stimulated for 20 min with 20 μM glutamate in the absence or presence of 50 μM AP5. Kv2.1 protein is shown to illustrate the characteristic downward shift in electrophoretic mobility in response to glutamate. Clathrin heavy chain (CHC) is included as a loading control. (b) Quantitative analysis of pro-Venus-NRG2 downregulation by glutamate. Data are normalized to untreated controls. N=13 (Ctrl), 12 (Glu) and 8 (Glu+AP5) from six independent experiments. (c,d) Glutamate triggers NMDAR- and alpha-secretase-dependent NRG2 ectodomain shedding. Western blot in (c) shows Venus-NRG2 ectodomain protein in conditioned medium (CM), after replacement of CM with CM from uninfected cells, and after 20 min of 20 μM glutamate. Dependence of ectodomain shedding on NMDAR- and alpha-secretase activity revealed by pretreatment of cells with 50 μM AP5 and 10 μM GM6001, respectively. By contrast, ectodomain shedding was not affected by the inclusion of the beta-secretase inhibitor BACE-IV (1 μM). (d) Quantitative analysis of Venus-NRG2 ectodomain. Data are normalized to glutamate. N=3 independent experiments. Data represent the mean±s.e.m. **P<0.01; ****P<0.0001 (one-way analysis of variance). Ctrl, control; Glut (or Glu), glutamate.

Figure 5. NRG2 promotes the association of ErbB4 with GluN2B-containing NMDARs.

Proteins interacting with ErbB4 were isolated from metabolizing rat brain SMs following stimulation with 10 nM NRG2, and analysed by tandem MS (see Methods section for details). (a) Left, Coomassie-stained SDS–PAGE showing proteins obtained after ErbB4 immunoprecipitation from unstimulated control (C) and NRG2-stimulated membranes (N). Right, western blot of the same samples showing tyrosine-phosphorylated proteins. M, marker lane. Areas excised for MS are marked by arrows (see Supplementary Table 1 for a complete list of identified proteins). (b) ErbB4 stimulation by NRG2 (10 nM for 10 min) markedly increases association with GluN2B in metabolizing SMs from the rat cortex. IgG, antibody heavy chain signal. (c) NRG2 also promotes the association of ErbB4 with GluN2B, and to a lesser extent with GluN2A, in cultured hippocampal neurons (>DIV 28). Note that ErbB4 interactions with the AMPAR subunit GluA1 are not affected by NRG2. Western blot of NRG2 included to ascertain binding of ErbB4 in stimulated membranes. (d) Summary analysis of results shown in c. Data represent the mean±s.e.m. of densitometric signal ratios of glutamate receptor subunits over ErbB4, normalized to unstimulated controls (set as 100%). N=4 from three independent experiment; *P<0.05 (Student's paired t-test).

Figure 6. NRG2 promotes the internalization of GluN2B-containing NMDARs and the downregulation of whole-cell NMDAR currents in cultured ErbB4⁺ interneurons.

(a–d) The effect of NRG2 on surface NMDAR expression was investigated in hippocampal neurons transfected with GFP-tagged GluN2A and GluN2B, and stimulated with NRG2 as described in Fig. 5. Surface NMDARs were labelled under non-permeabilizing conditions with anti-GFP (red), and surface+internal NMDARs were assessed using native GFP fluorescence (green). Cells were additionally labelled for ErbB4 (not shown) and nuclei were visualized with DAPI. (a,b) Representative images of total and surface GluN2B and GluN2A expression in vehicle and NRG2-stimulated ErbB4⁺ interneurons. Scale bars, 25 μm. (c,d) Quantitative analysis of relative surface expression of GFP-GluN2B (c) and GFP-GluN2A (d), expressed as arbitrary ratios of surface over total GFP fluorescence intensities. Data are mean±s.e.m. ****P<0.0001, N=6–12 neurons (Student's t-test). (e–h) Effect of NRG2 (10 nM) on whole-cell NMDAR currents in >DIV 28 ErbB4⁺ and ErbB4⁻ hippocampal neurons. Currents were isolated by tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and picrotoxin at +40 mV in the presence of 100 μM NMDA delivered via a Y-tube. (e) Representative image of a recorded ErbB4⁺ interneuron identified by post hoc labelling. Scale bar, 25 μm. (f,g) Representative traces of whole-cell NMDA currents before (black), and after (red), 10 min perfusion with 10 nM NRG2 in ErbB4⁺ (f) and ErbB4^- neurons (g). Duration of NMDA treatments denoted by corresponding black and red horizontal bars. Note that the left and right tails of the NMDA-elicited currents mark the beginning and end of agonist delivery via the Y-tube; the jitter at the end of each trace reflects a perfusion artifact. Scale bars, 5 s and 500 pA. (h) Summary analysis of NRG2 effects on NMDA currents. Data are relative to baseline (set as 100%) and represent the mean±s.e.m. N=5 ErbB4⁺ and 4 ErbB4⁻ neurons; ***P<0.001% (Student's t-test).

Figure 7. NRG2 acutely downregulates evoked NMDAR currents in cortical ErbB4⁺ interneurons but not pyramidal neurons.

(a) Representative confocal image of an aspiny, biocytin-filled ErbB4⁺ interneuron from the PFC of an Erbb4-2A-CreERT2 X Ai14 mouse used to record evoked NMDA and AMPA receptor currents. (b) Corresponding image of a biocytin-filled spiny PFC pyramidal neuron from a wild-type C57Bl/6J mouse. Scale bars, 25 μm. (c–f) Representative scatter plots of NMDA and AMPA receptor-mediated evoked excitatory postsynaptic currents (eEPSCs) recorded from an ErbB4⁺ interneuron (c,e) and from a pyramidal neuron (d,f). Duration of NRG2 treatment (10 nM) is indicated by horizontal bars. Arrows indicate the time points at which sample traces shown in the upper right corner were taken before (black) and after (red) NRG2. Scale bars in sample traces, 75 pA and 75 ms. (g,h) Summary plots of NRG2 effects on NMDA and AMPA eEPSCs in ErbB4⁺ interneurons (g) and pyramidal neurons (h). Data are normalized to baseline (set as 100%). N=6 (ErbB4⁺), 4 (pyramidal neurons). **P<0.01 (Student's paired t-test).

Figure 8. Reciprocal signalling between NMDA and NRG2/ErbB4 receptor systems.

Working model illustrating the intricate relationship between NMDARs and NRG2/ErbB4 signalling in cortical interneurons. At baseline, low glutamate concentrations and weak NMDAR activity permit the accumulation of pro-NRG2 as highly concentrated clusters atop subsurface cisternae. Sudden increases in glutamate, acting via synaptic and/or extrasynaptic NMDARs, promote the rapid dispersal of NRG2 puncta and the shedding of the NRG2 ectodomain by extracellular proteases. The NRG2 ectodomain then accumulates locally by binding to heparan sulfate proteoglycans. Dissociation from the extracellular matrix, possibly aided by additional proteolytic processing events as suggested for NRG1 (ref. 58), enables NRG2 to bind its cognate receptor ErbB4. In this manner, autocrine NRG2/ErbB4 signalling initiates downstream events, including the internalization of GluN2B-containing NMDARs themselves. Based on the NRG2-dependent augmentation of ErbB4-GluN2B interactions in detergent-soluble cortical membranes fractions, surface NMDAR internalization experiments and our prior study on the regulation of GABAa1 receptor surface expression by NRG2/ErbB4 (ref. 16), we hypothesize that NMDAR internalization occurs primarily at extrasynaptic sites. Reduction of NMDAR EPSCs may result from a net loss of synaptic receptors due to lateral diffusion out of the synapse combined with removal of extrasynaptic/perisynaptic receptors.