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. 2013 Aug 15;591(16):3935-47.
doi: 10.1113/jphysiol.2013.255075. Epub 2013 Jun 17.

SNARE proteins are essential in the potentiation of NMDA receptors by group II metabotropic glutamate receptors

Jia Cheng  1 , Wenhua Liu, Lara J Duffney, Zhen Yan
Affiliations

SNARE proteins are essential in the potentiation of NMDA receptors by group II metabotropic glutamate receptors

Jia Cheng et al. J Physiol. .

Abstract

The group II metabotropic glutamate receptors (group II mGluRs) have emerged as the new drug targets for the treatment of mental disorders like schizophrenia. To understand the potential mechanisms underlying the antipsychotic effects of group II mGluRs, we examined their impact on NMDA receptors (NMDARs), since NMDAR hypofunction has been implicated in schizophrenia. The activation of group II mGluRs caused a significant enhancement of NMDAR currents in cortical pyramidal neurons, which was associated with increased NMDAR surface expression and synaptic localization. We further examined whether these effects of group II mGluRs are through the regulation of NMDAR exocytosis via SNARE proteins, a family of proteins involved in vesicle fusion. We found that the enhancing effect of APDC, a selective agonist of group II mGluRs, on NMDAR currents was abolished when botulinum toxin was delivered into the recorded neurons to disrupt the SNARE complex. Inhibiting the function of two key SNARE proteins, SNAP-25 and syntaxin 4, also eliminated the effect of APDC on NMDAR currents. Moreover, the application of APDC increased the activity of Rab4, a small Rab GTPase mediating fast recycling from early endosomes to the plasma membrane, and enhanced the interaction between syntaxin 4 and Rab4. Knockdown of Rab4 or expression of dominant-negative Rab4 attenuated the effect of APDC on NMDAR currents. Taken together, these results have identified key molecules involved in the group II mGluR-induced potentiation of NMDAR exocytosis and function.

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Figures

Figure 1
Figure 1. Activation of Group II mGluRs increases NMDAR currents and NMDAR surface expression and synaptic localization
A, plot of peak NMDAR currents as a function of time and drug application in cultured cortical pyramidal neurons. APDC (50 μm): a selective group II mGluR agonist; LY341495 (0.5 μm): a selective group II mGluR antagonist. B, representative current traces taken from the recordings used to construct panel A (at time points denoted by #). Scale bars: 0.1 nA, 1 s. C, cumulative data (mean ± SEM) showing the percentage modulation of NMDAR currents by APDC in the absence or presence of LY341495. *P < 0.001, ANOVA. D–F, immunoblots (D) and quantitative analysis (E and F) of the surface and total NMDAR subunits in cortical slices treated without (con) or with APDC (50 μm, 10 min). *P < 0.001, **P < 0.01, t test. G, immunocytochemical images and quantitative analysis of surface NR1 in cortical cultures treated without (control) or with APDC (50 μm, 10 min). Enlarged versions of the boxed regions of dendrites are shown beneath each of the images. Surface NR1 cluster density, size and fluorescence intensity were analysed. *P < 0.05, t test. H, immunocytochemical images and quantitative analysis (cluster size) of total NR1 clusters (red puncta), PSD-95 clusters (green puncta) and synaptic NR1 (PSD-95 colocalized, yellow puncta) along dendrites of cortical cultures treated without or with APDC (50 μm, 10 min). *P < 0.05, t test.
Figure 2
Figure 2. Inhibiting the SNARE protein SNAP-25 blocks group II mGluR-induced enhancement of NMDAR currents
A, plot of normalized peak NMDAR currents showing the effect of APDC (50 μm) in neurons dialysed without or with botulinum toxin (BoTx, 0.5 μm). B, representative current traces taken from the records used to construct panel A (at time points denoted by #). Scale bars: 0.1 nA, 1 s. C, cumulative data (mean ± SEM) showing the percentage modulation of NMDAR currents by APDC in the absence or presence of BoTx. *P < 0.01, t test. D, plot of normalized peak NMDAR currents showing the effect of APDC (50 μm) in neurons dialysed with a scrambled peptide (1 mm), a SNAP-25 blocking peptide (1 mm) or a SNAP-23 blocking peptide (1 mm). E, representative current traces taken from the records used to construct D (at time points denoted by #). Scale bars: 0.1 nA, 1 s. F, cumulative data (mean ± SEM) showing the percentage modulation of NMDAR currents by APDC in the presence of various peptides. *P < 0.01, ANOVA.
Figure 3
Figure 3. Knockdown of syntaxin 4 blocks group II mGluR-induced enhancement of NMDAR currents
A, immunocytochemical staining of Stx4 in cultured cortical neurons co-transfected with syntaxin 4 shRNA (Stx4 shRNA) and GFP. B, plot of normalized peak NMDAR currents showing the effect of APDC (50 μm) in neurons transfected with GFP or Stx4 shRNA. C, representative current traces taken from the records used to construct panel B (at time points denoted by #). Scale bars: 0.1 nA, 1 s. D, cumulative data (mean ± SEM) showing the percentage modulation of NMDAR currents by APDC in neurons with different transfections. *P < 0.01, t test.
Figure 4
Figure 4. Rab4 is required for APDC enhancement of NMDAR currents
A, plot of normalized peak NMDAR currents showing the effect of APDC (50 μm) in neurons transfected with a scrambled siRNA or Rab4 siRNA. B, representative current traces taken from the records used to construct panel A (at time points denoted by #). Scale bars: 0.1 nA, 1 s. C, cumulative data (mean ± SEM) showing the percentage modulation of NMDAR currents by APDC in neurons transfected with a scrambled siRNA, Rab4 siRNA or dominant-negative Rab4 (DN-Rab4). *P < 0.01, ANOVA. D, representative co-immunoprecipitation blots showing the effect of APDC (50 μm, 10 min) on active (Rabaptin5-bound) Rab4 in cortical slices pretreated without or with the mGluR2/3 antagonist LY341495 (0.5 μm, 10 min). E, quantitative analysis (mean ± SEM) showing the level of active (Rabaptin5-bound) Rab4 with different treatments. *P < 0.01, ANOVA. F, representative co-immunoprecipitation blots showing the effect of APDC (50 μm, 10 min) on the interaction between syntaxin 4 and Rab4 in cortical slices. G, quantitative analysis (mean ± SEM) showing the level of Rab4-bound syntaxin 4 in control vs. APDC-treated cortical slices. *P < 0.01, t test.
Figure 5
Figure 5. Schematic diagram showing the potential mechanism underlying the regulation of NMDARs by group II mGluRs
Activation of group II mGluRs increases Rab4 activity and the interaction between Rab4 and syntaxin 4, facilitating the formation of SNARE complexes composed of SNAP-25, syntaxin 4 and VAMP at postsynaptic sites, which leads to the increased exocytosis of NMDARs. Consequently, NMDAR surface expression and synaptic function are up-regulated by the activation of group II mGluRs.
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