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Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity

Abstract

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca2+-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg2+ or a Ca2+-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

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Figure 1: NMDA-induced dendritic blebbing is differentially blocked by noncompetitive and competitive antagonists.
Figure 2: Disrupting NMDA ligand binding blocks the secondary current.
Figure 3: NMDA-induced ionic dysregulation occurs during pore block by physiological Mg2+.
Figure 4: NMDAR-Src-Panx1 colocalize in a metabotropic signaling complex.
Figure 5: Activation of NMDAR-Src-Panx1 signaling during excitotoxicity requires phosphorylation of Src and Panx1.
Figure 6: Disrupting metabotropic NMDAR signaling prevented Panx1-mediated Ca2+ dysregulation, MPT and neuronal death during in vitro ischemia.
Figure 7: Blocking the NMDAR metabotropic signalsome reduces lesion size and sensorimotor deficits induced by stroke in vivo.

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Acknowledgements

We thank J. Bains (University of Calgary) for critical reading of the manuscript and C. Sank for technical assistance. Plasmids encoding Panx1Y308F, GluN1WT and GluN1N616R were generated by F. Visser in the Hotchkiss Brain Institute Molecular Core Facility. Dominant active/inactive constructs for Src kinase and Src48 peptides were from D. Fujita (University of Calgary) and M. Salter (Sick Kids Research Institute), respectively. Myc-tagged Panx1 constructs were from C. Naus (University of British Columbia). This work was supported by Canadian Institutes for Health Research (CIHR) grants 136812 (R.J.T.), 110967 (I.R.W.), 201022 (M.A.C.) and 130495 (G.C.T.). R.J.T., G.C.T., M.A.C. and I.R.W. received grants from Alberta Innovates–Health Solutions (AI-HS), the Canadian Foundation for Innovation and the Natural Sciences and Engineering Research Council of Canada. R.J.T. and I.R.W. received funds from AI-HS. Additional support was provided to R.J.T. by the Cumming School of Medicine via the Ronald and Irene Ward Foundation and the Gwendolyn McLean Fund. N.L.W. is supported by an AI-HS scholarship and Dr. T. Chen Fong scholarship from the Hotchkiss Brain Institute. A.W.L. is supported by AI-HS and CIHR.

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Contributions

N.L.W., A.W.L. and R.J.T. designed the experiments. N.L.W. performed and analyzed the electrophysiological and laser-imaging experiments, and wrote the initial draft of the paper that all authors then edited. B.D.R. designed, performed and analyzed the in vivo surgeries, lesion volumes and behavioural tests. A.W.L. performed and analyzed the molecular biology and biochemistry experiments. E.M.M.M. performed and analyzed the calcein dye-efflux experiments. J.B. analyzed behavioural recordings (in a blinded fashion). V.M. and T.R. performed the initial characterization of the phospho-specific pY308Panx1 antibody. M.V.B., N.T.I. and I.R.W. performed and analyzed in vivo cerebral blood flow imaging experiments. L.S. and M.A.C. assisted with the development and maintenance of primary cell cultures. G.C.T. supervised the in vivo stroke experiments. R.J.T. supervised the study and wrote the paper with contributions from all authors.

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Correspondence to Roger J Thompson.

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Integrated supplementary information

Supplementary Figure 1 Acute Block of Panx1 does not inhibit NMDAR currents.

a) Raw sample traces (data from Figure 2) illustrating the transient NMDAR response. Inward currents were recorded in voltage-clamp in response to 100 μM NMDA. Peak current amplitude was measured as the first peak following the acute NMDAR response. b) Application of antagonists for Panx1, 10panx and TAT-Panx308 did not attenuate the peak inward current compared to their respective inactive controls (SCpanx and TAT-PanxSC) or MK-801 alone.

Supplementary Figure 2 Activation of Excitotoxic Panx1 currents requires both ligand binding sites of NMDARs.

a) Representative whole-cell recordings from neurons in acute brain slices pre-blocked with either CGP-78608 (1μM) or APV (50μM) in 0Mg2+ aCSF. After a 15-minute baseline, the ligand for the unblocked site (100μM NMDA with CGP and 10μM glycine with APV) was applied to test sufficiency for Panx1 activation. Block of either ligand-binding site was sufficient to prevent Panx1 opening. b) Quantitative summary of charge transfer over time between treatment groups. c) Representative 2-photon images of dendrites from patched cells in “a&b”. Dendritic blebbing was not observed when either the glutamate or glycine ligand binding sites were blocked. d&e) Quantifications of changes in dendrite size (swelling) and the formation of dendritic blebs. f) Raw trace for a CGP washout experiment in the presence of NMDA. As an important control, pre-treatment of CGP prior to NMDA stim did not induce internalization of NMDA receptors and excitotoxic currents were present upon CGP washout. g) Average charge accumulation over time after CGP washout. h) Representative images illustrating the dendritic swelling/blebbing following CGP washout. i&j) Quantification of dendritic swelling/blebbing after CGP washout. All data are presented as mean±sem, * = p<0.05.

Supplementary Figure 3 Pre-blocked NMDARs signal metabotropically to Panx1 during excitotoxicity.

To exclude the possibility that Ca2+ influx through unblocked NMDARs was sufficient to recruit Panx1, we pre-blocked with MK-801 prior to excitotoxic NMDA stimulation. a) Transient responses of patched CA1 hippocampal neurons to 0.1 Hz puffed NMDA (0.5 s) are blocked by the un-competitive antagonist MK-801. NMDA was puffed throughout the recording to probe receptor availability during the course of the experiment. Traces are averages from the neuron in B (top trace) prior to the application of 20 μM MK-801 and show that all NMDAR’s were blocked before exposure to excitotoxic (100 μM) NMDA. b) Quantification of 10 μM NMDA puff evoked responses, showing the NMDAR currents were completely blocked by MK-801. c) Bath application of 10 μM NMDA induced an inward current that was completely blocked by MK-801 (note return to baseline). d) Regularly puffing NMDA did not lead to run-down of the NMDAR evoked response in the absence of MK-801 block. (Top panel) representative recording with raw traces (grey) in inset. (Bottom panel) Averaged evoke responses over time. e) The uncompetitive open channel blocker, MK-801 fails to prevent secondary currents induced by 100 μM NMDA (n=9). Timing of agonist and antagonist application is indicated above each trace. Note that 10 μM NMDA puffs (0.1Hz) were maintained throughout the recording to ensure blocked NMDARs. Addition of the competitive NMDAR antagonist, 50 μM APV was sufficient to prevent secondary current activation (middle trace, n=8). The secondary current was blocked by an intracellular antibody, α-Panx1 against the pannexin-1 channel (lower trace, n=6). f) Quantification of the excitotoxic depolarization as total membrane charge transfer (i.e. ionic dysregulation) shows APV significantly reduced ionic dysregulation but MK-801 and boiled α-Panx1 (n=7) are ineffective.

Supplementary Figure 4 GluN1 N616R pore mutation renders NMDARs Ca2+-impermeable.

a) Representative I-V traces from GluN1WT and GluN1N616R transfected N2a cells in normal (2mM) and high (40mM) extracellular Ca2+. Cells were voltage-clamped in whole-cell configuration (Vhold = -60mV), and voltage ramps were applied from -80mV →; +80mV in the presence of NMDA(100μM)/Gly(10μM). Ca2+ permeability was assessed by increasing bath concentrations from 2mM to 40mM. Insets show expression of GluN1WT- and GluN1N616R–mCerulean+ constructs. b) Quantification of average shift in reversal potential in different [Ca2+]o.

Supplementary Figure 5 NMDAR activation induces Panx1 phosphorylation at tyrosine 308.

a) Overlay and intensity line-scan of pY308Panx1 and Myc signals identifying the higher molecule weight species of Panx1 as the preferentially phosphorylated form. b) Phosphatase treatment of NMDA:Gly stimulated N2a cells expressing Panx1WT. Cell lysates were treated with or without lambda phosphatase for 1 hour at 60°C to dephosphorylate Panx1. c) Inhibition of Panx1 phosphorylation at Y308 by the SFK inhibitor PP2 (10µM) in cells co-expressing Panx1WT and SrcY530F. *=p<0.05 compared to non treated cells (lane 1). d) Western blot analysis of Panx1 phosphorylation at Y308 in N2a cells co-expressing Panx1WT or Panx1Y308F and the kinase dead SrcK298M or constitutively active SrcY530F. ** and ***=p<0.01 and 0.005 respectively. e) Immunoprecipitation efficiency for GluN1 in hippocampal slices. Pre=whole cell lysate before IP; Post=remaining fraction of GluN1 in unbound lysate (flow through) following IP. Full length blots are presented in supplementary figure 10.

Supplementary Figure 6 Src mediated phosphorylation of Panx1 is Ca2+-independent.

a&b) Co-immunoprecipitation assays (a) and quantification b) of phosphorylated Src and Panx1 in NMDAR complexes from hippocampal slices treated with excitotoxic NMDA/Glycine (100µM/10µM) in the presence of MK801 (20µM), BAPTA (100µM) or combined MK801/BAPTA application. **=p<0.01, ***=p<0.005 and ****=p<0.001 compared to unstimulated controls (lane 1). Full length blots are presented in supplementary figure 10.

Supplementary Figure 7 TAT-Panx308 is a specific inhibitor of Src-mediated activation of Panx1.

Averaged NMDAR currents in response to 10µM NMDA puff. Recordings were in voltage clamp, holding at -60mV in 10µM glycine / 0mM Mg2+ aCSF. Bath applied TAT-Panx308 (1µM) treatments were compared against the Src inhibitor PP2 (10µM a known de-potentiator of NMDAR activity), and its inactive control PP3 (10µM). Data were quantified as peak amplitude elicited by NMDAR stimulation normalized to baseline. Bath application of 1 μM TAT-Panx308 did not reduce NMDAR currents, suggesting that TAT-Panx308 is not a general antagonist of Src.

Supplementary Figure 8 Panx1 opening in the plasma membrane induces mitochondrial dysfunction during ischemia.

a) Images showing that calcein-green loading is colocalized with mitochondria specific stain, mitotracker red (upper panels). Co-loading of neurons with calcein green and calcein red/orange in the presence of CoCl2 allowed for simultaneous imaging of mPTP as green dye destaining and Panx1 opening as loss of the red dye from the cytosol. b) Families of raw data traces of calcein-green fluorescence showing mitochondrial destaining (decreased fluorescence) following the onset of OGD. c) Mean ± sem for calcein green fluorescence loss during OGD. d) Statistical comparison of mitochondrial destaining at 60 minutes following OGD application. e) Demonstration that the mPTP antagonist did not directly alter efflux of calcein red/orange from the cytosol through Panx1.

Supplementary Figure 9 TAT-Panx308 does not alter cerebral blood flow under physiological conditions.

a) Maximum projection images of cortical cerebral vasculature before and following intraperitoneal injection of 5 mg/kg TAT-Panx308. b) Quantification of changes in arteriole diameter and blood flow velocity following intraperitoneal injection of 5 mg/kg TAT-Panx308.

Supplementary Figure 10 Full length blots cropped for representative figures.

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Weilinger, N., Lohman, A., Rakai, B. et al. Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity. Nat Neurosci 19, 432–442 (2016). https://doi.org/10.1038/nn.4236

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