Ogden KK et al. (2017), Molecular Mechanism of Disease-Associated Mutat...

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PLoS Genet 2017 Jan 17;131:e1006536. doi: 10.1371/journal.pgen.1006536.

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Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology.

Ogden KK, Chen W, Swanger SA, McDaniel MJ, Fan LZ, Hu C, Tankovic A, Kusumoto H, Kosobucki GJ, Schulien AJ, Su Z, Pecha J, Bhattacharya S, Petrovski S, Cohen AE, Aizenman E, Traynelis SF, Yuan H.

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N-methyl-D-aspartate receptors (NMDARs), ligand-gated ionotropic glutamate receptors, play key roles in normal brain development and various neurological disorders. Here we use standing variation data from the human population to assess which protein domains within NMDAR GluN1, GluN2A and GluN2B subunits show the strongest signal for being depleted of missense variants. We find that this includes the GluN2 pre-M1 helix and linker between the agonist-binding domain (ABD) and first transmembrane domain (M1). We then evaluate the functional changes of multiple missense mutations in the NMDAR pre-M1 helix found in children with epilepsy and developmental delay. We find mutant GluN1/GluN2A receptors exhibit prolonged glutamate response time course for channels containing 1 or 2 GluN2A-P552R subunits, and a slow rise time only for receptors with 2 mutant subunits, suggesting rearrangement of one GluN2A pre-M1 helix is sufficient for rapid activation. GluN2A-P552R and analogous mutations in other GluN subunits increased the agonist potency and slowed response time course, suggesting a functionally conserved role for this residue. Although there is no detectable change in surface expression or open probability for GluN2A-P552R, the prolonged response time course for receptors that contained GluN2A-P552R increased charge transfer for synaptic-like activation, which should promote excitotoxic damage. Transfection of cultured neurons with GluN2A-P552R prolonged EPSPs, and triggered pronounced dendritic swelling in addition to excitotoxicity, which were both attenuated by memantine. These data implicate the pre-M1 region in gating, provide insight into how different subunits contribute to gating, and suggest that mutations in the pre-M1 helix can compromise neuronal health. Evaluation of FDA-approved NMDAR inhibitors on the mutant NMDAR-mediated current response and neuronal damage provides a potential clinical path to treat individuals harboring similar mutations in NMDARs.

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Species referenced: Xenopus
Genes referenced: avd grin2a grin2b mapt tf

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	Fig 1. Intolerance analysis of genetic variation across functional domains of GluN1, GluN2A and GluN2B.A, Ribbon structure of homology model of a tetrameric NMDAR and a single GluN subunit [10,12]. B, A cartoon illustrating the domain arrangement of an individual GluN subunit. ATD—amino terminal domain (in GREEN), S1 and S2 –first and second polypeptide sequences comprising the agonist binding domain (ABD, in BLUE), linker regions (S1-M1 linker, M3-S2 linker, and S2-M4 linker; in GRAY), M1, M3, and M4 –transmembrane domains (TM, in ORANGE), M2 –re-entrant pore loop (in ORANGE), and CTD—carboxy-terminal domain (in PINK). C, GRIN1; D, GRIN2A; E, GRIN2B: Sliding window OE-ratio estimates (black full line), Neutrality expected OE-ratio estimate (blue full line), Median OE-ratio for the gene (dark grey dashed line), 25th percentile of OE-ratio (green dashed line), 5th percentile of OE-ratio (red dashed line).
	Fig 2. Locations of pre-M1 mutations.A, Domain architecture of NMDARs and protein sequence alignment showing pre-M1 helix across NMDAR subunits. B-D, Ribbon structure of homology model of GluN1/GluN2A built from GluN1/GluN2B [10,12]. Five residues harboring mutations in human patients are highlighted: GluN1-D552E (in GOLD), GluN1-P557R (in LIGHT BLUE), GluN2A-A548T (in BLUE), GluN2A-P552R (in MAGENTA), and GluN2B-P553L (in MAGENTA). The mutation labels refer to GluN1 as N1 and GluN2 as N2. ATD—amino terminal domain, S1 and S2 –first and second polypeptide sequences comprising the agonist binding domain (ABD), M1, M3, and M4 –transmembrane helices, and M2 –re-entrant pore loop. The mutation information is given in Table 2.
	Fig 3. Pre-M1 mutations affect current amplitudes and NMDAR surface expression.A,B, Summary of current amplitudes evoked by 1 mM glutamate and 100 μM glycine in whole cell voltage clamp recordings from HEK293 cells expressing human NMDARs (holding at -60 mV). p < 0.05, see Table 3 and S4 Table for statistical analyses. The graph legends refer to GluN1 as N1 and GluN2 as N2. C-F, The surface proteins of HEK293 cells transiently expressing WT or mutated human NMDARs were labeled with biotin and pulled down with avidin-conjugated beads. The total and surface protein fractions were immunoblotted for GluN1, GluN2A or GluN2B, transferrin receptor (TfR), and tubulin. Representative blots are shown for HEK293 cells expressing GluN1/GluN2A and GluN1-P557R/GluN2A (C), and GluN1/GluN2B and GluN1-P557R/GluN2B (D). Chemiluminescence signals were quantified by densitometry, and the ratio of surface-to-total protein and total protein levels for each mutant were plotted as the fold-change from WT (dashed line). Total protein levels were normalized to tubulin levels. The data were analyzed by one-way ANOVA and paired t-tests between respective mutant and WT controls, with Benjamini-Hochberg correction for multiple comparisons (E, GluN1/GluN2A surface/total: F(7,28) = 5.081, p < 0.001; p = 0.019; GluN1-D552E/GluN2A p = 0.475; 2A-A548T p = 0.453; GluN1/GluN2A-P552R p = 0.404; GluN1/GluN2A total (lower panel): F(7,28) = 2.050, p = 0.084; F, GluN1/GluN2B surface/total: F(5,28) = 7.585, p < 0.001; p = 0.025; #p = 0.018; GluN1-P557R/GluN2B p = 0.102; GluN1/GluN2B total (lower panel): F(5,28) = 4.903, p = 0.002; p = 0.018; GluN1-D552E/GluN2B p = 0.473; GluN1/GluN2B-P553L p = 0.649). See also S1 Fig.
	Fig 4. GluN2A-P552R increases agonist potency and alters the NMDAR response time course.A, Steady-state concentration-response relationship for glutamate activation of human di-heteromeric (left panel) GluN1/GluN2A receptors and rat tri-heteromeric GluN1/GluN2A (right panel) containing 0, 1, or 2 copies of GluN2A-P552R receptors (labelled N2A/N2A, N2A/N2A-P552R, N2A-P552/N2A-P552R) expressed in Xenopus oocytes with 100 μM glycine present in all solutions. B, Concentration-effect relationship for glycine activation of human di-heteromeric (left panel) and rat tri-heteromeric (right panel) GluN1/GluN2A and GluN1/GluN2A-P552R receptors with 100 μM glutamate present in all solutions. C, Whole cell voltage clamp recording of di-heteromeric (left panels) and tri-heteromeric (right panels) GluN1/GluN2A-P552R receptor responses to rapid application of long (1.5 sec, upper panels) and brief (5 ms, lower panels) application of 1000 μM glutamate; saturating glycine (30 μM) was present in all of the solutions. Fitted parameters describing the time course of the response to long glutamate application are given in Table 4. The rise time and weighted tau describing the response to brief 5 ms application of glutamate were 7 ± 0.7 and 42 ± 4.3 ms for WT GluN1/GluN2A and 146 ± 20 and 689 ± 49 ms for diheteromeric GluN1/GluN2A-P552R, respectively (p < 0.0001 for both, t-test, n = 7–9). The rise time and weighted tau for receptors with 0, 1, or 2 copies of GluN2A-P552R were 8 ± 1and 38 ± 3.4 ms, 9 ± 1 and 201 ± 30 ms, 210 ± 16 and 1283 ± 153 ms, respectively (n = 8). D, Whole cell voltage clamp recording of di-heteromeric (left panels) and tri-heteromeric (right panels) GluN1/GluN2A-P552R receptor responses to rapid application (1.5 sec) of glycine; saturating glutamate (100 μM) was present in all of the solutions. See Table 4 for all mean ± s.e.m. values and S5 Table for statistical analyses for panels A-D. E, Single channel currents were recorded from HEK293 Tet-On cells selectively expressing GluN1/GluN2A receptors with 0, 1 or 2 copies of GluN2A-P552R. Currents were recorded in the outside-out configuration at -80 mV. The closed state (C) is indicated by a dashed line and openings are downwards deflections of current and the open state is indicated by an O. F, Normalized open time histograms from the three current recordings shown in (E) reveal an increase in the slower open time component of GluN1/GluN2A-P552R. Smooth lines show fitted dual component exponential functions. See also S2 Fig.
	Fig 5. Conserved effects of the Pro552Arg mutation across GluN2 subunits.A,B, Composite concentration-response curves of glutamate in the presence of 100 μM glycine (A) and glycine in the presence of 100 μM glutamate (B) for human GluN1-P557R/GluN2A, GluN1-P557R/GluN2B, GluN1/GluN2B-P553R, and rat GluN1/GluN2C-P550R, and GluN1/GluN2D-P577R. The graph legends refer to GluN1 as N1 and GluN2 as N2. Fitted EC50 values are summarized in Tables 3 and 5. C,D, human GluN1-P557R/GluN2A significantly prolongs deactivation time course after removal of glutamate (C) or removal of glycine (D) on transfected HEK293 cells, but does not slow the rise time when the receptors were activated by the agonists. E, GluN1/GluN2B-P553R significantly slows rise time and prolongs deactivation time course. Fitted parameters describing the response time course are given in Table 6.
	Fig 6. Assessment of alternative Pro552 substitutions in GluN2A.A,B, the composite glutamate (in the presence of 100 μM glycine) and glycine (in the presence of 100 μM glutamate) concentration-response curves of GluN2A- P552A, P552G, P552I, P552K, P552Q, P552L constructs. Error bars are SEM and shown when larger than symbol. C,D,E, The response time courses are shown of GluN1/GluN2A(P552K), GluN1/GluN2A(P552G), and GluN1/GluN2A(P552L) receptors activated by rapid application of 100 μM glutamate; 100 μM glycine was present in all solutions. For panel D the rise time is expanded as an inset. The data (mean ± SEM) are given in Table 7.
	Fig 7. Effect of GluN2B-P553R on the NMDAR component of the EPSP.A, Red shows the presynaptic neuron expressing a channelrhodopsin variant (CheRiff) and green shows postsynaptic neuron transfected with GFP-GluN2B or GFP-GluN2B-P553R and a QuasAr voltage indicator (inset). Scale bars, 30 μm. B, Optically-evoked and optically-monitored EPSPs. The mean postsynaptic EPSP is given for neurons expressing GFP-GluN2B-P553R (red) or GFP-GluN2B (black). Shading represents s.e.m; n = number of neurons. EPSPs are shown normalized to peak (see Methods).
	Fig 8. Neurotoxic consequences of GluN2A-P552R expression and rescue pharmacology.A, Schematic of experimental timeline indicates the relative dates of neuronal cell culture from embryonic day 16/17 (E16/17), transfection along with memantine/vehicle treatment, and toxicity studies (luciferase assays, cell counts, and confocal imaging). B, Confocal images of cortical neurons transfected with a GFP-expressing construct, with various concentrations of the human GluN2A-P552R-expressing plasmid illustrate the cell morphology. Images were acquired 24 h post-transfection at 20× magnification (scale bars 10 μm). C, Confocal images of cortical neurons transfected with GFP-expressing construct with either 0.6 μg cDNA per well (40% of total transfection cDNA) of WT GluN2A or GluN2A-P552R, the latter in the absence or presence of memantine (50 μM). Images were acquired 24 h post-transfection at 20× magnification, with the exception of the bottom left panel (40×), which highlights GluN2A-P552R-induced dendritic swelling and blebs (scale bar 10 μm). D, The mean cell viability values are shown as a percent of control. Luciferase assays: neuronal cultures were transfected with GFP-N1 plasmid (0.525 μg or 0.825 μg per well) luciferase cDNA (0.375 μg/well) for cell viability assaying, with varied concentrations (0.3 μg or 0.6 μg per well) of pCIneo-vector, WT GluN2A, or GluN2A-P552R cDNA (1.5 μg total DNA per well). Each transfection condition was performed in pairs, either supplemented with vehicle (–) or memantine (20 μM for 0.3 μg; 50 μM for 0.6 μg) treatment (+). Luciferase assays were performed 48 h following transfection and treatment. Experiments were performed in quadruplicate, and independent experiments were repeated (0.3 μg cDNA, n = 7; 0.6 μg cDNA, n = 8). Each condition was normalized to its relevant vector-transfected group to obtain relative viability values, expressed as % control. Data are mean ± SEM of viability (% control) for each condition (ANOVA/Bonferroni; p <0.05, p < 0.01, p < 0.001). Cell counts: Neuronal cultures were transfected with GFP-N1 plasmid for cell visualization, with either 0.6 μg pCIneo including vector, WT GluN2A, or GluN2A-P552R cDNA (40% of total transfection cDNA). Each transfection condition was performed in duplicate. Cell counts were performed 48 h post-transfection (Methods). Data are mean ± SEM of viability (% control) for each condition in 6 independent experiments. ANOVA/Bonferroni (p < 0.01). See S9 Table for statistics.
	Fig 9. Potential interaction between the pre-M1 and M3 helices.A,B, Ribbon structures of the GluN1/GluN2A (A) and GluN1/GluN2B (B) receptors without the amino terminal domain is shown. GluN1 is tan and GluN2 is light blue; regions with an OE-ratio below the 5th percentile are colored purple, and indicate the regions under the strongest purifying selection. C, Side and top down view of the pore forming elements M1, M3, M4 in GluN1/GluN2A receptors colored as in (A), with regions of purifying selection shown in purple. D, Expanded view of the pre-M1 helix for GluN1 (left panel) and for GluN2A (right panel).

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