Research ArticleNEURODEVELOPMENT

l-Serine dietary supplementation is associated with clinical improvement of loss-of-function GRIN2B-related pediatric encephalopathy

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Science Signaling  18 Jun 2019:
Vol. 12, Issue 586, eaaw0936
DOI: 10.1126/scisignal.aaw0936
  • Fig. 1 Identification of GRIN2B(P553T) mutation associated with the case study and predicted structural consequences.

    (A) Left: Familial pedigree of the GRIN2B(P553T) case study. Right: Chromatograms of GRIN2B(c.1657C > A) mutated nucleotide (indicated by an arrow) using forward and reverse oligonucleotides. (B) Structure of heterotetrameric (GluN1)2-(GluN2B)2 NMDAR [Protein Data Bank (PDB) ID: 4PE5], according to Karakas and Furukawa (61), showing the N-terminal domain (NTD), the ligand-binding domain (LBD), and the transmembrane domain (TMD; containing the mutated amino acid P553T). Bottom: Magnification of the transmembrane domain, showing the topological position of Pro553 residue at the beginning of the M1 (P553; green) of the transmembrane domain, facing Phe653 residue (F653; light blue) at M3 (blue). (C) Top: Transmembrane domain structural molecular model of wild-type (GluN1)2-(GluN2B)2 receptor (from the extracellular domain). Inset: Magnification of residues Pro553(M1)-F653(M3) proximity. Bottom: Transmembrane domain structural molecular model of mutant (GluN1)2-[GluN2B(P553T)]2 receptor (from the extracellular domain). Inset: Magnification of residues Pro553(M1)-F653(M3) distance. (D) GluN2B protein sequence alignment around residue Pro553. Representative sequences from a larger alignment containing 147 proteins from 12 metazoan species spanning seven phyla are shown. Displayed protein sequences are from the following species: Homo sapiens GluN2B (UniProt ref. Q13224), Mus musculus GluN2B (UniProt ref. Q01097), Danio rerio GluN2Bb (Ensembl ref. ENSDARG00000030376), Branchiostoma belcheri 254360R.t1 (from the database B.belcheri_v18h27.r3_ref_protein included in LanceletDB Genome browser; Sun Yat-sen University), Saccoglossus kowalevskii Sakowv30010297m (Metazome database), and Strigamia maritima SmarNMDAR2b, Apis melifera GB48097, Capitela teleta CapteT179505, and Lottia gigantea LotgiT137890 (all from Ensembl Metazoa).

  • Fig. 2 Biophysical characterization of (GluN1)2-(GluN2B(P533T))2channel properties.

    (A) Representative whole-cell currents evoked by rapid application of 1 mM glutamate + 50 μM glycine (0.5-s duration; −60 mV) in HEK-293T cells expressing GluN1-GluN2B (black trace) or GluN1-GluN2B(P533T) (red trace) receptors. n = 19 and 21 cells from six and five experiments, respectively. (B) Average of raw peak currents from HEK-293T cells expressing GluN2B and GluN2B(P533T). n = 19 and 21 cells from six and five experiments, respectively. ***P < 0.001 by Mann-Whitney U test. (C) Normalized peak currents (in pA/pF) in HEK-293T cells expressing GluN1-GluN2B and GluN1-GluN2B(P533T), with values from a representative experiment superimposed. Data are from six and five experiments, respectively. ****P < 0.0001 by Mann-Whitney U test. (D) Traces recorded at −60 mV in an HEK-293T cell expressing GluN1-GluN2B(P533T) with Mg2+ block of the NMDAR. Data are representative of five and seven cells from three independent cultures. (E) Percentage of current blocked at −60 mV by Mg2+ (1 mM) for GluN2Bwt- and GluN2B(P533T)-containing NMDARs. Single-value experiments are denoted as open circles for each condition. n = 5 and 7 cells, respectively, from three independent experiments per condition. n.s. (not significant) by Mann-Whitney U test. wt, wild-type. (F) Current-voltage relationship for GluN2B- and GluN2B(P533T)-containing NMDARs. n = 3 and 4, respectively, from two independent experiments. (G) Representative peak-scaled responses to 1 mM glutamate + 50 μM glycine (0.5-s agonists application; −60 mV) for GluN1-GluN2B (black trace) and GluN1-GluN2B(P553T) (red trace). n = 16 and 17 cells from six and five experiments, respectively. (H) Average deactivation time constant (τw; fitted to a double exponential) fitted from tail currents for GluN1-GluN2B and GluN1-GluN2B(P553T). Values from a representative experiment are shown as open circles for each condition. n = 16 and 17 cells from six and five independent experiments per condition, respectively. ****P < 0.0001 and n.s. by Mann-Whitney U test. (I) Representative peak-scaled responses to 1 mM glutamate + 50 μM glycine (long jumps of 5-s duration; −60 mV) in HEK-293T cells expressing GluN1-GluN2B or GluN1-GluN2B(P553T), for the comparison of desensitization rates. n = 14 cells from three independent experiments. (J) Desensitization weighted time constant (τw) for GluN2Bwt and GluN2B(P553T). Values from a representative experiment are shown as open circles for each condition. n = 14 from three independent experiments. **P < 0.01 by Mann-Whitney U test. (K and L) Whole-cell currents activated by rapid application of 1 mM glutamate + 50 μM glycine (0.5 s; −60 mV) from HEK-293T cells expressing GluN1-GluN2Bwt (K) or GluN1-GluN2B(P553T) (L). Gray traces represent single responses, and black lines are the average of 69 (wild-type) or 33 (P553T) responses. Insets: Current variance versus mean current plot calculated from the deactivating tail current. (M and N) Bar graph showing single-channel conductance values (M) and peak open probability (N) in GluN1-GluN2Bwt– and GluN1-GluN2B(P553T)–containing NMDARs expressed in HEK-293T cells. n = 12 and 9 cells, respectively, from four independent experiments. *P < 0.05 by Mann-Whitney U test. Single cells are shown as open circles superimposed to bar graph.

  • Fig. 3 d-Serine coapplication effect on wild-type and mutant (GluN1)2-[GluN2B(P533T)]2NMDARs.

    (A) Representative traces evoked by physiological concentrations of 1 mM glutamate + 1 μM glycine (0.5 s; −60 mV) from GluN1-GluN2Bwt– or GluN1-GluN2B(P553T)–expressing HEK-293T cells, either in the absence (black traces) or in the presence (red traces) of d-serine at different concentrations. n ≥ 6 cells from at least two independent experiments. (B) Average peak current evoked in transfected HEK-293T cells by application of 1 mM glutamate + 1 μM glycine in the presence of different d-Ser concentrations (gray bars) normalized to that of 1 mM glutamate + 1 μM glycine without d-Ser (white bars). Numbers inside the bars denote the recordings for each condition, from at least two independent experiments. *P < 0.05 and **P < 0.01 by Mann-Whitney U test. (C) Raw peak current responses from data shown in (B), indicating the percentage increase in current due to 100 μM d-Ser. n = 10 cells from at least two experiments. (D) Representative recordings in HEK-293T cells expressing GluN1-GluN2Bwt or GluN1-GluN2B(P553T) receptors evoked by physiological applications (5 s; −60 mV) of 1 mM glutamate + 1 or 100 μM glycine. n = 8 cells per condition from five independent experiments. (E) Bar graph representing peak current potentiation induced by high glycine concentration (100 μM versus 1 μM) with coapplication of 1 mM glutamate. n = 8 from five independent experiments for each condition. *P < 0.05 and n.s. by Student’s t test. (F) d-Serine concentration-response curves recorded in GluN1-GluN2Bwt and GluN1-GluN2B(P553T) NMDARs expressed in HEK-293T cells, constructed from responses to 1 mM glutamate + the specified d-serine concentration in the absence of glycine. n = 5 cells per construct from two independent experiments. (G) Glutamate concentration-response curves recorded in GluN1-GluN2Bwt and GluN1-GluN2B(P553T) NMDARs expressed in HEK-293T cells. The dose-response curve was constructed from responses to 1 mM d-serine plus the specified glutamate concentration in the absence of glycine. n = 5 and 7 cells, respectively, from two independent experiments.

  • Fig. 4 Synaptic outcomes and d-serine effects on Gly-induced chemical long-term potentiation (Gly-cLTP) in GluN2B(P553T)-expressing primary hippocampal neurons.

    (A) Top left: Representative images of murine primary hippocampal neurons transfected with GFP-GluN2Bwt or GFP-GluN2B(P553T). Insets: Immunodecoration to visualize spines, indicated by yellow arrowheads. Top right: Immunofluorescence detection of GFP-GluN2B to analyze dendritic arborization by Sholl analysis. n = 16 to 18 neurons per condition from three independent experiments; F = 0.71, P = 0.884 by repeated measures two-way analysis of variance (ANOVA) and Bonferroni post hoc test. Bottom: Quantification of spine density and morphology in basal neurons and neurons treated with glycine alone or glycine and 100 μΜ d-Ser. n = 27 to 48 dendrites per condition from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. by Student’s t test or Mann-Whitney U test for parametric or nonparametric analyses, respectively. (B) Representative traces from spontaneous activity–dependent NMDAR-mediated EPSCs recordings from GluN2Bwt- or GFP-GluN2B(P553T)–transfected murine hippocampal neuronal cultures, recorded at −70 mV in the presence of 50 μM NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione) and 10 μM picrotoxin and in the absence of tetrodotoxin, both under basal conditions (top traces) or after 100 μM d-serine application (bottom traces). Graphs show mean amplitudes (left) and mean time of interevent intervals (right) of the EPSCs recorded. n = 8 and 6 neurons, respectively, from three independent experiments. **P < 0.01 and #P < 0.05 by Mann-Whitney U test. (C) Representative images and immunofluorescence analysis of surface abundance of the AMPAR subunit GluA1 (red) in murine primary hippocampal neurons (green) at DIV16 that had been transiently transfected at DIV11 with GFP-GluN2Bwt or with GFP-GluN2B(P553T) assessed under basal conditions, after Gly-cLTP, and after simultaneous Gly-cLTP induction and 100 μM d-serine application (Gly + d-Ser cLTP). A.U., arbitrary units. Data are presented relative to the basal condition as means ± SEM from n = 30 to 40 spines per dendrite from 14 to 40 dendrites and three to seven neurons per condition, obtained from three independent experiments. **P < 0.01 and ***P < 0.001 by Student’s t test.

  • Fig. 5 Biochemical and behavioral assessment of GRIN2B(P553T) patient after l-serine dietary supplementation.

    (A) Top: Analysis of amino acid plasma concentration of d-l-serine, d-l-valine, d-l-isoleucine, d-l-leucine, glycine, taurine, and cysteine. Plasma samples analyzed correspond to the patient before and after l-serine treatment (light red and dark red dots, respectively) and the mean of controls (black lines; whiskers representation of mean-min-max values). Bottom: Chromatographic profiles of d-serine and l-serine enantiomers in plasma samples from the control individual (gray trace), the patient before treatment (light red trace), and the patient with l-serine dietary supplementation (dark red trace). (B) Top: Overlapping chromatographic profiles of d-serine and l-serine enantiomers (left and right, respectively) in CSF from a control (gray trace) and from the patient with GRIN2B(P553T) mutation, after l-serine dietary supplementation (red trace). Bottom: Serine enantiomers concentration analysis in CSF of controls (gray dots; whiskers representation of mean-min-max values; gray traces in the chromatogram) and the patient with l-serine dietary supplement (red dots and traces). (C) Clinical manifestations of the patient harboring a de novo GRIN2B(P553T) mutation, before and after 17 months of l-serine dietary supplementation.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/586/eaaw0936/DC1

    Fig. S1. Protein interactions and cellular trafficking of GluN2Bwt- and GluN2B(P553T)-containing NMDARs.

    Fig. S2. Altered biophysical properties of heterotrimeric GluN1-GluN2A-GluN2B(P553T) NMDARs.

    Fig. S3. GluN2B(P553T) mutation alters GluA1 abundance in hippocampal neurons.

    Fig. S4. Alignment of eumetazoan iGluRs showing the residues conservation of Pro553 and Phe653.

    Table S1. Untargeted analysis of plasma sphingolipid profile in the GRIN2B(P553T) patient before and after l-serine dietary supplementation.

    References (78, 79)

  • This PDF file includes:

    • Fig. S1. Protein interactions and cellular trafficking of GluN2Bwt- and GluN2B(P553T)-containing NMDARs.
    • Fig. S2. Altered biophysical properties of heterotrimeric GluN1-GluN2A-GluN2B(P553T) NMDARs.
    • Fig. S3. GluN2B(P553T) mutation alters GluA1 abundance in hippocampal neurons.
    • Fig. S4. Alignment of eumetazoan iGluRs showing the residues conservation of Pro553 and Phe653.
    • Table S1. Untargeted analysis of plasma sphingolipid profile in the GRIN2B(P553T) patient before and after L-serine dietary supplementation.
    • References (78, 79)

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