Positive Allosteric Modulation as a Potential Therapeutic Strategy in Anti-NMDA Receptor Encephalitis

Abstract

N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors important for synaptic plasticity, memory, and neuropsychiatric health. NMDAR hypofunction contributes to multiple disorders, including anti-NMDAR encephalitis (NMDARE), an autoimmune disease of the CNS associated with GluN1 antibody-mediated NMDAR internalization. Here we characterize the functional/pharmacological consequences of exposure to CSF from female human NMDARE patients on NMDAR function, and we characterize the effects of intervention with recently described positive allosteric modulators (PAMs) of NMDARs. Incubation (48 h) of rat hippocampal neurons of both sexes in confirmed NMDARE patient CSF, but not control CSF, attenuated NMDA-induced current. Residual NMDAR function was characterized by lack of change in channel open probability, indiscriminate loss of synaptic and extrasynaptic NMDARs, and indiscriminate loss of GluN2B-containing and GluN2B-lacking NMDARs. NMDARs tagged with N-terminal pHluorin fluorescence demonstrated loss of surface receptors. Thus, function of residual NMDARs following CSF exposure was indistinguishable from baseline, and deficits appear wholly accounted for by receptor loss. Coapplication of CSF and PAMs of NMDARs (SGE-301 or SGE-550, oxysterol-mimetic) for 24 h restored NMDAR function following 24 h incubation in patient CSF. Curiously, restoration of NMDAR function was observed despite washout of PAMs before electrophysiological recordings. Subsequent experiments suggested that residual allosteric potentiation of NMDAR function explained the persistent rescue. Further studies of the pathogenesis of NMDARE and intervention with PAMs may inform new treatments for NMDARE and other disorders associated with NMDAR hypofunction.SIGNIFICANCE STATEMENT Anti-N-methyl-d-aspartate receptor encephalitis (NMDARE) is increasingly recognized as an important cause of sudden-onset psychosis and other neuropsychiatric symptoms. Current treatment leaves unmet medical need. Here we demonstrate cellular evidence that newly identified positive allosteric modulators of NMDAR function may be a viable therapeutic strategy.

Keywords: NMDA receptor; autoimmune; glutamate; schizophrenia.

Conflict of interest statement

Conflict of interest: S.M.P., M.L., J.D., M.Q., and C.F.Z. have financial interests in Sage Therapeutics. The remaining authors declare no competing financial interests.

Copyright © 2018 the authors 0270-6474/18/383218-12$15.00/0.

Figures

Figure 1.

Depression of NMDA current in hippocampal neurons following patient CSF incubation. A, NMDARE-A CSF (1:20 dilution) labels mouse hippocampus in a pattern typical of NMDAR distribution. Human antibody binding to the tissue section was visualized with anti-human secondary antibody conjugated to AlexaFluor 555. B, CSF from an age- and sex-matched MS patient (1:20 dilution), failed to exhibit an NMDAR-like pattern of labeling. Scale bar: 500 μm. C, NMDA (10 μm) -elicited current from a hippocampal neuron following 48 h incubation with either aCSF (1:12; black trace) or NMDARE-A CSF (1:12; red trace). D, Summary of NMDA current density from cells treated as in C (t(46) = 6.951, *p = 1.08e−8; n = 21 for control and 27 for NMDARE-A). E, Comparison of effects of two dilutions of NMDARE-A CSF, 24 h in either 1:12 dilution (n = 10) or 1:24 dilution (n = 10) in sibling cultures. A one-way ANOVA showed a significant NMDARE-A CSF-mediated current depression (F(2,27) = 14.1, *p = 6.6e−5). Asterisks denote p < 0.05 versus aCSF, determined with Student's unpaired t test, Bonferroni corrected for multiple comparisons. F, Representative traces from an experiment examining NMDA (10 μm) -elicited current from a hippocampal neuron following 48 h incubation of hippocampal cultures in aCSF (1:12; black trace; n = 15) or CSF from MS-A (1:12; gray trace; n = 10). G, Control CSF from age- and sex-matched MS patients (MS-A, MS-B) and neurologically normal controls (NN-A, NN-B). There was no significant difference between any treated group and control; p > 0.05 uncorrected t tests.

Figure 2.

Lack of change in key functional properties of residual NMDARs. A, B, Evoked autaptic EPSCs from a hippocampal neuron incubated with aCSF (A) or NMDARE-A CSF (B). Dual-component AMPAR and NMDAR EPSCs (light traces) and pharmacologically isolated NMDAR EPSCs (dark traces) are shown. C, Summary of ratio of NMDAR to AMPAR peak EPSC (t(14) = 4.2, *p = 1e−9, Student's unpaired t test). D, Summary of weighted time constant values (τw) obtained from a biexponential fit to the decay phase of the NMDAR EPSCs (t(14) = 1.15, p = 0.27, Student's unpaired t test). E, F, The rapid onset/offset open-channel blocker memantine was used to probe changes in NMDAR channel open probability. G, H, Summary of the memantine onset and offset kinetics (obtained from exponential fits) revealed no change following NMDARE-A CSF-mediated depression; (G, t(7) = 1.15, p = 0.29; H, t(13) = 1.33, p = 0.21, respectively, Student's unpaired t tests).

Figure 3.

No evidence of selective effect of NMDARE-A CSF on proportion of extrasynaptic NMDARs or on the proportion of GluN2B-containing receptors. A, B, NMDA-elicited current from neurons without (black) and with (blue) preceding MK-801 challenge to block synaptic receptors. C, Summary of raw NMDA current density of respective ±MK-801 conditions. Analysis of raw current density revealed no interaction between condition (aCSF/NMDARE-A CSF) and treatment (±MK-801; F(1,52) = 0.36, two-way ANOVA, p > 0.55). D, Summary of MK-801 effect, normalized to the mean current density of the respective −MK-801 condition (right). Analysis of normalized data also revealed no change in the proportion of MK-801-insensitive current following NMDARE-A CSF incubation (t(42) = 0.93, p>0.36, Student's unpaired t test). E, F, Representative examples of ifenprodil (10 μm) sensitivity of NMDA-elicited current. G, Summary of raw NMDA current density in baseline and ifenprodil conditions. There was no statistical interaction between condition (aCSF/NMDARE-A CSF) and treatment (±ifenprodil; F(1,48) = 0.7209, p = 0.4, two-way ANOVA, p > 0.05). H, Summary of ifenprodil effect, normalized to the respective baseline current density. Analysis of normalized data also revealed no change in the proportion of ifenprodil-sensitive current following NMDARE-A CSF incubation; (t(24) = 0.74, p=0.47, Student's unpaired t test).

Figure 4.

NMDARE-A CSF depresses surface NMDAR presence. A, Fluorescence quenching associated with surface NMDARs. N2a cells transfected with GluN1/GluN2B-SEP were used to visualize NMDARs on the plasma membrane. Transient wash with cell-impermeant MES quenched fluorescence in control cells. Scale bar, 10 μm. B, Comparison of MES quenching in control cells and cells incubated in NMDARE-A CSF for 24 h. The absolute MES-induced change in fluorescence between cells treated with MS-A CSF (n = 21 cells) versus NMDARE-A CSF (n = 21 cells from 4 independent replicates) was significantly different (t(40) = 2.4, *p = 0.02, Student's unpaired t test). C–G, Surface receptors detected using anti-GFP labeling of fixed, non-permeabilized transfected N2a cells. Cells were transfected with GluN1/GluN2B-SEP subunits or with cytosolic GFP as a control. Analysis lines (yellow) through labeled cells in various conditions yielded evidence for surface receptors (red; anti-GFP antibody labeling) in GluN2B-SEP-transfected cells labeled with primary antibody but not in other control conditions. Pri, Primary antibody incubated. Significant membrane labeling was detected in SEP-transfected cells labeled with primary antibody, compared with other indicated conditions (Bonferroni corrected Student's unpaired t test, *p < 0.05). G, Numbers on graph bars indicate pooled cell number from four experiments. Scale bar, 10 μm. H–J, Anti-GFP used to label surface NMDARs in transfected N2a cells incubated for 24 h in MS-A CSF or in NMDARE-A CSF, before fixation. NMDARE-A CSF reduced anti-GFP membrane labeling (Bonferroni corrected Student's unpaired t test, *p < 0.05).

Figure 5.

Intervention by oxysterol-mimetic PAMs. A, B, Acute potentiation of NMDA-elicited current by SGE-301 (2 μm; A) and by SGE-550 (2 μm; B). PAM was pre-applied for 30 s before coapplication with NMDA (colored traces). C, Normalized current showed significant potentiation by both SGE-301(t(27) = 4.3, *p = 2e−4) and SGE-550 (t(63) = 4.3, *p < 1e−4, paired Student's t tests). D, E, Summary of NMDA-elicited current density recorded following 48 h of incubation with either aCSF or NMDARE-A CSF, with or without SGE-301 (D) or SGE-550 (E) treatment at 24 h. Two-way ANOVA with Bonferroni corrected post hoc t tests indicated significant potentiation in aCSF and CSF conditions for both SGE-301 and SGE-550 (*p < 0.05). There was no statistical interaction between CSF condition and either SGE-301 (D; F(1,56) = 0.5132, p = 0.48) or SGE-550 (E; F(1,125) = 2.121, p = 0.15) treatment. Currents were obtained in recording saline, following removal of CSF and PAM in the culture medium.

Figure 6.

Tests of residual PAM activity to explain persisting potentiation. A, B, Memantine test of NMDAR channel open probability following 24 h aCSF incubation (A) or incubation with SGE-301 (B). C, D, Summary of memantine onset and offset time constant values, obtained from exponential fits; (C) onset: t(13) = 3.1, *p = 1.6e−3; (C) offset: t(25) = 3.9, *p = 6e−4; (D) onset: t(12) = 2.8, *p = 1.6e−2; (D) offset: t(12) = 3.9, *p = 2e−3, Student's unpaired t tests. E, Sample traces from cultures incubated under control or in SGE-301 (2 μm) for 24 h then rinsed with saline or γ-CDX (500 μm) for 2 min before recording. F, G, Summary of effect of brief CDX incubation on residual SGE-301 (F) and SGE-550 (G). A two-way ANOVA showed a significant main effect of SGE-301 (F(13,39) = 11.35, *p < 1e−4). Post hoc testing revealed significant potentiation by SGE-301 and SGE-550 and significant reversal of potentiation by CDX (*p < 0.05, Bonferroni corrected Student's unpaired t tests).

Figure 7.

No effect of SGE-301 intervention on surface NMDAR presence. A–D, Examples of GluN1/GluN2B-SEP-transfected N2a cells incubated under the indicated conditions. Cells were incubated in CSF for 48 h and incubation with SGE-301 commenced 24 h following CSF initiation. Anti-GFP labeling (red) in non-permeabilized cells was used to quantify surface NMDARs. Green labeling represents SEP fluorescence following fixation. E, F, Two-way ANOVA with Bonferroni corrected post hoc t tests indicated significant depression of surface labeling after incubation in NMDARE-A CSF (E; t(38) = 3.0, *p = 4.8e−3). There was no statistical interaction between SGE-301 and CSF condition (E; F(1,76) = 0.75, p = 0.39). SGE-301 did not affect membrane (E) or intracellular anti-GFP labeling (F) in post hoc testing (p > 0.05, Bonferroni corrected t tests).

Figure 8.

Effect of SGE-301 intervention on depression induced by CSF from a second NMDARE patient. A–C, Staining performed as in Figure 1, A and B, for two additional patient samples and a control sample. Scale bar: 500 μm. D, Sample traces representative of the indicated conditions. NMDARE-B CSF was incubated for 48 h at 1:12 dilution. Intervention with 2 μm SGE-301 commenced at 24 h. E, Summary of effect of incubation with NMDARE-B CSF and intervention with SGE-301. A two-way ANOVA showed a significant main effect of SGE-301 (F(1,59) = 11.9, *p = 1.0e−3; asterisk associated with legend) but no interaction with CSF incubation condition (F(1,59) = 1.02, p = 0.32). Post hoc testing revealed significant depression of NMDA-mediated current induced by NMDARE-B CSF compared with the aCSF condition (t(29) = 2.38, *p = 0.02, Student's unpaired t test). F, Same protocol and statistics as depicted in D and E applied to patient NMDAR-C. A two-way ANOVA showed a significant main effect of SGE-301(F(1,48) = 9.71, *p = 3.1e−3) but no interaction with CSF incubation condition (F(1,48) = 0.26, p = 0.61). Post hoc testing revealed significant depression of NMDA-mediated current induced by NMDARE-B CSF compared with the aCSF condition (t(24) = 2.99, *p = 6.4e−3, Student's unpaired t test). One data point in the aCSF+SGE-301 condition >120 pA/pF is not shown on the graph for clarity but was used in statistics.