Human N-methyl D-aspartate receptor antibodies alter memory and behaviour in mice

Abstract

Anti-N-methyl D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder that associates with prominent memory and behavioural deficits. Patients' antibodies react with the N-terminal domain of the GluN1 (previously known as NR1) subunit of NMDAR causing in cultured neurons a selective and reversible internalization of cell-surface receptors. These effects and the frequent response to immunotherapy have suggested an antibody-mediated pathogenesis, but to date there is no animal model showing that patients' antibodies cause memory and behavioural deficits. To develop such a model, C57BL6/J mice underwent placement of ventricular catheters connected to osmotic pumps that delivered a continuous infusion of patients' or control cerebrospinal fluid (flow rate 0.25 µl/h, 14 days). During and after the infusion period standardized tests were applied, including tasks to assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviours (sucrose preference test), depressive-like behaviours (tail suspension, forced swimming tests), anxiety (black and white, elevated plus maze tests), aggressiveness (resident-intruder test), and locomotor activity (horizontal and vertical). Animals sacrificed at Days 5, 13, 18, 26 and 46 were examined for brain-bound antibodies and the antibody effects on total and synaptic NMDAR clusters and protein concentration using confocal microscopy and immunoblot analysis. These experiments showed that animals infused with patients' cerebrospinal fluid, but not control cerebrospinal fluid, developed progressive memory deficits, and anhedonic and depressive-like behaviours, without affecting other behavioural or locomotor tasks. Memory deficits gradually worsened until Day 18 (4 days after the infusion stopped) and all symptoms resolved over the next week. Accompanying brain tissue studies showed progressive increase of brain-bound human antibodies, predominantly in the hippocampus (maximal on Days 13-18), that after acid extraction and characterization with GluN1-expressing human embryonic kidney cells were confirmed to be against the NMDAR. Confocal microscopy and immunoblot analysis of the hippocampus showed progressive decrease of the density of total and synaptic NMDAR clusters and total NMDAR protein concentration (maximal on Day 18), without affecting the post-synaptic density protein 95 (PSD95) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. These effects occurred in parallel with memory and other behavioural deficits and gradually improved after Day 18, with reversibility of symptoms accompanied by a decrease of brain-bound antibodies and restoration of NMDAR levels. Overall, these findings establish a link between memory and behavioural deficits and antibody-mediated reduction of NMDAR, provide the biological basis by which removal of antibodies and antibody-producing cells improve neurological function, and offer a model for testing experimental therapies in this and similar disorders.

Keywords: animal model; anti-NMDAR encephalitis; antibodies; mechanism; pathogenesis.

Figure 1

Experimental design and placement of ventricular catheters. (A) Representative coronal mouse brain section with catheter placement. Scale bar = 2 mm. (B and C) Coronal and sagittal mouse brain sections demonstrating cerebroventricular diffusion of methylene blue after ventricular infusion. Scale bars = 2 mm. (D) Schedule of cognitive testing and animal sacrifice. At Day 0, catheters and osmotic pumps were placed and bilateral ventricular infusion of patients’ or control CSF started. Infusion lasted for 14 days. Memory [novel object recognition (NOR)], anhedonia [sucrose preference test (ANH)], depressive-like behaviour [tail suspension test (TST) and forced swimming test (FST)], anxiety [black and white test (BW) and elevated plus maze test (EPM)], aggressiveness [resident intruder test (RI)] and locomotor activity (LOC) were assessed blinded to treatment at the indicated days. The novel object recognition was assessed in open field and V-maze paradigms in two different cohorts of mice. Animals were habituated for 1 to 4 days before surgery (baseline) to novel object recognition, anhedonia, and locomotor activity. Red arrowheads indicate the days of sacrifice for studies of effects of antibodies in brain.

Figure 2

Infusion of CSF from patients with NMDAR antibodies causes deficits in memory, anhedonia and depressive-like behaviour. (A and B) Novel object recognition index in open field (A) or V-maze paradigms (B) in animals treated with patients’ CSF (grey circles) or control CSF (white circles). A high index indicates better object recognition memory. (C) Preference for sucrose-containing water in animals infused with patients' CSF (grey) or control CSF (white). Lower percentages indicate anhedonia. (D and E) Total time of immobility in tail-suspension test during the infusion period (D, Day 12) and in forced swimming test after the infusion period (E, Day 20). Data are presented as mean ± SEM (median ± IQR in D). Number of animals: patients’ CSF n = 18 (open field novel object recognition n = 8), control CSF n = 20 (open field novel object recognition n = 10). Significance of treatment effect was assessed by two-way ANOVA (A–C) with an α-error of 0.05 and post hoc testing with Sidak-Holm adjustment (asterisks), unpaired t-test (E) or Mann-Whitney U test (D). *P < 0.05, ***P < 0.001. See Supplementary Table 1 for detailed statistics.

Figure 3

Infusion of CSF from patients with NMDAR antibodies does not alter the tests of anxiety, aggression and locomotor activity. (A and B) Number of entries into bright/open compartments during a 5 min period in a standard black and white (A, Day 6) or elevated plus maze test (B, Day 14) in animals treated with patients’ CSF (filled circles) or control CSF (open circles). (C) Number of aggressive events over a 4-min period in a resident intruder paradigm in both treatment groups. (D) Horizontal (solid lines) and vertical (dashed lines) movement count over a 10 min period in both treatment groups. Data are presented as mean ± SEM. Number of animals: patients’ CSF n = 18, control CSF n = 20. Statistical assessment as indicated in Fig. 2 and Supplementary Table 1.

Figure 4

Animals infused with patient’s CSF have a progressive increase of human IgG bound to hippocampus. (A and B) Immunostaining of human IgG in sagittal brain sections (A) and hippocampus (B) of representative animals infused with patients’ CSF (left) and control CSF (right), sacrificed at the indicated experimental days. In animals infused with patients’ CSF there is a gradual increase of IgG immunostaining until Day 18, followed by decrease of immunostaining. Scale bars: A = 2 mm; B = 200 µm. (C) Quantification of intensity of human IgG immunolabelling in hippocampus of mice infused with patients’ CSF (dark grey columns) and control CSF (light grey columns) sacrificed at the indicated time points. (D–H) Confocal microscopy analysis of IgG bound to the hippocampus on Day 18. (D) Sagittal section of the hippocampus with areas examined at higher magnification in E (arrow in CA1), F (arrow heads in CA3) and G (asterisks in dentate gyrus). Note the fine punctate IgG immunolabelling surrounding neuronal bodies in mice infused with patients’ CSF; this immunolabelling is similar to that reported in brain sections directly incubated with patients’ antibodies, as in Dalmau et al. (2008). Scale bars: D = 200 µm; E–G = 10 µm. (H) Quantification of the intensity of human IgG immunofluorescence in the indicated areas in animals infused with patients’ CSF (dark grey columns) or control CSF (light grey columns). For all quantifications, mean intensity of IgG immunostaining in the group with the highest value (animals treated with patients’ CSF and sacrificed at Day 18) was defined as 100%. All data are presented as mean ± SEM. For each time point five animals infused with patients’ CSF and five with control CSF were examined. Significance of treatment effect was assessed by two-way ANOVA with an α-error of 0.05 (*) and post hoc testing with Sidak-Holm adjustment ($). ***^{, $$$}P < 0.001; ^$P < 0.05. See Supplementary Table 2 for detailed statistics.

Figure 5

The human IgG extracted from brain of mice infused with patients’ CSF is specific for NMDARs. (A and B) HEK293 cells expressing the GluN1 subunit of the NMDAR immunolabelled with acid-extracted IgG fractions (top row in A) or pre-extraction fractions (top row in B) from hippocampus of mice infused with patients’ CSF and sacrificed on the indicated days. The maximal reactivity with GluN1-expressing cells was noted in acid-extracted IgG fractions from Days 13 and 18 (A); none of the pre-extraction fractions showed GluN1 reactivity (B) indicating that the reactivity of acid-extracted fractions corresponds to IgG antibodies that were bound to brain NMDAR receptors. The second row in A and B shows the reactivity with a monoclonal GluN1 antibody, and the third row the colocalization of immunolabelling. Scale bars = 10 µm. (C) Quantification of NMDAR antibody titre in IgG-extracted fractions from hippocampus of animals treated with patients’ CSF (n = 5 mice per each time point, except four mice for Day 5). Solid line = median. Significance was tested by Kruskal-Wallis with an α-error of 0.05 (asterisks) and post hoc testing with Dunn’s test ($). **^{, $$}P < 0.01, ***^{, $$$}P < 0.001. See Supplementary Table 2 for detailed statistics. (D and E) HEK293 cells expressing the GluN1 subunit of the NMDAR immunolabelled with acid-extracted IgG fractions (D) and pre-extraction fractions (E) from hippocampus (Hippo), cerebral cortex (Ctx) and cerebellum (Cb) of mice infused with patients’ CSF (Day 18). The acid-extracted IgG fraction from hippocampus showed higher level of NMDAR antibodies than those extracted from cerebral cortex (Ctx) and cerebellum (Cb). Scale bars = 10 µm. n.s = not significant.

Figure 6

Patients’ NMDAR antibodies selectively reduce the density of total and synaptic NMDAR clusters in hippocampus of mice. (A) Hippocampus of mice infused for 14 days (Day 18) with patients’ CSF (upper row) or control CSF (lower row) immunolabelled for PSD95 and NMDAR. Images were merged (merge) and post-processed to demonstrate co-localizing clusters (co-localization). Squares in ‘co-localization’ indicate the analysed areas in CA1, CA3 and dentate gyrus. Scale bar = 200 µm. (B) Three-dimensional projection and analysis of the density of total clusters of PSD95 and NMDAR, and synaptic clusters of NMDAR (defined as NMDAR clusters colocalizing with PSD95) in a representative CA3 region (square in A ‘co-localization’). Merged images (merge, PSD95 green, NMDAR red) were post-processed and used to calculate the density of clusters (density = spots/µm³). Scale bar = 2 µm. (C–F) Quantification of the density of total (C) and synaptic (D) NMDAR clusters, PSD95 clusters (E), and total/synaptic AMPAR and PSD95 clusters (Day 18 only, F) in a pooled analysis of hippocampal subregions (CA1, CA3, dentate gyrus) in animals treated with patients’ CSF (dark grey) or control CSF (light grey) on the indicated days. Mean density of clusters in control CSF treated animals was defined as 100%. Data are presented as mean ± SEM. For each time point five animals infused with patients’ CSF and five with control CSF were examined. Significance of treatment effect was assessed by two-way ANOVA with an α-error of 0.05 (asterisks) and post hoc testing with Sidak-Holm adjustment ($) (C–E) or unpaired t-test (F). *^{, $}P < 0.05; **^{, $$}P < 0.01; ***^{, $$$}P < 0.001. See Supplementary Table 2 for detailed statistics.

Figure 7

Patients’ NMDAR antibodies selectively reduce the protein concentration of NMDAR in hippocampus of mice. (A) Representative immunoblots of proteins extracted from hippocampus of animals infused with patients’ CSF (P) or control CSF (C) sacrificed at the indicated time points and probed for expression of GluN1 (NMDAR), PSD95 and β-actin (loading control). Note that there is less visible GluN1 expression on Days 13 and 18. (B, D and E) Quantification of total NMDAR (B), AMPAR (D) or PSD95 (E) protein in animals treated with patients’ CSF (filled columns) or control CSF (open columns) sacrificed at the indicated time points (AMPAR Day 18 only, D). Results were normalized to β-actin (loading control). Mean band density of animals treated with control CSF was defined as 100%. Data are presented as mean ± SEM. For each time point six animals infused with patients’ CSF and six with control CSF were examined (for Days 26 and 46, only five animals treated with patient’s CSF were available). Significance of treatment effect was assessed by two-way ANOVA with an α-error of 0.05 (asterisks) and post hoc testing with Sidak-Holm adjustment ($). ^$$P < 0.01; ***P < 0.001. See Supplementary Table 2 for detailed statistics. (C) Correlation between concentration of human IgG bound to hippocampus (x-axis, highest hippocampal IgG intensity was defined as 100%) and hippocampal NMDAR protein concentration in mice sacrificed on Day 18 (R² = 0.69, P = 0.003). Filled circles: mice infused with patients’ CSF (n = 5), open circles: mice infused with control CSF (n = 5).

Figure 8

Absence of neuronal apoptosis, deposits of complement, and lymphocytic infiltrates in the hippocampus of mice infused with patients’ CSF. (A and B) TUNEL and cleaved caspase 3 immunolabelling of a representative area of CA3 (area with maximal IgG binding and lower NMDAR concentration) of an animal infused with patients’ CSF, showing lack of apoptotic cells. A section of the same region in an animal with transient middle cerebral artery occlusion (stroke model) shows apoptotic cells in the penumbra (left). (C) Same CA3 region as in (A) immunostained for C5b-9 showing lack of deposit of complement. A section of the same region in the indicated stroke model shows presence of complement in the penumbra (left). (D and E) Same CA3 region as in (A) immunostained for T (CD3) and B (CD45R) lymphocytes showing absence of inflammatory infiltrates. A section of spleen was used as control tissue showing the presence of CD3 (green) and CD45R (red) cells. Scale bar = 10 µm. Total number of animals examined: patients’ CSF n = 5; control CSF n = 5. Scale bars = 20 µm.