Autoimmune receptor encephalitis in mice induced by active immunization with conformationally stabilized holoreceptors

Abstract

Autoimmunity to membrane proteins in the central nervous system has been increasingly recognized as a cause of neuropsychiatric disease. A key recent development was the discovery of autoantibodies to N-methyl-d-aspartate (NMDA) receptors in some cases of encephalitis, characterized by cognitive changes, memory loss, and seizures that could lead to long-term morbidity or mortality. Treatment approaches and experimental studies have largely focused on the pathogenic role of these autoantibodies. Passive antibody transfer to mice has provided useful insights but does not produce the full spectrum of the human disease. Here, we describe a de novo autoimmune mouse model of anti-NMDA receptor encephalitis. Active immunization of immunocompetent mice with conformationally stabilized, native-like NMDA receptors induced a fulminant encephalitis, consistent with the behavioral and pathologic characteristics of human cases. Our results provide evidence for neuroinflammation and immune cell infiltration as components of the autoimmune response in mice. Use of transgenic mice indicated that mature T cells and antibody-producing cells were required for disease induction. This active immunization model may provide insights into disease induction and a platform for testing therapeutic approaches.

Conflict of interest statement

Competing interests: All authors declare that they have no competing interests.

Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Figure 1.. Clinical phenotype in proteoliposome-treated mice

(A) Timeline of treatment and behavioral testing. Adult wild-type mice received subcutaneous injections at day 0 and at day 15 with proteoliposome (purple) or control - liposome (green) or saline (blue). (B) Clinical signs were plotted from the first injection. (C) Clinical signs in proteoliposome-treated mice (D) Kaplan-Meier survival plot for proteoliposome- and control-treated mice.

Figure 2.. Behavioral assessment

(A) Representative movement traces in the open field from a control mouse (field outlined in green) and two proteoliposome treated-mice (field outlined in purple) to examine hyperactivity. Total distance moved in the treatment groups are plotted at right: proteoliposome (purple), liposome (green) and saline (blue) purple. (B) Representative images of a nest created by a control mouse compared to nests in two proteoliposome treated-mice. Nests were scored at 24 and 48 hours and quantified as shown at right. (C) Mice were assessed in the zero maze for time spent in the open area (left) and for the total distance moved in the open and closed areas.

Figure 3.. Histological assessment

(A) Representative Images of perivascular cuffing (black arrow, right panels) in hippocampal and neocortical tissue from a proteoliposome-treated mouse. Matched samples from liposome-treated controls are shown at left. (B) Representative tissue sections were used to examine for areas of cell loss including karyolysis (single white arrow) and pyknosis (double white arrow) in a proteoliposome- treated mouse (right), compared to control (left). Scale bar: 100µm.

Figure 4.. Glial cell labeling in proteoliposome-treated mice

(A) Immunofluorescence for GFAP (white) in proteoliposome-treated mice (right) compared to controls (left). The higher magnification insets from the hippocampus show individual astrocytes. The histogram shows quantification of GFAP immunofluorescence (see methods). (B) Labeling with the microglial marker Iba1 (white) in proteoliposome-treated mice (right) compared to control (left). Higher magnification inset shows microglia in the hippocampus of proteoliposome-treated mouse compared to control. Immunofluorescence was quantified for Iba1 as for GFAP. Scale bar: 1000µm (inset 100µm).

Figure 5.. Immunohistochemical labeling of CNS immune cell infiltrates

(A) Labeling with the pan-leukocyte marker, CD45R in coronal sections from a proteoliposome treated-mouse (right) and control mouse (left). Insets show labeling of individual CD45R+ cells (white) from the indicated region of the hippocampus. CD45R+ cells in controls (left). Scale bar: 1000µm (inset 100µm). (B) Immunohistochemical labeling for a battery of immune cell markers in brains of proteoliposome-treated mice and control mice (CD8+, CD4+, CD20+, CD138+, Gal3+). Proteoliposome treated-mice showed immune cell infiltrates as indicated with the insets from the hippocampal region; [Scale bar: 500µm (inset 200µm). (C) Left: CD45R+ density (cells/µm3) in striatum (Str), cortex (Ctx), amygdala (Amyg), hippocampus (Hipp) and thalamus (Thal) in proteoliposome-treated (black) and control mice (gray). Right: CD8+, CD4+, CD20+, CD138+, Galectin3+ cell densities (cells/µm3) in the hippocampus of proteoliposome treated (black) and control mice (gray). Only Gal3+ cells were of sufficient density to be apparent above zero on the histogram in control mice.

Figure 6.. Detection of NMDA receptor subtype-specific serum antibodies and localization of IgG binding to NMDA receptors

(A) HEK293FT cells transfected with rat GluN½A subunits were labeled with anti-GluN1 antibody (left, top and bottom, green) or with IgG from proteoliposome-treated mice (bottom, middle, red). Merge panel shows colocalization (bottom, right, yellow). IgG derived from control mice (top middle panel, red) showed no labeling. Scale bar: 15µm. (B) Dendrites of cultured hippocampal neurons with the expected punctate pattern of synapses using an anti-GluN1 antibody (green, left) compared to IgG from proteoliposome-treated mice (bottom row, middle, red). The merge image is shown at bottom row, right, yellow. IgG from liposome-treated mice (top row, middle) compared to puncta observed with anti-GluN1 (top row, left and right, green). Dendritic shafts were labeled with anti-MAP2 antibody (gray). Scale bar: 5µm. (C) The pattern of immunoreactivity in hippocampal tissue sections for a commercial NMDA receptor antibody (GluN2A) in an untreated wildtype mouse (top) compared to labeling using purified IgG derived from a proteoliposome-treated mouse (bottom) and purified IgG derived from a liposome-treated mouse. (Scale bar: 500µm). (D) A battery of recombinant GFP-tagged NMDA receptor subunits from Xenopus (XI) and Rat (R) were blotted and detected with anti-GFP antibodies (red, top panel). Incubation with serum (1:100) derived from a liposome-treated mouse (green, middle panel). Serum (1:100) derived from a proteoliposome-treated mouse showed bands corresponding to GluN1 subunit isoforms in Xenopus and rat (Xl.GluN1–3a, R.GluN1–1a, R.GluN1–1b), Xenopus GluN2B as well as a Xenopus GluN1 lacking the ATD domain (XI.NR1–3a ΔATD). Blots are from representative liposome-treated control mice as well as proteoliposome-treated mice with clinical signs of disease.

Figure 7.. Electrophysiological effects in neurons following acute and chronic exposure to serum from proteoliposome-treated mice

(A) Representative whole-cell currents in a neuron evoked by acute flow-pipe application of NMDA + serum from liposome-treated, or NMDA + serum from proteoliposome-treated mice, respectively. Serum dilution was 1:100. (B) Quantification of evoked whole-cell NMDA currents in neurons following acute serum application. (C, D) Spontaneous excitatory postsynaptic currents were recorded following 24-hour incubation (“chronic”) in serum from liposome or proteoliposome-treated mice. (C) Representative sEPSC traces in cells treated with serum from liposome-treated before and after D-AP5 application (top, left and right traces, respectively) and cells treated with proteoliposome-derived serum (bottom, left and right traces, respectively). (D) Quantification of D-AP5 induced reduction in sEPSCs shown in histogram. (E) Immunocytochemical labeling of dendrites, PSD-95, and GluN1 puncta in cultured hippocampal neurons following 24hr incubated in serum from liposome and proteoliposome-treated mice. PSD-95 immunoreactivity (left, top & bottom, red). GluN1 labeled puncta from control- and proteoliposome-treated mice (middle, top and bottom, green; respectively). Right panels show overlap of GluN1 and PSD-95 immunolabeling (right, top and bottom, yellow). Dendrites are labeled with anti-MAP antibody (gray). Scale bar: 10µm. Quantification of PSD-95 and GluN1 positive synaptic puncta per µm of dendrite shown in left and right histograms (liposome = gray; proteoliposome = black). Quantification of PSD-95 and GluN1 positive synaptic puncta per µm of dendrite shown in left and right histograms (liposome = gray; proteoliposome = black).

Figure 8.. Response of T cells mutant mice to proteoliposome treatment

(A) Tcrα− mutant mice were immunized in parallel with a cohort of wildtype mice. Graphs show clinical observations and mortality rates by 12 weeks post-immunization (liposome = green; proteoliposome = purple). (B) CD45R (top panels), GFAP (middle panels) or Iba1 (bottom panels) immunolabeling in proteoliposome- and liposome-treated TCRα− mice. Scale bar: 500µm (C)) Immunocytochemical colabeling of GluN1 and purified IgG from control- and proteoliposome-treated TCRα− mice in hippocampal neuronal cultures (top panels) and HEK cells expressing GluN1-GluN2A subunits (bottom panels). The upper two rows show GluN1 positive puncta (left, green) along a dendrite (MAP2, gray) or lack of staining for IgG from liposome-treated mice (top middle, red) or IgG from proteoliposome treated-mice (bottom middle panel, red). Scale bar = 5µm. The lower two rows show anti-GluN1 antibody labeling of HEK293FT cells expressing rat GluN½A subunits (left, green), and absent labeling for IgG from proteoliposome-treated or liposome-treated mice (bottom two rows, middle). Scale bar: 15µm.