Neuro-immunobiology and treatment assessment in a mouse model of anti-NMDAR encephalitis

Abstract

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a disorder mediated by autoantibodies against the GluN1 subunit of NMDAR. It occurs with severe neuropsychiatric symptoms that often improve with immunotherapy. Clinical studies and animal models based on patients' antibody transfer or NMDAR immunization suggest that the autoantibodies play a major pathogenic role. Yet, there is an important need for models offering an all-inclusive neuro-immunobiology of the disease together with a clinical course long enough to facilitate the assessment of potential new treatments. To develop such a model, eight-week-old female mice (C57BL/6J) were immunized (Days 1 and 28) with GluN1356-385 peptide or saline with AddaVaxTM adjuvant and pertussis toxin. After developing behavioural alterations (∼Day 35), subsets of mice were treated with an anti-CD20 (Day 35), a positive allosteric modulator (PAM) of NMDAR (PAM-NMDAR, SGE-301) from Days 45 to 71, or both. GluN1-antibody synthesis, epitope spreading, effects of antibodies on density and function of NMDAR, brain immunological infiltrates, microglial activation and NMDAR phagocytosis, and antibody synthesis in cultured inguinal and deep cervical lymph nodes were assessed with techniques including immunohistochemistry, calcium imaging, confocal and super-resolution microscopy, electrophysiology and flow cytometry. Changes in memory and behaviour were assessed with a panel of behavioural tests and clinical/subclinical seizures with brain-implanted electrodes. Immunized mice, but not controls, developed serum and CSF NMDAR-antibodies (100% IgG1, 40% IgG2) against the immunizing peptide and other GluN1 regions (epitope spreading) resulting in a decrease of synaptic and extrasynaptic NMDAR clusters and reduction of hippocampal plasticity. These findings were associated with brain inflammatory infiltrates, mainly B- and plasma cells, microglial activation, co-localization of NMDAR-IgG complexes with microglia, and presence of these complexes within microglial endosomes. Cultures of DCLC showed GluN1-antibody synthesis. These findings were associated with psychotic-like behaviour (predominant at disease onset), memory deficit, depressive-like behaviour, abnormal movements (14% of mice), and lower threshold for developing pentylenetetrazole-induced seizures (hypoactivity, myoclonic jerks, continuous tonic-clonic), which correlated with regional cFOS expression. Most behavioural signs and neurobiological alterations were reversed by the anti-CD20 and PAM-NMDAR, alone or combined. Initial repopulation of B cells, by the end of the study, was associated with re-emergence of clinical-neurobiological alterations, which were abrogated by PAM-NMDAR. This model offers an all-inclusive neuro-immunobiology of the disease, allowing for the testing of novel treatments, supporting the potential therapeutic role of PAM-NMDAR, and suggesting an immunological paradigm of systemic antigen presentation and brain NMDAR epitope spreading, which along the deep cervical lymph nodes, might contribute to fine-tune the polyclonal immune response.

Keywords: NMDA receptor encephalitis; SGE-301; active immunization; animal model; immunotherapy; treatments.