N-Methyl-D-Aspartate Receptor (NMDAR) Antibodies Induce Diverse Calcium Activity Patterns on Cultured Astrocytes and Astrocyte–Neuron Cocultures

Background

Preliminary evidence suggests that astrocytes might contribute to the disease mechanisms of N-methyl-D-aspartate receptor (NMDAR) encephalitis. However, the precise role of astrocytes in the pathogenesis of encephalitis is poorly understood.

Methods

To characterize the impact of NMDAR antibodies on the activity of astrocytes, we isolated and purified serum IgG from three patients with NMDAR encephalitis and three healthy controls. Primary cultured mouse astrocytes and neuron–astrocyte cocultures were incubated with 5 μg patient or control IgG for 12 and 24 h. Cell viability and astrocyte activity were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and calcium (Ca²⁺) imaging studies (using a vector expressing a genetically encoded indicator under the GFAP promoter), respectively. Supernatant levels of TNF-α, a mediator of astrocyte–neuron interactions, were measured by ELISA.

Results

Purified serum IgG from N-methyl-D-aspartate receptor-antibody positive patients (NMDAR-Ab+ IgG) and healthy controls did not alter the viability of isolated astrocytes and cocultures. NMDAR-Ab+ IgG suppressed the duration and amplitude of Ca²⁺ activity of isolated astrocytes at 12 and/or 24 h, whereas all parameters of Ca²⁺ activity were enhanced by NMDAR-Ab+ IgG in astrocyte–neuron cocultures at 12 h. TNF-α levels were increased in supernatants of NMDAR-Ab+ IgG–administered astrocyte–neuron cocultures but not isolated astrocytes.

Discussion

This study provides the first preliminary evidence on the impact of patient-derived NMDAR-Ab+ IgG on astrocytic activity. Our results imply that the interaction of NMDAR-Abs with neurons and astrocytes creates a positive feedback loop reinforcing the activation of astrocytes. TNF-α might be one of the perpetrators of this loop.