SEE ZEKERIDOU AND LENNON DOI101093/AWW213 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a recently discovered autoimmune syndrome associated with psychosis, dyskinesias, and seizures. Little is known about the cerebrospinal fluid autoantibody repertoire. Antibodies against the NR1 subunit of the NMDAR are thought to be pathogenic; however, direct proof is lacking as previous experiments could not distinguish the contribution of further anti-neuronal antibodies. Using single cell cloning of full-length immunoglobulin heavy and light chain genes, we generated a panel of recombinant monoclonal NR1 antibodies from cerebrospinal fluid memory B cells and antibody secreting cells of NMDAR encephalitis patients. Cells typically carried somatically mutated immunoglobulin genes and had undergone class-switching to immunoglobulin G, clonally expanded cells carried identical somatic hypermutation patterns. A fraction of NR1 antibodies were non-mutated, thus resembling 'naturally occurring antibodies' and indicating that tolerance induction against NMDAR was incomplete and somatic hypermutation not essential for functional antibodies. However, only a small percentage of cerebrospinal fluid-derived antibodies reacted against NR1. Instead, nearly all further antibodies bound specifically to diverse brain-expressed epitopes including neuronal surfaces, suggesting that a broad repertoire of antibody-secreting cells enrich in the central nervous system during encephalitis. Our functional data using primary hippocampal neurons indicate that human cerebrospinal fluid-derived monoclonal NR1 antibodies alone are sufficient to cause neuronal surface receptor downregulation and subsequent impairment of NMDAR-mediated currents, thus providing ultimate proof of antibody pathogenicity. The observed formation of immunological memory might be relevant for clinical relapses.

Objective: Maternal autoantibodies are a risk factor for impaired brain development in offspring. Antibodies (ABs) against the NR1 (GluN1) subunit of the N-methyl-D-aspartate receptor (NMDAR) are among the most frequently diagnosed anti-neuronal surface ABs, yet little is known about effects on fetal development during pregnancy. Methods: We established a murine model of in utero exposure to human recombinant NR1 and isotype-matched nonreactive control ABs. Pregnant C57BL/6J mice were intraperitoneally injected on embryonic days 13 and 17 each with 240μg of human monoclonal ABs. Offspring were investigated for acute and chronic effects on NMDAR function, brain development, and behavior. Results: Transferred NR1 ABs enriched in the fetus and bound to synaptic structures in the fetal brain. Density of NMDAR was considerably reduced (up to −49.2%) and electrophysiological properties were altered, reflected by decreased amplitudes of spontaneous excitatory postsynaptic currents in young neonates (−34.4%). NR1 AB-treated animals displayed increased early postnatal mortality (+27.2%), impaired neurodevelopmental reflexes, altered blood pH, and reduced bodyweight. During adolescence and adulthood, animals showed hyperactivity (+27.8% median activity over 14 days), lower anxiety, and impaired sensorimotor gating. NR1 ABs caused long-lasting neuropathological effects also in aged mice (10 months), such as reduced volumes of cerebellum, midbrain, and brainstem. Interpretation: The data collectively support a model in which asymptomatic mothers can harbor low-level pathogenic human NR1 ABs that are diaplacentally transferred, causing neurotoxic effects on neonatal development. Thus, ABmediated network changes may represent a potentially treatable neurodevelopmental congenital brain disorder contributing to lifelong neuropsychiatric morbidity in affected children.

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1–3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6–8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.