Autoantibodies against neuronal membrane proteins can manifest in autoimmune encephalitis, inducing seizures, cognitive dysfunction and psychosis. Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is the most dominant autoimmune encephalitis; however, insights into how autoantibodies recognize and alter receptor functions remain limited. Here we determined structures of human and rat NMDARs bound to three distinct patient-derived antibodies using single-particle electron cryo-microscopy. These antibodies bind different regions within the amino-terminal domain of the GluN1 subunit. Through electrophysiology, we show that all three autoantibodies acutely and directly reduced NMDAR channel functions in primary neurons. Antibodies show different stoichiometry of binding and antibody-receptor complex formation, which in one antibody, 003-102, also results in reduced synaptic localization of NMDARs. These studies demonstrate mechanisms of diverse epitope recognition and direct channel regulation of anti-NMDAR autoantibodies underlying autoimmune encephalitis. Anti-NMDA receptor encephalitis is the most common autoimmune encephalitis. Michalski et al. reveal epitope diversity, conformational changes and functional impacts of the autoantibodies using cryo-EM and electrophysiology.

Sepsis-associated encephalopathy (SAE) is a frequent complication of severe systemic infection resulting in delirium, premature death, and long-term cognitive impairment. We closely mimicked SAE in a murine peritoneal contamination and infection (PCI) model. We found long-lasting synaptic pathology in the hippocampus including defective long-term synaptic plasticity, reduction of mature neuronal dendritic spines, and severely affected excitatory neurotransmission. Genes related to synaptic signaling, including the gene for activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and members of the transcription-regulatory EGR gene family, were downregulated. At the protein level, ARC expression and mitogen-activated protein kinase signaling in the brain were affected. For targeted rescue we used adeno-associated virus-mediated overexpression of ARC in the hippocampus in vivo. This recovered defective synaptic plasticity and improved memory dysfunction. Using the enriched environment paradigm as a non-invasive rescue intervention, we found improvement of defective long-term potentiation, memory, and anxiety. The beneficial effects of an enriched environment were accompanied by an increase in brain-derived neurotrophic factor (BDNF) and ARC expression in the hippocampus, suggesting that activation of the BDNF-TrkB pathway leads to restoration of the PCI-induced reduction of ARC. Collectively, our findings identify synaptic pathomechanisms underlying SAE and provide a conceptual approach to target SAE-induced synaptic dysfunction with potential therapeutic applications to patients with SAE.

Sepsis-associated encephalopathy (SAE) is a severe and frequent complication of sepsis causing delirium, coma, and long-term cognitive dysfunction. We identified microglia and C1q complement activation in hippocampal autopsy tissue of patients with sepsis and increased C1q-mediated synaptic pruning in a murine polymicrobial sepsis model. Unbiased transcriptomics of hippocampal tissue and isolated microglia derived from septic mice revealed an involvement of the innate immune system, complement activation, and up-regulation of lysosomal pathways during SAE in parallel to neuronal and synaptic damage. Microglial engulfment of C1q-tagged synapses could be prevented by stereotactic intrahippocampal injection of a specific C1q-blocking antibody. Pharmacologically targeting microglia by PLX5622, a CSF1-R inhibitor, reduced C1q levels and the number of C1q-tagged synapses, protected from neuronal damage and synapse loss, and improved neurocognitive outcome. Thus, we identified complement-dependent synaptic pruning by microglia as a crucial pathomechanism for the development of neuronal defects during SAE.

Anti-NMDA receptor autoantibodies (NMDAR-Abs) in patients with NMDAR encephalitis cause severe dis-ease symptoms resembling psychosis and cause cognitive dysfunction. After passive transfer of patients' cerebrospinal fluid or human monoclonal anti-GluN1-autoantibodies in mice, we find a disrupted excit-atory-inhibitory balance resulting from CA1 neuronal hypoexcitability, reduced AMPA receptor (AMPAR) signaling, and faster synaptic inhibition in acute hippocampal slices. Functional alterations are also reflected in widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. NMDAR-Abs amplify network g oscillations and disrupt q -g coupling. A data-informed network model reveals that lower AMPAR strength and faster GABAA receptor current kinetics chiefly ac-count for these abnormal oscillations. As predicted in silico and evidenced ex vivo, positive allosteric mod-ulation of AMPARs alleviates aberrant g activity, reinforcing the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-Ab-induced aberrant synaptic, cellular, and network dynamics provide con-ceptual insights into NMDAR-Ab-mediated pathomechanisms and reveal promising therapeutic targets that merit future in vivo validation.

In comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings, allowing translation of gene expression information into their spatial context. This enables to unravel complex interactions of neighboring cells or to link cell morphology to transcriptome data. The 10× Genomics Visium platform offers to combine spatial transcriptomics with immunofluorescent staining of cryo-sectioned tissue slices. We applied this technique to fresh frozen mouse brain slices and developed a protocol that still protects RNA quality while improving buffers for immunofluorescent staining. We investigated the impact of various parameters, including fixation time and buffer composition, on RNA quality and antibody binding. Here, we propose an improved version of the manufacturer protocol, which does not alter RNA quality and facilitates the use of multiple additional antibodies that were not compatible with the manufacturer protocol before. Finally, we discuss the influence of various staining parameters, which contribute to the development of application specific staining protocols.

AbstractSpecific autoantibodies against the NMDA-receptor (NMDAR) GluN1 subunit cause severe and debilitating NMDAR-encephalitis. Autoantibodies induce prototypic disease symptoms resembling schizophrenia, including psychosis and cognitive dysfunction. Using a mouse passive transfer model applying human monoclonal anti-GluN1-autoantibodies, we observed CA1 pyramidal neuron hypoexcitability, reduced AMPA-receptor (AMPAR) signaling, and faster synaptic inhibition resulting in disrupted excitatory-inhibitory balance. Functional alterations were supported by widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. At the network level, anti-GluN1-autoantibodies amplified gamma oscillations and disrupted theta-gamma coupling. A data-informed network model revealed that lower AMPAR strength and faster GABAA-receptor current kinetics chiefly account for these abnormal oscillations. As predicted by our model and evidenced experimentally, positive allosteric modulation of AMPARs alleviated aberrant gamma activity and thus reinforced the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-hypofunction-induced aberrant synaptic, cellular, and network dynamics provide new mechanistic insights into disease symptoms in NMDAR-encephalitis and schizophrenia.

Center for Sepsis Control and Care, 2 Section Translational Neuroimmunology Department of Neurology, Department of Anesthesiology and Intensive Care, Jena University Hospital, Am Klinikum 1, 07747 Jena, Germany; 4 Institute of Pathophysiology, Johannes Gutenberg University Mainz, 55131 Mainz, Germany, 5 Department of Psychiatry, Psychosomatic Medicine and Psychotherapy, University Hospital of Frankfurt, 60528 Frankfurt, Germany, 6 Institute of Precision Medicine, Medical and Life Sciences Faculty, Furtwangen University, 78054 Villingen-Schwenningen, Germany

Sepsis-associated encephalopathy (SAE) is a frequent complication in patients with severe systemic infection resulting in acute brain dysfunction and incapacitating long-term sequelae. SAE includes delirium, premature death, post-traumatic stress disorder, and major long-term cognitive impairment. The underlying pathophysiology of SAE is largely unresolved and specific treatment options are missing. We induced the peritoneal contamination and infection (PCI) sepsis model in 769 mice and compared these with 259 control mice. We found that experimental sepsis causes synaptic pathology in the brain characterized by severely disordered synaptic plasticity with reduced long-term potentiation, changes in CA1 pyramidal neuron dendritic spines, and behavioral abnormalities indicating cognitive dysfunction. Using electrophysiology, we found reduced frequency of quantal and spontaneous excitatory postsynaptic currents whereas amplitudes of miniature, spontaneous, and evoked excitatory currents were increased, pointing towards a homeostatic synaptic scaling mechanism. Corresponding to dysfunctional excitatory synaptic function we discovered downregulation of genes related to neuronal and synaptic signaling in the brain, including the gene for activity-regulated cytoskeleton-associated protein ( Arc/Arg3.1 ), members of the transcription-regulatory EGR gene family, and the gene for dual-specificity phosphatase 6 ( Dusp6 ). At the protein level, ARC expression and MAP kinase signaling in the brain were affected. For targeted rescue of dysfunctional synaptic signaling and plasticity, we overexpressed ARC in the hippocampus by microinjection of an adeno-associated virus containing a neuron-specific plasmid of the ARC transgene. Hereby we achieved recovery of defective synaptic plasticity in the hippocampal Schaffer collateral-CA1 pathway and improvement of memory dysfunction. Using a different rescue paradigm, PCI mice were subjected to enriched environment providing multiple activating stimuli. Enriched environment led to restoration of disordered long-term potentiation and memory, thus demonstrating the potential for activity-induced improvement. Together, we identified synaptic pathomechanisms of SAE after severe systemic infection and provide a conceptual approach to treat SAE-related disease mechanisms which may be applicable to patients afflicted with SAE.

We investigated the pathogenic role of anti-N-Methyl-D-aspartate receptor (NMDAR) antibodies in neurons and in a mouse model of autoimmune encephalitis using LSM and super-resolution microscopy. We found a functional reduction of synaptic NMDARs.

We present an approach for fabrication of reproducible, chemically and mechanically robust functionalized layers based on MgF2 thin films on thin glass substrates. These show great advantages for use in super-resolution microscopy as well as for multi-electrode-array fabrication and are especially suited for combination of these techniques. The transparency of the coated substrates with the low refractive index material is adjustable by the layer thickness and can be increased above 92%. Due to the hydrophobic and lipophilic properties of the thin crystalline MgF2 layers, the temporal stable adhesion needed for fixation of thin tissue, e.g. cryogenic brain slices is given. This has been tested using localization-based super-resolution microscopy with currently highest spatial resolution in light microscopy. We demonstrated that direct stochastic optical reconstruction microscopy revealed in reliable imaging of structures of central synapses by use of double immunostaining of post- (homer1 and GluA2) and presynaptic (bassoon) marker structure in a 10 µm brain slice without additional fixing of the slices. Due to the proven additional electrical insulating effect of MgF2 layers, surfaces of multi-electrode-arrays were coated with this material and tested by voltage-current-measurements. MgF2 coated multi-electrode-arrays can be used as a functionalized microscope cover slip for combination with live-cell super-resolution microscopy.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common autoimmune encephalitis related to autoantibody-mediated synaptic dysfunction. Cerebrospinal fluid-derived human monoclonal NR1 autoantibodies showed low numbers of somatic hypermutations or were unmutated. These unexpected germline-configured antibodies showed weaker binding to the NMDAR than matured antibodies from the same patient. In primary hippocampal neurons, germline NR1 autoantibodies strongly and specifically reduced total and synaptic NMDAR currents in a dose- and time-dependent manner. The findings suggest that functional NMDAR antibodies are part of the human naïve B cell repertoire. Given their effects on synaptic function, they might contribute to a broad spectrum of neuropsychiatric symptoms. Ann Neurol 2019;85:771-776.

AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor sub-units, providing a framework to explain disease symptoms.