Pathomechanisms of AMPA receptor signalling in chronic CNS inflammation

Autoimmune Encephalitis (AE) is a new group of disorders characterized by autoantibodies (aABs) to synaptic surface antigens. Currently, there are 16 different subformes of AE described with aABs to postsynaptic and presynaptic, to vesicular proteins as well as to synaptic anchoring proteins. AE can be treated by immunotherapy, but recovery is often prolonged and takes several weeks. Intensive care is needed in severe cases. Specific treatment is difficult since for most subforms of AE the molecular mechanisms of aAB action are still unknown. This work focuses on investigating the molecular mechanisms of aABs in AE. Therefore, high resolution imaging, electrophysiological recordings, and behavioral tests in in-vitro models and in-vivo animal models were used. With these new methods, several molecular mechanisms of aAB action in AE were uncovered: In AMPA receptor AE aABs lead to synaptic scaling by replacing the synaptic AMPA receptor subunit composition leading to changes in synaptic transmission, impairment in long term potentiation, and defects in learning and memory. In an osmotic pump infusion animal model of NMDA receptor AE the internalization of NMDA receptors by aABs can be antagonized by addition of the EphrineB2 receptor agonist ephrine-B2 leading to a rescue of disease symptoms. Taken together, this work elucidates the mechanisms of different subtypes of AE and demonstrates new and antigen-specific treatment approaches that may become therapeutic options in patients in the future.Die autoimmune Enzephalitis ist eine neue Gruppe von Krankheiten die durch Autoantikörper gegen synaptische Membranproteine gekennzeichnet ist. Zum heutigen Stand sind 16 verschiedene Typen von autoimmuner Enzephalitis mit Autoantikörpern gegen postsynaptische, präsynaptische, vesikuläre und synaptische Ankerproteine bekannt. Autoimmune Enzephalitis kann durch Immunotherapie behandelt werden, die Genesung von dieser Krankheit dauert allerdings oft mehrere Wochen und in schweren Fällen ist mitunter eine intensivmedizinische Behandlung notwendig. Eine spezifische Behandlung für viele Typen der autoimmunen Enzephalitis ist schwierig, da bisher die molekulare Wirkweise der Autoantikörper noch immer unbekannt ist. Die vorliegende Arbeit fokussiert sich auf die Untersuchung der molekularen Mechanismen von Autoantikörpern mittels hochauflösender Mikroskopie, elektrophysiologischer Messungen und Verhaltenstests in in-vitro Zellmodellen und in-vivo Tiermodellen. Durch diese neuen Methoden konnten Wirkmechanismen von Autoantikörpern bei der autoimmunen Enzephalitis aufgeklärt werden: Bei der AMPA Rezeptor Autoimmunenzephalitis bewirken Autoantikörper die synaptische Reorganisation durch die Änderung der AMPA Rezeptorzusammensetzung. Dies führt zu Änderungen in der synaptischen Transmission, Störungen in der Langzeitpotenzierung und Defiziten beim Lernen und der Gedächtnisleistung. Bei der NMDA Rezeptor Autoimmunenzephalitis kann die Internalisierung von NMDA Rezeptoren durch die Gabe des EphrineB2 Rezeptor Agonisten Ephrin-B2 im Tiermodell verhindert werden. Zusammengefasst untersucht diese Arbeit die pathogenen Mechanismen von Autoantikörpern in verschiedenen Subtypen autoimmuner Enzephalitiden und zeigt erste Möglichkeiten für die Entwicklung potentieller neuer und Antigen-spezifischer Behandlungsmöglichkeiten beim Patienten

LGI1 antibodies alter Kv1.1 and AMPA receptors changing synaptic excitability, plasticity and memory

Leucine-rich glioma-inactivated 1 (LGI1) is a secreted neuronal protein that forms a trans-synaptic complex that includes the presynaptic disintegrin and metalloproteinase domain-containing protein 23 (ADAM23), which interacts with voltage-gated potassium channels Kv1.1, and the postsynaptic ADAM22, which interacts with AMPA receptors. Human autoantibodies against LGI1 associate with a form of autoimmune limbic encephalitis characterized by severe but treatable memory impairment and frequent faciobrachial dystonic seizures. Although there is evidence that this disease is immune-mediated, the underlying LGI1 antibody-mediated mechanisms are unknown. Here, we used patient-derived immunoglobulin G (IgG) antibodies to determine the main epitope regions of LGI1 and whether the antibodies disrupt the interaction of LGI1 with ADAM23 and ADAM22. In addition, we assessed the effects of patient-derived antibodies on Kv1.1, AMPA receptors, and memory in a mouse model based on cerebroventricular transfer of patient-derived IgG. We found that IgG from all patients (n = 25), but not from healthy participants (n = 20), prevented the binding of LGI1 to ADAM23 and ADAM22. Using full-length LGI1, LGI3, and LGI1 constructs containing the LRR1 domain (EPTP1-deleted) or EPTP1 domain (LRR3-EPTP1), IgG from all patients reacted with epitope regions contained in the LRR1 and EPTP1 domains. Confocal analysis of hippocampal slices of mice infused with pooled IgG from eight patients, but not pooled IgG from controls, showed a decrease of total and synaptic levels of Kv1.1 and AMPA receptors. The effects on Kv1.1 preceded those involving the AMPA receptors. In acute slice preparations of hippocampus, patch-clamp analysis from dentate gyrus granule cells and CA1 pyramidal neurons showed neuronal hyperexcitability with increased glutamatergic transmission, higher presynaptic release probability, and reduced synaptic failure rate upon minimal stimulation, all likely caused by the decreased expression of Kv1.1. Analysis of synaptic plasticity by recording field potentials in the CA1 region of the hippocampus showed a severe impairment of long-term potentiation. This defect in synaptic plasticity was independent from Kv1 blockade and was possibly mediated by ineffective recruitment of postsynaptic AMPA receptors. In parallel with these findings, mice infused with patient-derived IgG showed severe memory deficits in the novel object recognition test that progressively improved after stopping the infusion of patient-derived IgG. Different from genetic models of LGI1 deficiency, we did not observe aberrant dendritic sprouting or defective synaptic pruning as potential cause of the symptoms. Overall, these findings demonstrate that patient-derived IgG disrupt presynaptic and postsynaptic LGI1 signalling, causing neuronal hyperexcitability, decreased plasticity, and reversible memory deficits

Combining super-resolution microcopy with neuronal network recording using magnesium fluoride thin films as cover layer for multi-electrode array technology

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

Mechanosensing in actin stress fibers revealed by a close correlation between force

The mechanics of the actin cytoskeleton have a central role in the regulation of cells and tissues, but the details of how molecular sensors recognize deformations and forces are elusive. By performing cytoskeleton laser nanosurgery in cultured epithelial cells and fibroblasts, we show that the retraction of stress fibers (SFs) is restricted to the proximity of the cut and that new adhesions form at the retracting end. This suggests that SFs are attached to the substrate. A new computational model for SFs confirms this hypothesis and predicts the distribution and propagation of contractile forces along the SF. We then analyzed the dynamics of zyxin, a focal adhesion protein present in SFs. Fluorescent redistribution after laser nanosurgery and drug treatment shows a high correlation between the experimentally measured localization of zyxin and the computed localization of forces along SFs. Correlative electron microscopy reveals that zyxin is recruited very fast to intermediate substrate anchor points that are highly tensed upon SF release. A similar acute localization response is found if SFs are mechanically perturbed with the cantilever of an atomic force microscope. If actin bundles are cut by nanosurgery in living Drosophila egg chambers, we also find that zyxin redistribution dynamics correlate to force propagation and that zyxin relocates at tensed SF anchor points, demonstrating that these processes also occur in living organisms. In summary, our quantitative analysis shows that force and protein localization are closely correlated in stress fibers, suggesting a very direct force-sensing mechanism along actin bundles

Human NMDAR autoantibodies disrupt excitatory-inhibitory balance, leading to hippocampal network hypersynchrony

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

Ephrin-B2 prevents N-methyl-D-aspartate receptor antibody effects on memory and neuroplasticity

OBJECTIVE: To demonstrate that ephrin-B2 (the ligand of EphB2 receptor) antagonizes the pathogenic effects of patients' N-methyl-D-aspartate receptor (NMDAR) antibodies on memory and synaptic plasticity. METHODS: One hundred twenty-two C57BL/6J mice infused with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, with or without ephrin-B2, were investigated. CSF was infused through ventricular catheters connected to subcutaneous osmotic pumps over 14 days. Memory, behavioral tasks, locomotor activity, presence of human antibodies specifically bound to hippocampal NMDAR, and antibody effects on the density of cell-surface and synaptic NMDAR and EphB2 were examined at different time points using reported techniques. Short- and long-term synaptic plasticity were determined in acute brain sections; the Schaffer collateral pathway was stimulated and the field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. RESULTS: Mice infused with patients' CSF, but not control CSF, developed progressive memory deficit and depressive-like behavior along with deposits of NMDAR antibodies in the hippocampus. These findings were associated with a decrease of the density of cell-surface and synaptic NMDAR and EphB2, and marked impairment of long-term synaptic plasticity without altering short-term plasticity. Administration of ephrin-B2 prevented the pathogenic effects of the antibodies in all the investigated paradigms assessing memory, depressive-like behavior, density of cell-surface and synaptic NMDAR and EphB2, and long-term synaptic plasticity. INTERPRETATION: Administration of ephrin-B2 prevents the pathogenic effects of anti-NMDAR encephalitis antibodies on memory and behavior, levels of cell-surface NMDAR, and synaptic plasticity. These findings reveal a strategy beyond immunotherapy to antagonize patients' antibody effects. Ann Neurol 2016; 80: 388-400.This study was supported, in part, by Instituto Carlos III/FEDER (FIS 15/00377 [to F.G.], FIS 14/00203 and CIBERER [to J.D.], and RETICS-RTA and RD12/0028/0023 [to R.M.]), NIH RO1NS077851 (to J.D.), MINECO (SAF2014-59648-P; to R.M.), European Commission (HEALTH-F2-2013-602891; to R.M.), Fundació Cellex (to J.D.), the Netherlands Organisation for Scientific Research (NWO, Veni incentive; to M.T.), an Erasmus MC fellowship (to M.T.), and the German Research Council (DFG; GE 2519/3-1 and CRC-TR 166/1 B2 [to C.G.]

Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction

AMPA receptors are essential for fast excitatorytransmission in the CNS. Autoantibodies to AMPAreceptors have been identified in humans withautoimmune encephalitis and severe defects ofhippocampal function. Here, combining electrophysiologyand high-resolution imaging withneuronal culture preparations and passive-transfermodels in wild-type and GluA1-knockout mice,we analyze how specific human autoantibodiesagainst the AMPA receptor subunit GluA2 affect receptorfunction and composition, synaptic transmission,and plasticity. Anti-GluA2 antibodiesinduce receptor internalization and a reduction ofsynaptic GluA2-containing AMPARs followed bycompensatory ryanodine receptor-dependent incorporationof synaptic non-GluA2 AMPARs. Furthermore,application of human pathogenic anti-GluA2antibodies to mice impairs long-term synaptic plasticityin vitro and affects learning and memoryin vivo. Our results identify a specific immuneneuronalrearrangement of AMPA receptor subunits,providing a framework to explain diseasesymptoms

Ephrin-B2 prevents N-methyl-D-aspartate receptor antibody effects on memory and neuroplasticity

OBJECTIVE: To demonstrate that ephrin-B2 (the ligand of EphB2 receptor) antagonizes the pathogenic effects of patients' N-methyl-D-aspartate receptor (NMDAR) antibodies on memory and synaptic plasticity. METHODS: One hundred twenty-two C57BL/6J mice infused with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, with or without ephrin-B2, were investigated. CSF was infused through ventricular catheters connected to subcutaneous osmotic pumps over 14 days. Memory, behavioral tasks, locomotor activity, presence of human antibodies specifically bound to hippocampal NMDAR, and antibody effects on the density of cell-surface and synaptic NMDAR and EphB2 were examined at different time points using reported techniques. Short- and long-term synaptic plasticity were determined in acute brain sections; the Schaffer collateral pathway was stimulated and the field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. RESULTS: Mice infused with patients' CSF, but not control CSF, developed progressive memory deficit and depressive-like behavior along with deposits of NMDAR antibodies in the hippocampus. These findings were associated with a decrease of the density of cell-surface and synaptic NMDAR and EphB2, and marked impairment of long-term synaptic plasticity without altering short-term plasticity. Administration of ephrin-B2 prevented the pathogenic effects of the antibodies in all the investigated paradigms assessing memory, depressive-like behavior, density of cell-surface and synaptic NMDAR and EphB2, and long-term synaptic plasticity. INTERPRETATION: Administration of ephrin-B2 prevents the pathogenic effects of anti-NMDAR encephalitis antibodies on memory and behavior, levels of cell-surface NMDAR, and synaptic plasticity. These findings reveal a strategy beyond immunotherapy to antagonize patients' antibody effects. Ann Neurol 2016; 80: 388-400.This study was supported, in part, by Instituto Carlos III/FEDER (FIS 15/00377 [to F.G.], FIS 14/00203 and CIBERER [to J.D.], and RETICS-RTA and RD12/0028/0023 [to R.M.]), NIH RO1NS077851 (to J.D.), MINECO (SAF2014-59648-P; to R.M.), European Commission (HEALTH-F2-2013-602891; to R.M.), Fundació Cellex (to J.D.), the Netherlands Organisation for Scientific Research (NWO, Veni incentive; to M.T.), an Erasmus MC fellowship (to M.T.), and the German Research Council (DFG; GE 2519/3-1 and CRC-TR 166/1 B2 [to C.G.]

Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation.

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses

Cancer Cell-Autonomous TRAIL-R Signaling Promotes KRAS-Driven Cancer Progression, Invasion, and Metastasis

Many cancers harbor oncogenic mutations of KRAS . Effectors mediating cancer progression, invasion, and metastasis in KRAS- mutated cancers are only incompletely understood. Here we identify cancer cell-ex- pressed murine TRAIL-R, whose main function ascribed so far has been the induction of apoptosis as a crucial mediator of KRAS-driven cancer progression, invasion, and metastasis and in vivo Rac-1 activation. Cancer cell-restricted genetic ablation of murine TRAIL-R in autochthonous KRAS-driven models of non- small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) reduces tumor growth, blunts metastasis, and prolongs survival by inhibiting cancer cell-autonomous migration, proliferation, and inva- sion. Consistent with this, high TRAIL-R2 expression correlates with invasion of human PDAC into lymph ves- sels and with shortened metastasis-free survival of KRAS- mutated colorectal cancer patients