Allosteric Modulation of NMDARs Reverses Patients' Autoantibody Effects in Mice

Background and Objectives To demonstrate that an analog (SGE-301) of a brain-derived cholesterol metabolite, 24(S)- hydroxycholesterol, which is a selective positive allosteric modulator (PAM) of NMDA re- ceptors (NMDARs), is able to reverse the memory and synaptic alterations caused by CSF from patients with anti-NMDAR encephalitis in an animal model of passive transfer of antibodies. Methods Four groups of mice received (days 1-14) patients' or controls' CSF via osmotic pumps connected to the cerebroventricular system and from day 11 were treated with daily sub- cutaneous injections of SGE-301 or vehicle (no drug). Visuospatial memory, locomotor activity (LA), synaptic NMDAR cluster density, hippocampal long-term potentiation (LTP), and paired-pulse facilitation (PPF) were assessed on days 10, 13, 18, and 26 using reported techniques. Results On day 10, mice infused with patients' CSF, but not controls' CSF, presented a significant visuospatial memory deficit, reduction of NMDAR clusters, and impairment of LTP, whereas LA and PPF were unaffected. These alterations persisted until day 18, the time of maximal deficits in this model. In contrast, mice that received patients' CSF but from day 11 were treated with SGE-301 showed memory recovery (day 13), and on day 18, all paradigms (memory, NMDAR clusters, and LTP) had reversed to values similar to those of controls. On day 26, no differences were observed among experimental groups. Discussion An oxysterol biology-based PAM of NMDARs is able to reverse the synaptic and memory deficits caused by CSF from patients with anti-NMDAR encephalitis. These findings suggest a novel adjuvant treatment approach that deserves future clinical evaluation

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

Placental transfer of NMDAR antibodies causes reversible alterations in mice

Objective: To determine whether maternofetal transfer of NMDA receptor (NMDAR) antibodies has pathogenic effects on the fetus and offspring, we developed a model of placental transfer of antibodies. Methods: Pregnant C57BL/6J mice were administered via tail vein patients' or controls' immunoglobulin G (IgG) on days 14-16 of gestation, when the placenta is able to transport IgG and the immature fetal blood-brain barrier is less restrictive to IgG crossing. Immunohistochemical and DiOlistic (gene gun delivery of fluorescent dye) staining, confocal microscopy, standardized developmental and behavioral tasks, and hippocampal long-term potentiation were used to determine the antibody effects. Results: In brains of fetuses, patients' IgG, but not controls' IgG, bound to NMDAR, causing a decrease in NMDAR clusters and cortical plate thickness. No increase in neonatal mortality was observed, but offspring exposed in utero to patients' IgG had reduced levels of cell-surface and synaptic NMDAR, increased dendritic arborization, decreased density of mature (mushroom-shaped) spines, microglial activation, and thinning of brain cortical layers II-IV with cellular compaction. These animals also had a delay in innate reflexes and eye opening and during follow-up showed depressive-like behavior, deficits in nest building, poor motor coordination, and impaired social-spatial memory and hippocampal plasticity. Remarkably, all these paradigms progressively improved (becoming similar to those of controls) during follow-up until adulthood. Conclusions: In this model, placental transfer of patients' NMDAR antibodies caused severe but reversible synaptic and neurodevelopmental alterations. Reversible antibody effects may contribute to the infrequent and limited number of complications described in children of patients who develop anti-NMDAR encephalitis during pregnancy

Allosteric modulation of NMDA receptors prevents the antibody effects of patients with anti-NMDAR encephalitis

Anti-N-Methyl-D-Aspartate Receptor (NMDAR) encephalitis is an immune-mediated disease characterized by a complex neuropsychiatric syndrome in association with an antibody-mediated decrease of NMDAR. About 85% of patients respond to immunotherapy (and removal of an associated tumor if it applies), but it often takes several months or more than 1 year for patients to recover. There are no complementary treatments, beyond immunotherapy, to accelerate this recovery. Previous studies showed that SGE-301, a synthetic analog of 24(S)-hydroxycholesterol, which is a potent, and selective positive allosteric modulator of NMDAR, reverted the memory deficit caused by phencyclidine (a non-competitive antagonist of NMDAR), and prevented the NMDAR dysfunction caused by patients' NMDAR antibodies in cultured neurons. An advantage of SGE-301 is that it is optimized for systemic delivery such that plasma and brain exposures are sufficient to modulate NMDAR activity. Here, we used SGE-301 to confirm that in cultured neurons it prevented the antibody-mediated reduction of receptors, and then we applied it to a previously reported mouse model of passive cerebroventricular transfer of patients' CSF antibodies. Four groups were established: mice receiving continuous (14-day) infusion of patients' or controls' CSF, treated with daily subcutaneous administration of SGE-301 or vehicle (no drug). The effects on memory were examined with the novel object location (NOL) test at different time points, and the effects on synaptic levels of NMDAR (assessed with confocal microscopy) and plasticity (long-term potentiation [LTP]) were examined in the hippocampus on day 18, which in this model corresponds to the last day of maximal clinical and synaptic alterations. As expected, mice infused with patients' CSF antibodies, but not those infused with controls' CSF, and treated with vehicle developed severe memory deficit without locomotor alteration, accompanied by a decrease of NMDAR clusters and impairment of LTP. All antibody-mediated pathogenic effects (memory, synaptic NMDAR, LTP) were prevented in the animals that were treated with SGE-301, despite that this compound did not antagonize antibody binding. Additional investigations on the potential mechanisms related to these SGE-301 effects showed that (1) in cultured neurons SGE-301 prolonged the decay time of NMDAR-dependent spontaneous excitatory postsynaptic currents suggesting a prolonged open time of the channel, and (2) it significantly decreased the internalization of antibody-bound receptors suggesting that additional, yet unclear mechanisms, contribute in keeping unchanged the surface NMDAR density. Overall, these findings suggest that SGE-301, or similar modulators of NMDAR, could potentially serve as complementary treatment for anti-NMDAR encephalitis and deserve future investigations

Human CASPR2 antibodies reversibly alter memory and the CASPR2 protein complex

Objective: The encephalitis associated with antibodies against contactin-associated proteinlike 2 (CASPR2) is presumably antibody-mediated, but the antibody effects and whether they cause behavioral alterations are not well known. Here, we used a mouse model of patients' immunoglobulin G (IgG) transfer and super-resolution microscopy to demonstrate the antibody pathogenicity. Methods: IgG from patients with anti-CASPR2 encephalitis or healthy controls was infused into the cerebroventricular system of mice. The levels and colocalization of CASPR2 with transient axonal glycoprotein 1 (TAG1) were determined with stimulated emission depletion microscopy (40-70μm lateral resolution). Hippocampal clusters of Kv1.1 voltage-gated potassium channels (VGKCs) and GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were quantified with confocal microscopy. Behavioral alterations were assessed with standard behavioral paradigms. Cultured neurons were used to determine the levels of intracellular CASPR2 and TAG1 after exposure to patients' IgG. Results: Infusion of patients' IgG, but not controls' IgG, caused memory impairment along with hippocampal reduction of surface CASPR2 clusters and decreased CASPR2/TAG1 colocalization. In cultured neurons, patients' IgG led to an increase of intracellular CASPR2 without affecting TAG1, suggesting selective CASPR2 internalization. Additionally, mice infused with patients' IgG showed decreased levels of Kv1.1 and GluA1 (two CASPR2-regulated proteins). All these alterations and the memory deficit reverted to normal after removing patients' IgG. Interpretation: IgG from patients with anti-CASPR2 encephalitis causes reversible memory impairment, inhibits the interaction of CASPR2/TAG1, and decreases the levels of CASPR2 and related proteins (VGKC, AMPAR). These findings fulfill the postulates of antibody-mediated disease and provide a biological basis for antibody-removing treatment approaches. ANN NEUROL 2022;91:801-813

Passive experimental autoimmune encephalomyelitis in C57BL/6 with MOG: evidence of involvement of B cells

Experimental autoimmune encephalomyelitis (EAE) is the most relevant animal model to study demyelinating diseases such as multiple sclerosis. EAE can be induced by active (active EAE) or passive (at-EAE) transfer of activated T cells in several species and strains of rodents. However, histological features of at-EAE model in C57BL/6 are poorly described. The aim of this study was to characterize the neuroinflammatory and neurodegenerative responses of at-EAE in C57BL/6 mice by histological techniques and compare them with that observed in the active EAE model. To develop the at-EAE, splenocytes from active EAE female mice were harvested and cultured in presence of MOG 35-55 and IL-12, and then injected intraperitoneally in recipient female C57BL6/J mice. In both models, the development of EAE was similar except for starting before the onset of symptoms and presenting a higher EAE cumulative score in the at-EAE model. Spinal cord histological examination revealed an increased glial activation as well as more extensive demyelinating areas in the at-EAE than in the active EAE model. Although inflammatory infiltrates composed by macrophages and T lymphocytes were found in the spinal cord and brain of both models, B lymphocytes were significantly increased in the at-EAE model. The co-localization of these B cells with IgG and their predominant distribution in areas of demyelination would suggest that IgG-secreting B cells are involved in the neurodegenerative processes associated with at-EAE

Tcf20 deficiency is associated with increased liver fibrogenesis and alterations in mitochondrial metabolism in mice and humans

Transcription co-activator factor 20 (TCF20) is a regulator of transcription factors involved in extracellular matrix remodelling. In addition, TCF20 genomic variants in humans have been associated with impaired intellectual disability. Therefore, we hypothesized that TCF20 has several functions beyond those described in neurogenesis, including the regulation of fibrogenesis.We acknowledge the support from grants PDI2019-105502RB-100 and PID2022-138243OB-I00 to MM-R, PID2019-111669RB, BES-2017-080823 to IP "ESF Investing in your future", and RTI2018-094734-B-C21 to WJ and PM-L, from MCIN/AEI/10.13039/501100011033. RedFibro (RED2022-134485-T) of the 2022 caLL for aid to «RESEARCH NETWORKS», within the framework of the Programa Estatal del Plan Estatal de Investigación Científica, Técnica y de Innovación 2021–2023. Consolidated Research Group of the Generalitat de Catalunya AGAUR (2021 SGR 00881 to MM-R) and PM-L financed ESF Investing in your future, support from AGAUR of the Generalitat de Catalunya. Fundación Mutua Madrileña AP171442019, European Cooperation in Science & Technology (COST) ACTION CA17112 Prospective European Drug-Induced Liver Injury Network, and the Project 201 916/31 Contribution of mitochondrialoxysterol and bile acid metabolism to liver carcinogenesis 2019 by Fundació Marató TV3. PM-L was additionally supported by a fellowship from the Ramon y Cajal Program (RYC2018-0Z23971-I) and a grant (PID2021-123426OB-I00) from the Spanish Ministerio de Ciencia, Innovación y Universidades. CIBERehd is financed by the Instituto de Salud Carlos III.Peer reviewe

Ricerca di mutazioni nel gene JUP, candidato per ARVD/C: analisi della regione codificante per la porzione N-terminale

Risultati del tirocinio durante il quale è stato effettuato lo screening della regione del gene JUP codificante per la porzione N-terminale della placoglobina in 21 pazienti affetti da ARVD classica, già sottoposti e risultati negativi all’analisi per la ricerca di mutazioni negli altri geni desmosomali noti. Lo screening è stato effettuato mediante DHPLC e sequenziamento diretto

A novel approach for internet traffic classification based on multi-objective evolutionary fuzzy classifiers

Internet traffic classification has moved in the last years from traditional port and payload-based approaches towards methods employing statistical measurements and machine learning techniques. Despite the success achieved by these techniques, they are not able to explain the relation between the features, which describe the traffic flow, and the corresponding traffic classes. This relation can be extremely useful to network managers for quickly handling possible network drawback. In this paper, we propose to tackle the traffic classification problem by using multi-objective evolutionary fuzzy classifiers (MOEFCs). MOEFCs are characterised by good trade-offs between accuracy and interpretability. We adopt two Internet traffic datasets extracted from two real-world networks. We discuss the results obtained both by applying a cross validation on each single dataset, and by using a dataset as training set and the other as test set. We show that, in both cases, MOEFCs can achieve satisfactory accuracy in the face of low complexity and, therefore, high interpretability

NMDAR encephalitis: passive transfer from man to mouse by a recombinant antibody

Objective: Autoimmune encephalitis is most frequently associated with anti-NMDAR autoantibodies. Their pathogenic relevance has been suggested by passive transfer of patients' cerebrospinal fluid (CSF) in mice in vivo. We aimed to analyze the intrathecal plasma cell repertoire, identify autoantibody-producing clones, and characterize their antibody signatures in recombinant form. Methods: Patients with recent onset typical anti-NMDAR encephalitis were subjected to flow cytometry analysis of the peripheral and intrathecal immune response before, during, and after immunotherapy. Recombinant human monoclonal antibodies (rhuMab) were cloned and expressed from matching immunoglobulin heavy- (IgH) and light-chain (IgL) amplicons of clonally expanded intrathecal plasma cells (cePc) and tested for their pathogenic relevance. Results: Intrathecal accumulation of B and plasma cells corresponded to the clinical course. The presence of cePc with hypermutated antigen receptors indicated an antigen-driven intrathecal immune response. Consistently, a single recombinant human GluN1-specific monoclonal antibody, rebuilt from intrathecal cePc, was sufficient to reproduce NMDAR epitope specificity in vitro. After intraventricular infusion in mice, it accumulated in the hippocampus, decreased synaptic NMDAR density, and caused severe reversible memory impairment, a key pathogenic feature of the human disease, in vivo. Interpretation: A CNS-specific humoral immune response is present in anti-NMDAR encephalitis specifically targeting the GluN1 subunit of the NMDAR. Using reverse genetics, we recovered the typical intrathecal antibody signature in recombinant form, and proved its pathogenic relevance by passive transfer of disease symptoms from man to mouse, providing the critical link between intrathecal immune response and the pathogenesis of anti-NMDAR encephalitis as a humorally mediated autoimmune disease