Receptor ephrin-B2 Research Articles

Mechanisms of autoimmune encephalitis.

To provide an overview of the pathogenic mechanisms involved in autoimmune encephalitides mediated by antibodies against neuronal surface antigens, with a focus on NMDAR and LGI1 encephalitis. In antibody-mediated encephalitides, binding of IgG antibodies to neuronal surface antigens results in different pathogenic effects depending on the type of antibody, IgG subclass and epitope specificity. NMDAR IgG1 antibodies cause crosslinking and internalization of the target, synaptic and brain circuitry alterations, as well as alterations of NMDAR expressing oligodendrocytes, suggesting a link with white matter lesions observed in MRI studies. LGI1 IgG4 antibodies, instead, induce neuronal dysfunction by disrupting the interaction with cognate proteins and altering AMPAR-mediated signaling. In-vitro findings have been corroborated by memory and behavioral changes in animal models obtained by passive transfer of patients' antibodies or active immunization. These models have been fundamental to identify targets for innovative therapeutic strategies, aimed at counteracting or preventing antibody effects, such as the use of soluble ephrin-B2, NMDAR modulators (e.g., pregnenolone, SGE-301) or chimeric autoantibody receptor T cells (CAART) in models of NMDAR encephalitis. A deep understanding of the pathogenic mechanisms underlying antibody-mediated encephalitides is crucial for the development of new therapeutic approaches targeting brain autoimmunity.

In silico prediction of interaction between Nipah virus attachment glycoprotein and host cell receptors Ephrin-B2 and Ephrin-B3 in domestic and peridomestic mammals

Nipah virus (NiV) is a lethal bat-borne zoonotic virus that causes mild to acute respiratory distress and neurological manifestations in humans with a high mortality rate. NiV transmission to humans occurs via consumption of bat-contaminated fruit and date palm sap (DPS), or through direct contact with infected individuals and livestock. Since NiV outbreaks were first reported in pigs from Malaysia and Singapore, non-neutralizing antibodies against NiV attachment Glycoprotein (G) have also been detected in a few domestic mammals. NiV infection is initiated after NiV G binds to the host cell receptors Ephrin-B2 and Ephrin-B3. In this study, we assessed the degree of NiV host tropism in domestic and peridomestic mammals commonly found in Bangladesh that may be crucial in the transmission of NiV by serving as intermediate hosts. We carried out a protein-protein docking analysis of NiV G complexes (n = 52) with Ephrin-B2 and B3 of 13 domestic and peridomestic species using bioinformatics tools. Protein models were generated by homology modelling and the structures were validated for model quality. The different protein-protein complexes in this study were stable, and their binding affinity (ΔG) scores ranged between −8.0 to −19.1 kcal/mol. NiV Bangladesh (NiV-B) strain displayed stronger binding to Ephrin receptors, especially with Ephrin-B3 than the NiV Malaysia (NiV-M) strain, correlating with the observed higher pathogenicity of NiV-B strains. From the docking result, we found that Ephrin receptors of domestic rat (R. norvegicus) had a higher binding affinity for NiV G, suggesting greater susceptibility to NiV infections compared to other study species. Investigations for NiV exposure to domestic/peridomestic animals will help us knowing more the possible role of rats and other animals as intermediate hosts of NiV and would improve future NiV outbreak control and prevention in humans and domestic animals.

Bioinformatic investigation of Nipah virus surface protein mutations: Molecular docking with Ephrin B2 receptor, molecular dynamics simulation, and structural impact analysis.

The SARS-CoV-2 outbreak resulted in significant challenges and loss of life. The Nipah virus, known for its high infectivity and severity, was designated an emergency concern by the World Health Organization. To understand its mutations, the Nipah virus proteins were analyzed extensively, with a focus on the essential G and F proteins responsible for viral entry into host cells. Our bioinformatics analysis unveiled multiple mutations, including simultaneous mutations within a single sequence. Notably, the G273S mutation in the F protein was identified as a potential cause of structural damage, which carries significant implications for vaccine development. Comparing the docking scores of G and F proteins with the Ephrin B2 receptor, it was found that the Y228H mutation in the G protein and the D252G mutation in the F protein likely affect virus entry into host cells. Moreover, our investigation into stability and deformability highlighted the impact of the Y228H mutation in the G protein complex. Molecular dynamics simulations revealed increased flexibility and conformational changes in the G protein complex with the Y228H mutation compared with the known complex. Furthermore, evaluating the root mean square deviation variation demonstrated greater dynamic behavior in the G protein complex and the Ephrin B2 receptor complex. This comprehensive study provides valuable insights into Nipah virus mutations, their significance for vaccine development, and the importance of understanding protein complex behavior in drug discovery. The identified mutations, especially G273S and Y228H, hold crucial implications for future research and potential interventions against the Nipah virus.

Computer-assisted identification of potential quinolone derivatives targeting Nipah virus glycoprotein attachment with human cell surface receptor ephrin-B2: Multistep virtual screening

Nipah Virus (NiV) is a single-stranded, negative-sense, highly lethal RNA virus. Even though NiV has close to 70–80% of mortality in India and Bangladesh, still there is no available US FDA-approved drug or vaccine. NiV attachment glycoprotein (NiV-G) is critical for NiV to invade the human cell where ephrinB2 which is a crucial membrane-bound ligand that acts as a target of NiV. Most of the research has been performed targeting NiV or human ephrin-B to date. Quinolone derivatives are proven scaffolds for many approved drugs used to treat various bacterial, viral respiratory tract, and urinary tract infections, and rheumatologic disorders such as systemic lupus erythematosus, rheumatoid arthritis. Therefore, we have tried to find potential drug molecules employing quinolone scaffold-based derivatives from PubChem targeting both NiV-G and ephrin-B2 protein. A total of 1500+ quinolone derivatives were obtained from PubChem which were screened based on Drug Likeness followed by being subjected to XP docking employing Schrödinger software. The top ten best molecules were then chosen for their absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling based on the docking score ranking. Further, the top five molecules were selected for 200ns molecular dynamics (MD) simulation study with Desmond module followed by MM-GBSA study by Prime module of Schrödinger. The exhaustive analysis leads us to the top three probable lead drug molecules for NiV are PubChem CID 23646770, an analog of PubChem CID 67726448, and PubChem CID 10613168 which have predicted Ki values of 0.480 μm, 0.785 μm, and 0.380 μm, respectively. These proposed molecules can be the future drugs targeting NiV-G and human ephrin-B2 which requires further in vivo validation.

An investigation of binding interactions of tumor-targeted peptide conjugated polyphenols with the kinase domain of ephrin B4 and B2 receptors.

Recent studies have shown that Ephrin receptors may be upregulated in several types of cancers including breast, ovarian and endometrial cancers, making them a target for drug design. In this work, we have utilized a target-hopping approach to design new natural product-peptide conjugates and examined their interactions with the kinase-binding domain of EphB4 and EphB2 receptors. The peptide sequences were generated through point mutations of the known EphB4 antagonist peptide TNYLFSPNGPIA. Their anticancer properties and secondary structures were analyzed computationally. Conjugates of most optimum of peptides were then designed by binding the N-terminal of the peptides with the free carboxyl group of the polyphenols sinapate, gallate and coumarate, which are known for their inherent anticancer properties. To investigate if these conjugates have a potential to bind to the kinase domain, we carried out docking studies and MMGBSA free energy calculations of the trajectories based on the molecular dynamics simulations, with both the apo and the ATP bound kinase domains of both receptors. In most cases binding interactions occurred within the catalytic loop region, while in some cases the conjugates were found to spread out across the N-lobe and the DFG motif region. The conjugates were further tested for prediction of pharmacokinetic properties using ADME studies. Our results indicated that the conjugates were lipophilic and MDCK permeable with no CYP interactions. These findings provide an insight into the molecular interactions of these peptides and conjugates with the kinase domain of the EphB4 and EphB2 receptor. As a proof of concept, we synthesized and carried out SPR analysis with two of the conjugates (gallate-TNYLFSPNGPIA and sinapate-TNYLFSPNGPIA). Results indicated that the conjugates showed higher binding with the EphB4 receptor and minimal binding to EphB2 receptor. Sinapate-TNYLFSPNGPIA showed inhibitory activity against EphB4. These studies reveal that some of the conjugates may be developed for further investigation into in vitro and in vivo studies and potential development as therapeutics.

Evaluation of the interaction between potent small molecules against the Nipah virus Glycoprotein in Malaysia and Bangladesh strains, accompanied by the human Ephrin-B2 and Ephrin-B3 receptors; a simulation approach.

Malaysia reported the first human case of Nipah virus (NiV) in late September 1998 with encephalitis and respiratory symptoms. As a result of viral genomic mutations, two main strains (NiV-Malaysia and NiV-Bangladesh) have spread around the world. There are no licensed molecular therapeutics available for this biosafety level 4 pathogen. NiV attachment glycoprotein plays a critical role in viral transmission through its human receptors (Ephrin-B2 and Ephrin-B3), so identifying small molecules that can be repurposed to inhibit them is crucial to developing anti-NiV drugs. Consequently, in this study annealing simulations, pharmacophore modeling, molecular docking, and molecular dynamics were used to evaluate seven potential drugs (Pemirolast, Nitrofurantoin, Isoniazid Pyruvate, Eriodictyol, Cepharanthine, Ergoloid, and Hypericin) against NiV-G, Ephrin-B2, and Ephrin-B3 receptors. Based on the annealing analysis, Pemirolast for efnb2 protein and Isoniazid Pyruvate for efnb3 receptor were repurposed as the most promising small molecule candidates. Furthermore, Hypericin and Cepharanthine, with notable interaction values, are the top Glycoprotein inhibitors in Malaysia and Bangladesh strains, respectively. In addition, docking calculations revealed that their binding affinity scores are related to efnb2-pem (- 7.1kcal/mol), efnb3-iso (- 5.8kcal/mol), gm-hyp (- 9.6kcal/mol), gb-ceph (- 9.2kcal/mol). Finally, our computational research minimizes the time-consuming aspects and provides options for dealing with any new variants of Nipah virus that might emerge in the future.

Glycoprotein attachment with host cell surface receptor ephrin B2 and B3 in mediating entry of nipah and hendra virus: a computational investigation.

The viral attachment and fusion of Nipah and Hendra virus was explored through the interaction between viral glycoprotein and the host cell surface ephrin protein. The MD simulation results displayed more stability in Nipah and Hendra glycoprotein with EFNB2 as compared to EFNB3. The residue Glu533 in the central cavity of HeV/NiV glycoprotein protein identified as the potential hotspot in binding with the G-H loop of EFNB2. The online version contains supplementary material available at 10.1007/s12039-022-02110-9.

GluN1 antibody causes behavioral deficits in prepulse inhibition and memory through CaMKIIβ signaling

Accumulating evidence suggests that some patients with schizophrenia have high production of autoantibodies against the N-methyl-d-aspartate receptor (NMDAR) subunit GluN1 and that these antibodies lead to cognitive impairment. However, the molecular mechanisms of the deficits seen in these patients are largely unknown. In the present study, we found that passive infusion of GluN1 antibody into the hippocampus of mice for 7 days led to decreased expression of GluN1, phosphor-Ser897-GluN1, and EphrinB2 receptor (EphB2R); deficits in long-term potentiation (LTP) and synaptic transmission in the hippocampal CA1 area; impairment in prepulse inhibition (PPI); and deterioration of recognition memory in novel object recognition test. We also found decreased expression of CaMKIIβ, ERK1/2, CREB, and NF-κB after 7 days of GluN1 antibody exposure, as was the phosphorylation of these signaling molecules. The decrease in GluN1 and phosphor-Ser897-GluN1 expression and the deficits in LTP, PPI, and recognition memory were ameliorated by CaMKIIβ overexpression. These results suggest that downregulation of CaMKIIβ–ERK1/2–CREB–NF-κB signaling is responsiable for GluN1 antibody-associated impairment in PPI and memory and that GluN1 antibody-induced NMDAR hypofunction is the underlying mechanism of this impairment. Our findings indicate possible strategies to ameliorate NMDAR antibody-associated cognitive impairment in neuropsychiatric disease. They also provide evidence that NMDAR hypofunction is an underlying mechanism for cognitive impairment in schizophrenia.

Detection of Nipah and Hendra Viruses Using Recombinant Human Ephrin B2 Capture Virus in Immunoassays.

Nipah virus (NiV) and Hendra virus (HeV) are classified as high-consequence zoonotic viruses characterized by high pathogenicity and high mortality in animals and humans. Rapid diagnosis is essential to containing the outbreak. In this study, the henipavirus receptor ephrin B2 was examined to determine whether it could be used as a universal ligand for henipavirus detection in immunoassays. Enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant ephrin B2 as the capture ligand and two monoclonal antibodies (mAbs) as detection reagents. Using mAb F27NiV-34, which cross-reacts with NiV and HeV, we were able to detect NiV and HeV, while mAb F20NiV-65 was used to detect NiV. Therefore, using these two ELISAs, we were able to differentiate between NiV and HeV. Furthermore, we developed a rapid lateral flow strip test for NiV detection using ephrin B2 as the capture ligand combined with mAb F20NiV-65 as the detector. Taken together, our results show that the combination of ephrin B2 and a specific mAb provides an excellent pairing for NiV and HeV detection.

Recombinant vesicular stomatitis vaccine against Nipah virus has a favorable safety profile: Model for assessment of live vaccines with neurotropic potential.

Nipah virus (NiV) disease is a bat-borne zoonosis responsible for outbreaks with high lethality and is a priority for vaccine development. With funding from the Coalition of Epidemic Preparedness Innovations (CEPI), we are developing a chimeric vaccine (PHV02) composed of recombinant vesicular stomatitis virus (VSV) expressing the envelope glycoproteins of both Ebola virus (EBOV) and NiV. The EBOV glycoprotein (GP) mediates fusion and viral entry and the NiV attachment glycoprotein (G) is a ligand for cell receptors, and stimulates neutralizing antibody, the putative mediator of protection against NiV. PHV02 is identical in construction to the registered Ebola vaccine (Ervebo) with the addition of the NiV G gene. NiV ephrin B2 and B3 receptors are expressed on neural cells and the wild-type NiV is neurotropic and causes encephalitis in affected patients. It was therefore important to assess whether the NiV G alters tropism of the rVSV vector and serves as a virulence factor. PHV02 was fully attenuated in adult hamsters inoculated by the intramuscular (IM) route, whereas parental wild-type VSV was 100% lethal. Two rodent models (mice, hamsters) were infected by the intracerebral (IC) route with graded doses of PHV02. Comparator active controls in various experiments included rVSV-EBOV (representative of Ebola vaccine) and yellow fever (YF) 17DD commercial vaccine. These studies showed PHV02 to be more neurovirulent than both rVSV-EBOV and YF 17DD in infant animals. PHV02 was lethal for adult hamsters inoculated IC but not for adult mice. In contrast YF 17DD retained virulence for adult mice inoculated IC but was not virulent for adult hamsters. Because of the inconsistency of neurovirulence patterns in the rodent models, a monkey neurovirulence test (MNVT) was performed, using YF 17DD as the active comparator because it has a well-established profile of quantifiable microscopic changes in brain centers and a known reporting rate of neurotropic adverse events in humans. In the MNVT PHV02 was significantly less neurovirulent than the YF 17DD vaccine reference control, indicating that the vaccine will have an acceptable safety profile for humans. The findings are important because they illustrate the complexities of phenotypic assessment of novel viral vectors with tissue tropisms determined by transgenic proteins, and because it is unprecedented to use a heterologous comparator virus (YF vaccine) in a regulatory-enabling study. This approach may have value in future studies of other novel viral vectors.

Cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein.

SUMMARYHendra virus and Nipah virus (NiV), members of the Henipavirus (HNV) genus, are zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. We isolated a panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior exposure to equine Hendra virus (HeV) vaccine, targeting distinct antigenic sites. The most potent class of cross-reactive antibodies achieves neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3, with a second class being enhanced by receptor binding. mAbs from both classes display synergistic activity in vitro. In a stringent hamster model of NiV Bangladesh (NiVB) infection, antibodies from both classes reduce morbidity and mortality and achieve synergistic protection in combination. These candidate mAbs might be suitable for use in a cocktail therapeutic approach to achieve synergistic potency and reduce the risk of virus escape.

Loss of ephrin B2 receptor (EPHB2) sets lipid rheostat by regulating proteins DGAT1 and ATGL inducing lipid droplet storage in prostate cancer cells

Lipid droplet (LD) accumulation in cancer results from aberrant metabolic reprograming due to increased lipid uptake, diminished lipolysis and/or de novo lipid synthesis. Initially implicated in storage and lipid trafficking in adipocytes, LDs are more recently recognized to fuel key functions associated with carcinogenesis and progression of several cancers, including prostate cancer (PCa). However, the mechanisms controlling LD accumulation in cancer are largely unknown. EPHB2, a tyrosine kinase (TKR) ephrin receptor has been proposed to have tumor suppressor functions in PCa, although the mechanisms responsible for these effects are unclear. Given that dysregulation in TRK signaling can result in glutaminolysis we postulated that EPHB2 might have potential effects on lipid metabolism. Knockdown strategies for EPHB2 were performed in prostate cancer cells to analyze the impact on the net lipid balance, proliferation, triacylglycerol-regulating proteins, effect on LD biogenesis, and intracellular localization of LDs. We found that EPHB2 protein expression in a panel of human-derived prostate cancer cell lines was inversely associated with in vivo cell aggressiveness. EPHB2 silencing increased the proliferation of prostate cancer cells and concurrently induced de novo LD accumulation in both cytoplasmic and nuclear compartments as well as a “shift” on LD size distribution in newly formed lipid-rich organelles. Lipid challenge using oleic acid exacerbated the effects on the LD phenotype. Loss of EPHB2 directly regulated key proteins involved in maintaining lipid homeostasis including, increasing lipogenic DGAT1, DGAT2 and PLIN2 and decreasing lipolytic ATGL and PEDF. A DGAT1-specific inhibitor abrogated LD accumulation and proliferative effects induced by EPHB2 loss. In conclusion, we highlight a new anti-tumor function of EPHB2 in lipid metabolism through regulation of DGAT1 and ATGL in prostate cancer. Blockade of DGAT1 in EPHB2-deficient tumors appears to be effective in restoring the lipid balance and reducing tumor growth.EPHB2 deficiency in prostate cancer cells leads to alterations of the triacylglyceride (TAG) synthesis pathway involved in lipogenesis and lipolysis. These changes initiate de novo cytoplasmic and intranuclear lipid droplets accumulation associated with increased proliferative capacity. Targeting the TAG pathway molecule DGAT1 diminishes the deleterious effects exerted by EPHB2 silencing.

EphrinB2 and GRIP1 stabilize mushroom spines during denervation-induced homeostatic plasticity

SummaryDespite decades of work, much remains elusive about molecular events at the interplay between physiological and structural changes underlying neuronal plasticity. Here, we combined repetitive live imaging and expansion microscopy in organotypic brain slice cultures to quantitatively characterize the dynamic changes of the intracellular versus surface pools of GluA2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) across the different dendritic spine types and the shaft during hippocampal homeostatic plasticity. Mechanistically, we identify ephrinB2 and glutamate receptor interacting protein (GRIP) 1 as mediating AMPAR relocation to the mushroom spine surface following lesion-induced denervation. Moreover, stimulation with the ephrinB2 specific receptor EphB4 not only prevents the lesion-induced disappearance of mushroom spines but is also sufficient to shift AMPARs to the surface and rescue spine recovery in a GRIP1 dominant-negative background. Thus, our results unravel a crucial role for ephrinB2 during homeostatic plasticity and identify a potential pharmacological target to improve dendritic spine plasticity upon injury.

Dysregulation of the EphrinB2\u2212EphB4 ratio in pediatric cerebral arteriovenous malformations is associated with endothelial cell dysfunction in vitro and functions as a novel noninvasive biomarker in patients

We investigated (1) EphrinB2 and EphB4 receptor expression in cerebral AVMs, (2) the impact of an altered EphrinB2:EphB4 ratio on brain endothelial cell function and (3) potential translational applications of these data. The following parameters were compared between AVM endothelial cells (AVMECs) and human brain microvascular endothelial cells (HBMVECs): quantified EphrinB2 and EphB4 expression, angiogenic potential, and responses to manipulation of the EphrinB2:EphB4 ratio via pharmacologic stimulation/inhibition. To investigate the clinical relevance of these in vitro data, Ephrin expression was assessed in AVM tissue (by immunohistochemistry) and urine (by ELISA) from pediatric patients with AVM (n = 30), other cerebrovascular disease (n = 14) and control patients (n = 29), and the data were subjected to univariate and multivariate statistical analyses. Compared to HBMVECs, AVMECs demonstrated increased invasion (p = 0.04) and migration (p = 0.08), impaired tube formation (p = 0.06) and increased EphrinB2:EphB4 ratios. Altering the EphrinB2:EphB4 ratio (by increasing EphrinB2 or blocking EphB4) in HBMVECs increased invasion (p = 0.03 and p < 0.05, respectively). EphrinB2 expression was increased in AVM tissue, which correlated with increased urinary EphrinB2 levels in AVM patients. Using the optimal urinary cutoff value (EphrinB2 > 25.7 pg/μg), AVMs were detected with high accuracy (80% vs. controls) and were distinguished from other cerebrovascular disease (75% accuracy). Post-treatment urinary EphrinB2 levels normalized in an index patient. In summary, AVMECs have an EphrinB2:EphB4 ratio that is increased compared to that of normal HBMVECs. Changing this ratio in HBMVECs induces AVMEC-like behavior. EphrinB2 is clinically relevant, and its levels are increased in AVM tissue and patient urine. This work suggests that dysregulation of the EphrinB2:EphB4 signaling cascade and increases in EphrinB2 may play a role in AVM development, with potential utility as a diagnostic and therapeutic target.

High Pathogenicity of Nipah Virus from Pteropus lylei Fruit Bats, Cambodia.

We conducted an in-depth characterization of the Nipah virus (NiV) isolate previously obtained from a Pteropus lylei bat in Cambodia in 2003 (CSUR381). We performed full-genome sequencing and phylogenetic analyses and confirmed CSUR381 is part of the NiV-Malaysia genotype. In vitro studies revealed similar cell permissiveness and replication of CSUR381 (compared with 2 other NiV isolates) in both bat and human cell lines. Sequence alignments indicated conservation of the ephrin-B2 and ephrin-B3 receptor binding sites, the glycosylation site on the G attachment protein, as well as the editing site in phosphoprotein, suggesting production of nonstructural proteins V and W, known to counteract the host innate immunity. In the hamster animal model, CSUR381 induced lethal infections. Altogether, these data suggest that the Cambodia bat-derived NiV isolate has high pathogenic potential and, thus, provide insight for further studies and better risk assessment for future NiV outbreaks in Southeast Asia.

Fusogenicity of the Ghana Virus (Henipavirus: Ghanaian bat henipavirus) Fusion Protein is Controlled by the Cytoplasmic Domain of the Attachment Glycoprotein.

The Ghana virus (GhV) is phylogenetically related to the zoonotic henipaviruses Nipah (NiV) and Hendra virus. Although GhV uses the highly conserved receptor ephrin-B2, the fusogenicity is restricted to cell lines of bat origin. Furthermore, the surface expression of the GhV attachment glycoprotein (G) is reduced compared to NiV and most of this protein is retained in the endoplasmic reticulum (ER). Here, we generated truncated as well as chimeric GhV G proteins and investigated the influence of the structural domains (cytoplasmic tail, transmembrane domain, ectodomain) of this protein on the intracellular transport and the fusogenicity following coexpression with the GhV fusion protein (F). We demonstrate that neither the cytoplasmic tail nor the transmembrane domain is responsible for the intracellular retention of GhV G. Furthermore, the cytoplasmic tail of GhV G modulates the fusogenicity of GhV F and therefore controls the species-restricted fusogenicity of the GhV surface glycoproteins.

38.1 IMPACT OF ANTI-NMDA RECEPTOR AUTOANTIBODIES FROM PSYCHOTIC PATIENTS ON THE GLUTAMATE SYNAPSE

BackgroundThe flourishing identification of circulating autoantibodies against neuronal receptors in neuropsychiatric disorders has fostered new conceptual and clinical frameworks. However, their putative presence in different diseases, as well as in healthy subjects, has raised questions about detection reliability and pathogenic role.MethodsUsing a combination of single molecule-based imaging approaches, cell calcium imaging, and single-cell electrophysiological recordings, we investigated in hippocampal networks the impact of autoantibodies against glutamate NMDA receptor (NMDAR-Ab) on several aspects of the glutamate synapse.ResultsWe ascertain the presence of circulating autoantibodies against glutamate NMDA receptor (NMDAR-Ab) in about 20% of psychotic patients diagnosed with schizophrenia and very few healthy subjects. NMDAR-Ab from patients and healthy subjects do not compete for binding on native receptor. Strikingly, NMDAR-Ab from patients, but not from healthy subjects, specifically alter the surface dynamics and nanoscale organization of synaptic NMDAR and its anchoring partner the EphrinB2 receptor. Functionally, only patients’ NMDAR-Ab prevent long-term potentiation at glutamatergic synapses while leaving NMDAR-mediated calcium influx intact. Furthermore, we unveil that NMDAR-Ab from first episode psychotic patients produced similar effects.DiscussionBy taking advantage of the single molecule imaging and complementary ensemble approaches, we unveil that NMDAR-Ab from psychotic patients (schizophrenic and first episode) profoundly alter NMDAR synaptic transmission and NMDAR-dependent synaptic functions, supporting a pathogenically relevant role.

Rescue and characterization of recombinant cedar virus, a non-pathogenic Henipavirus species

BackgroundHendra virus and Nipah virus are zoonotic viruses that have caused severe to fatal disease in livestock and human populations. The isolation of Cedar virus, a non-pathogenic virus species in the genus Henipavirus, closely-related to the highly pathogenic Hendra virus and Nipah virus offers an opportunity to investigate differences in pathogenesis and receptor tropism among these viruses.MethodsWe constructed full-length cDNA clones of Cedar virus from synthetic oligonucleotides and rescued two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that expresses a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. Replication kinetics of both viruses and stimulation of the interferon pathway were characterized in vitro. Cellular tropism for ephrin-B type ligands was qualitatively investigated by microscopy and quantitatively by a split-luciferase fusion assay.ResultsSuccessful rescue of recombinant Cedar virus expressing a green fluorescent protein did not significantly affect virus replication compared to the recombinant wild-type Cedar virus. We demonstrated that recombinant Cedar virus stimulated the interferon pathway and utilized the established Hendra virus and Nipah virus receptor, ephrin-B2, but not ephrin-B3 to mediate virus entry. We further characterized virus-mediated membrane fusion kinetics of Cedar virus with the known henipavirus receptors ephrin-B2 and ephrin-B3.ConclusionsThe recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals. Moreover, these experiments can be conducted safely under BSL-2 conditions.

Dynamic disorganization of synaptic NMDA receptors triggered by autoantibodies from psychotic patients

The identification of circulating autoantibodies against neuronal receptors in neuropsychiatric disorders has fostered new conceptual and clinical frameworks. However, detection reliability, putative presence in different diseases and in health have raised questions about potential pathogenic mechanism mediated by autoantibodies. Using a combination of single molecule-based imaging approaches, we here ascertain the presence of circulating autoantibodies against glutamate NMDA receptor (NMDAR-Ab) in about 20% of psychotic patients diagnosed with schizophrenia and very few healthy subjects. NMDAR-Ab from patients and healthy subjects do not compete for binding on native receptor. Strikingly, NMDAR-Ab from patients, but not from healthy subjects, specifically alter the surface dynamics and nanoscale organization of synaptic NMDAR and its anchoring partner the EphrinB2 receptor in heterologous cells, cultured neurons and in mouse brain. Functionally, only patients’ NMDAR-Ab prevent long-term potentiation at glutamatergic synapses, while leaving NMDAR-mediated calcium influx intact. We unveil that NMDAR-Ab from psychotic patients alter NMDAR synaptic transmission, supporting a pathogenically relevant role.

GRIP1 Binds to ApoER2 and EphrinB2 to Induce Activity-Dependent AMPA Receptor Insertion at the Synapse

SummaryRegulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in response to neuronal activity is critical for synaptic function and plasticity. Here, we show that neuronal activity induces the binding of ephrinB2 and ApoER2 receptors at the postsynapse to regulate de novo insertion of AMPA receptors. Mechanistically, the multi-PDZ adaptor glutamate-receptor-interacting protein 1 (GRIP1) binds ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors. Phosphorylation of ephrinB2 in a serine residue (Ser-9) is essential for the stability of such a complex. In vivo, a mutation on ephrinB2 Ser-9 in mice results in a complete disruption of the complex, absence of ApoER2 downstream signaling, and impaired activity-induced and ApoER2-mediated AMPA receptor insertion. Using compound genetics, we show the requirement of this complex for long-term potentiation (LTP). Together, our findings uncover a cooperative ephrinB2 and ApoER2 signaling at the synapse, which serves to modulate activity-dependent AMPA receptor dynamic changes during synaptic plasticity.