Neuron-microelectrode junction induced by an engineered synapse organizer.

The conventional microelectrodes for recording neuronal activities do not have innate selectivity to cell type, which is one of the critical limitations for the detailed analysis of neuronal circuits. In this study, we engineered a downsized variant of the artificial synapse organizer based on neurexin1ß and a peptide-tag, fabricated gold microelectrodes functionalized with the receptor for the organizer, and performed validation experiments in primary cultured neurons. Successful inductions of synapse-like junctions were detected at the sites of contact between neurons expressing the engineered synapse organizer and functionalized microelectrodes, but not in the negative control experiment in which the electrode functionalization was omitted. Such a molecularly inducible neuron-microelectrode junction could be the basis for the next-generation electrophysiological technique enabling cell type-selective recording.

Cancer-associated point mutations within the extracellular domain of PTPRD affect protein stability and HSPG interaction.

PTPRD, a well-established tumor suppressor gene, encodes the protein tyrosine phosphatase-type D. This protein consists of three immunoglobulin-like (Ig) domains, four to eight fibronectin type 3 (FN) domains, a single transmembrane segment, and two cytoplasmic tandem tyrosine phosphatase domains. PTPRD is known to harbor various cancer-associated point mutations. While it is assumed that PTPRD regulates cellular functions as a tumor suppressor through the tyrosine phosphatase activity in the intracellular region, the function of its extracellular domain (ECD) in cancer is not well understood. In this study, we systematically examined the impact of 92 cancer-associated point mutations within the ECD. We found that 69.6% (64 out of 92) of these mutations suppressed total protein expression and/or plasma membrane localization. Notably, almost all mutations (20 out of 21) within the region between the last FN domain and transmembrane segment affected protein expression and/or localization, highlighting the importance of this region for protein stability. We further found that some mutations within the Ig domains adjacent to the glycosaminoglycan-binding pocket enhanced PTPRD's binding ability to heparan sulfate proteoglycans (HSPGs). This interaction is proposed to suppress phosphatase activity. Our findings therefore suggest that HSPG-mediated attenuation of phosphatase activity may be involved in tumorigenic processes through PTPRD dysregulation.

Differential contribution of canonical and noncanonical NLGN3 pathways to early social development and memory performance.

Neuroligin (NLGN) 3 is a postsynaptic cell adhesion protein organizing synapse formation through two different types of transsynaptic interactions, canonical interaction with neurexins (NRXNs) and a recently identified noncanonical interaction with protein tyrosine phosphatase (PTP) δ. Although, NLGN3 gene is known as a risk gene for neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID), the pathogenic contribution of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ pathways to these disorders remains elusive. In this study, we utilized Nlgn3 mutant mice selectively lacking the interaction with either NRXNs or PTPδ and investigated their social and memory performance. Neither Nlgn3 mutants showed any social cognitive deficiency in the social novelty recognition test. However, the Nlgn3 mutant mice lacking the PTPδ pathway exhibited significant decline in the social conditioned place preference (sCPP) at the juvenile stage, suggesting the involvement of the NLGN3-PTPδ pathway in the regulation of social motivation and reward. In terms of learning and memory, disrupting the canonical NRXN pathway attenuated contextual fear conditioning while disrupting the noncanonical NLGN3-PTPδ pathway enhanced it. Furthermore, disruption of the NLGN3-PTPδ pathway negatively affected the remote spatial reference memory in the Barnes maze test. These findings highlight the differential contributions of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ synaptogenic pathways to the regulation of higher order brain functions associated with ASD and ID.

Development of artificial synapse organizers liganded with a peptide tag for molecularly inducible neuron-microelectrode interface.

It has been proposed that cell-type-specific bioelectronic interfaces for neuronal circuits could be established by utilizing the function of synapse organizers. For this purpose, using neurexin-1ß and a peptide tag, we engineered compact synapse organizers that do not interact with the naturally occurring receptors but induce presynaptic differentiation upon contact with nanobody-decorated objects in cultured mammalian and chick forebrain neurons. In chick neurons, the engineered organizer exerted synaptogenesis typically in ∼4 h after the contact, even under an air atmosphere at room temperature, thereby providing a useful cellular model for establishing the molecularly inducible neuron-microelectrode interface.

Developmental synapse pathology triggered by maternal exposure to the herbicide glufosinate ammonium.

Environmental and genetic factors influence synapse formation. Numerous animal experiments have revealed that pesticides, including herbicides, can disturb normal intracellular signals, gene expression, and individual animal behaviors. However, the mechanism underlying the adverse outcomes of pesticide exposure remains elusive. Herein, we investigated the effect of maternal exposure to the herbicide glufosinate ammonium (GLA) on offspring neuronal synapse formation in vitro. Cultured cerebral cortical neurons prepared from mouse embryos with maternal GLA exposure demonstrated impaired synapse formation induced by synaptic organizer neuroligin 1 (NLGN1)-coated beads. Conversely, the direct administration of GLA to the neuronal cultures exhibited negligible effect on the NLGN1-induced synapse formation. The comparison of the transcriptomes of cultured neurons from embryos treated with maternal GLA or vehicle and a subsequent bioinformatics analysis of differentially expressed genes (DEGs) identified "nervous system development," including "synapse," as the top-ranking process for downregulated DEGs in the GLA group. In addition, we detected lower densities of parvalbumin (Pvalb)-positive neurons at the postnatal developmental stage in the medial prefrontal cortex (mPFC) of offspring born to GLA-exposed dams. These results suggest that maternal GLA exposure induces synapse pathology, with alterations in the expression of genes that regulate synaptic development via an indirect pathway distinct from the effect of direct GLA action on neurons.

Regional contributions of D-serine to Alzheimer's disease pathology in male AppNL-G-F/NL-G-F mice.

Background: Neurodegenerative processes in Alzheimer's disease (AD) are associated with excitotoxicity mediated by the N-methyl-D-aspartate receptor (NMDAR). D-Serine is an endogenous co-agonist necessary for NMDAR-mediated excitotoxicity. In the mammalian brain, it is produced by serine racemase (SRR) from L-serine, suggesting that dysregulation of L-serine, D-serine, or SRR may contribute to AD pathogenesis. Objective and methods: We examined the contributions of D-serine to AD pathology in the AppNL-G-F/NL-G-F gene knock-in (APPKI) mouse model of AD. We first examined brain SRR expression levels and neuropathology in APPKI mice and then assessed the effects of long-term D-serine supplementation in drinking water on neurodegeneration. To further confirm the involvement of endogenous D-serine in AD progression, we generated Srr gene-deleted APPKI (APPKI-SRRKO) mice. Finally, to examine the levels of brain amino acids, we conducted liquid chromatography-tandem mass spectrometry. Results: Expression of SRR was markedly reduced in the retrosplenial cortex (RSC) of APPKI mice at 12 months of age compared with age-matched wild-type mice. Neuronal density was decreased in the hippocampal CA1 region but not altered significantly in the RSC. D-Serine supplementation exacerbated neuronal loss in the hippocampal CA1 of APPKI mice, while APPKI-SRRKO mice exhibited attenuated astrogliosis and reduced neuronal death in the hippocampal CA1 compared with APPKI mice. Furthermore, APPKI mice demonstrated marked abnormalities in the cortical amino acid levels that were partially reversed in APPKI-SRRKO mice. Conclusion: These findings suggest that D-serine participates in the regional neurodegenerative process in the hippocampal CA1 during the amyloid pathology of AD and that reducing brain D-serine can partially attenuate neuronal loss and reactive astrogliosis. Therefore, regulating SRR could be an effective strategy to mitigate NMDAR-dependent neurodegeneration during AD progression.

Epitope-tag-mediated synaptogenic activity in an engineered neurexin-1ß lacking the binding interface with neuroligin-1.

Clustering of neurexin-1ß occurs through the formation of a trans-cellular complex with neuroligin-1, which promotes the generation of presynapse. While the extracellular region of neurexin-1ß functions to constitute the heterophilic binding interface with neuroligin-1, it has remained unclear whether the region could also play any key role in exerting the intracellular signaling for presynaptic differentiation. In this study, we generated neurexin-1ß lacking the binding site to neuroligin-1 and with a FLAG epitope at the N-terminus, and examined its activity in cultured neurons. The engineered protein still exhibited robust synaptogenic activities upon the epitope-mediated clustering, indicating that the region for complex formation and that for transmitting presynapse differentiation signals are structurally independent of each other. Using a fluorescence protein as an epitope, synaptogenesis was also induced by a gene-codable nanobody. The finding opens possibilities of neurexin-1ß as a platform for developing various molecular tools which may allow, for example, precise modifications of neural wirings under genetic control.

Neurexins play a crucial role in cerebellar granule cell survival by organizing autocrine machinery for neurotrophins.

Neurexins (NRXNs) are key presynaptic cell adhesion molecules that regulate synapse formation and function via trans-synaptic interaction with postsynaptic ligands. Here, we generate cerebellar granule cell (CGC)-specific Nrxn triple-knockout (TKO) mice for complete deletion of all NRXNs. Unexpectedly, most CGCs die in these mice, and this requirement for NRXNs for cell survival is reproduced in cultured CGCs. The axons of cultured Nrxn TKO CGCs that are not in contact with a postsynaptic structure show defects in the formation of presynaptic protein clusters and in action-potential-induced Ca2+ influxes. These cells also show impaired secretion of depolarization-induced, fluorescence-tagged brain-derived neurotrophic factor (BDNF) from their axons, and the cell-survival defect is rescued by the application of BDNF. These results suggest that CGC survival is maintained by autocrine neurotrophic factors and that NRXNs organize the presynaptic protein clusters and the autocrine neurotrophic-factor secretory machinery independent of contact with postsynaptic ligands.

d-Serine controls epidermal vesicle release via NMDA receptor, allowing tissue migration during the metamorphosis of the chordate Ciona.

d-Serine, a free amino acid synthesized by serine racemase, is a coagonist of N-methyl-d-aspartate-type glutamate receptor (NMDAR). d-Serine in the mammalian central nervous system modulates glutamatergic transmission. Functions of d-serine in mammalian peripheral tissues such as skin have also been described. However, d-serine's functions in nonmammals are unclear. Here, we characterized d-serine-dependent vesicle release from the epidermis during metamorphosis of the tunicate Ciona. d-Serine leads to the formation of a pocket that facilitates the arrival of migrating tissue during tail regression. NMDAR is the receptor of d-serine in the formation of the epidermal pocket. The epidermal pocket is formed by the release of epidermal vesicles' content mediated by d-serine/NMDAR. This mechanism is similar to observations of keratinocyte vesicle exocytosis in mammalian skin. Our findings provide a better understanding of the maintenance of epidermal homeostasis in animals and contribute to further evolutionary perspectives of d-amino acid function among metazoans.

BST1 regulates nicotinamide riboside metabolism via its glycohydrolase and base-exchange activities.

Nicotinamide riboside (NR) is one of the orally bioavailable NAD+ precursors and has been demonstrated to exhibit beneficial effects against aging and aging-associated diseases. However, the metabolic pathway of NR in vivo is not yet fully understood. Here, we demonstrate that orally administered NR increases NAD+ level via two different pathways. In the early phase, NR was directly absorbed and contributed to NAD+ generation through the NR salvage pathway, while in the late phase, NR was hydrolyzed to nicotinamide (NAM) by bone marrow stromal cell antigen 1 (BST1), and was further metabolized by the gut microbiota to nicotinic acid, contributing to generate NAD+ through the Preiss-Handler pathway. Furthermore, we report BST1 has a base-exchange activity against both NR and nicotinic acid riboside (NAR) to generate NAR and NR, respectively, connecting amidated and deamidated pathways. Thus, we conclude that BST1 plays a dual role as glycohydrolase and base-exchange enzyme during oral NR supplementation.

Prevalence of antibodies against human respiratory viruses potentially involving anthropozoonoses in wild bonobos.

One of the current threats to the bonobo (Pan paniscus), a highly endangered ape species only found in the Democratic Republic of the Congo, are anthropozoonoses caused by human respiratory viruses. To date, epidemiological information regarding respiratory viral infections in bonobos is limited. In this study, we examined fecal immunoglobulin A antibodies against human respiratory viruses in bonobos, which may help estimating the viral prevalence. A substantial proportion of bonobos were positive for the antiviral antibodies, including those against parainfluenza virus, respiratory syncytial virus, influenza virus, rhinovirus, and mumps virus. The prevalence of the antibodies was found to depend on the viral species and bonobo populations, suggesting that the bonobos had been exposed to these respiratory viruses. These results may indicate the need for an epidemiological evidence-based action plan for the protection of bonobos from anthropozoonoses.

Canonical versus non-canonical transsynaptic signaling of neuroligin 3 tunes development of sociality in mice.

Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality.

Roles of type IIa receptor protein tyrosine phosphatases as synaptic organizers.

Neurons establish circuits for brain functions such as cognition, emotion, learning, and memory. Their connections are mediated by synapses, which are specialized cell-cell adhesions responsible for neuronal signal transmission. During neurodevelopment, synapse formation is triggered by interactions of cell adhesion molecules termed synaptic organizers or synapse organizers. Type IIa receptor protein tyrosine phosphatases (IIa RPTPs; also known as leukocyte common antigen-related receptor tyrosine phosphatases or LAR-RPTPs) play important roles in axon guidance and neurite extension, and also serve as presynaptic organizers. IIa RPTPs transsynaptically interact with multiple sets of postsynaptic organizers, mostly in a splicing-dependent fashion. Here, we review and update research progress on IIa RPTPs, particularly regarding their functional roles in vivo demonstrated using conditional knockout approach and structural insights into their extracellular and intracellular molecular interactions revealed by crystallography and other biophysical techniques. Future directions in the research field of IIa RPTPs are also discussed, including recent findings of the molecular assembly mechanism underlying the formation of synapse-specific nanostructures essential for synaptic functions.

Characteristics of internalization of NMDA-type GluRs with antibodies to GluN1 and GluN2B.

To characterize internalization of NMDA-type glutamate receptors (GluRs) by antibodies to NMDA-type GluRs, we produced rabbit antibodies to N-terminals of human GluN1 and GluN2B, and examined internalization of NMDA-type GluRs in HEK293T cells using confocal microscopy. Internalization of NMDA-type GluRs occurred from at least 10 min after incubation with antibodies to GluN1 and or GluN2B and was temperature-dependent. These findings confirm that antibodies to N-terminals of GluN1 and GluN2B present in the cerebrospinal fluid of patients with NMDAR encephalitis can mediate prompt internalization of NMDA-type GluR complexes.

Functional characterization of rare NRXN1 variants identified in autism spectrum disorders and schizophrenia.

BACKGROUND: Rare genetic variants contribute to the etiology of both autism spectrum disorder (ASD) and schizophrenia (SCZ). Most genetic studies limit their focus to likely gene-disrupting mutations because they are relatively easier to interpret their effects on the gene product. Interpretation of missense variants is also informative to some pathophysiological mechanisms of these neurodevelopmental disorders; however, their contribution has not been elucidated because of relatively small effects. Therefore, we characterized missense variants detected in NRXN1, a well-known neurodevelopmental disease-causing gene, from individuals with ASD and SCZ. METHODS: To discover rare variants with large effect size and to evaluate their role in the shared etiopathophysiology of ASD and SCZ, we sequenced NRXN1 coding exons with a sample comprising 562 Japanese ASD and SCZ patients, followed by a genetic association analysis in 4273 unrelated individuals. Impact of each missense variant detected here on cell surface expression, interaction with NLGN1, and synaptogenic activity was analyzed using an in vitro functional assay and in silico three-dimensional (3D) structural modeling. RESULTS: Through mutation screening, we regarded three ultra-rare missense variants (T737M, D772G, and R856W), all of which affected the LNS4 domain of NRXN1α isoform, as disease-associated variants. Diagnosis of individuals with T737M, D772G, and R856W was 1ASD and 1SCZ, 1ASD, and 1SCZ, respectively. We observed the following phenotypic and functional burden caused by each variant. (i) D772G and R856W carriers had more serious social disabilities than T737M carriers. (ii) In vitro assay showed reduced cell surface expression of NRXN1α by D772G and R856W mutations. In vitro functional analysis showed decreased NRXN1α-NLGN1 interaction of T737M and D772G mutants. (iii) In silico 3D structural modeling indicated that T737M and D772G mutations could destabilize the rod-shaped structure of LNS2-LNS5 domains, and D772G and R856W could disturb N-glycan conformations for the transport signal. CONCLUSIONS: The combined data suggest that missense variants in NRXN1 could be associated with phenotypes of neurodevelopmental disorders beyond the diagnosis of ASD and/or SCZ.

[A Case of Gastric Schwannoma Diagnosed Preoperatively].

The patient was a 52-year-old woman, who was found to have an abnormality in the upper gastrointestinal(UGI)tract, via a contrast-imaging study; she had no symptoms.Computed tomography(CT)revealed a tumor, measuring approximately 100mm in diameter, in the antrum of the stomach.The tumor was diagnosed as gastric schwannoma using endoscopic ultrasonography-guided fine-needle aspiration(EUS-FNA).Preoperative CT revealed multiple lymph adenopathies around the antrum, which led to the suspicion of lymph node metastasis. The patient underwent a laparoscopic partial gastrectomy after the confirmation of the absence of lymph node metastasis by intraoperative rapid diagnosis.

TBX5 R264K acts as a modifier to develop dilated cardiomyopathy in mice independently of T-box pathway.

BACKGROUND: TBX5 is a transcription factor that has an important role in development of heart. TBX5 variants in the region encoding the T-box domain have been shown to cause cardiac defects, such as atrial septal defect or ventricular septal defect, while TBX5 variants have also been identified in a few cardiomyopathy patients and considered causative. We identified a TBX5 variant (c.791G>A, p.Arg264Lys), that is over-represented in cardiomyopathy patients. This variant is located outside of the T-box domain, and its pathogenicity has not been confirmed by functional analyses. OBJECTIVE: To investigate whether the TBX5 R264K is deleterious and could contribute to the pathogenesis of cardiomyopathy. METHODS AND RESULTS: We developed mice expressing Tbx5 R264K. Mice homozygous for this variant displayed compensated dilated cardiomyopathy; mild decreased fractional shortening, dilatation of the left ventricle, left ventricular wall thinning and increased heart weight without major heart structural disorders. There was no difference in activation of the ANF promotor, a transcriptional target of Tbx5, compared to wild-type. However, analysis of RNA isolated from left ventricular samples showed significant increases in the expression of Acta1 in left ventricle with concomitant increases in the protein level of ACTA1. CONCLUSIONS: Mice homozygous for Tbx5 R264K showed compensated dilated cardiomyopathy. Thus, TBX5 R264K may have a significant pathogenic role in some cardiomyopathy patients independently of T-box domain pathway.

Structural insights into selective interaction between type IIa receptor protein tyrosine phosphatases and Liprin-α.

Synapse formation is induced by transsynaptic interaction of neuronal cell-adhesion molecules termed synaptic organizers. Type IIa receptor protein tyrosine phosphatases (IIa RPTPs) function as presynaptic organizers. The cytoplasmic domain of IIa RPTPs consists of two phosphatase domains, and the membrane-distal one (D2) is essential for synapse formation. Liprin-α, which is an active zone protein critical for synapse formation, interacts with D2 via its C-terminal domain composed of three tandem sterile alpha motifs (tSAM). Structural mechanisms of this critical interaction for synapse formation remain elusive. Here, we report the crystal structure of the complex between mouse PTPδ D2 and Liprin-α3 tSAM at 1.91 Å resolution. PTPδ D2 interacts with the N-terminal helix and the first and second SAMs (SAM1 and SAM2, respectively) of Liprin-α3. Structure-based mutational analyses in vitro and in cellulo demonstrate that the interactions with Liprin-α SAM1 and SAM2 are essential for the binding and synaptogenic activity.

Roles of Collagen XXV and Its Putative Receptors PTPσ/δ in Intramuscular Motor Innervation and Congenital Cranial Dysinnervation Disorder.

Intramuscular motor innervation is an essential process in neuromuscular development. Recently, mutations in COL25A1, encoding CLAC-P/collagen XXV, have been linked to the development of a congenital cranial dysinnervation disorder (CCDD). Yet the molecular mechanisms of intramuscular innervation and the etiology of CCDD related to COL25A1 have remained elusive. Here, we report that muscle-derived collagen XXV is indispensable for intramuscular innervation. In developing skeletal muscles, Col25a1 expression is tightly regulated by muscle excitation. In vitro and cell-based assays reveal a direct interaction between collagen XXV and receptor protein tyrosine phosphatases (PTPs) σ and δ. Motor explant assays show that expression of collagen XXV in target cells attracts motor axons, but this is inhibited by exogenous PTPσ/δ. CCDD mutations attenuate motor axon attraction by reducing collagen XXV-PTPσ/δ interaction. Overall, our study identifies PTPσ/δ as putative receptors for collagen XXV, implicating collagen XXV and PTPσ/δ in intramuscular innervation and a developmental ocular motor disorder.

Intragranular three-dimensional stress tensor fields in plastically deformed polycrystals.

The failure of polycrystalline materials used in infrastructure and transportation can be catastrophic. Multiscale modeling, which requires multiscale measurements of internal stress fields, is the key to predicting the deformation and failure of alloys. We determined the three-dimensional intragranular stress tensor fields in plastically deformed bulk steel using a high-energy x-ray microbeam. We observed intragranular local stresses that deviated greatly from the grain-averaged stresses and exceeded the macroscopic tensile strength. Even under deformation smaller than the uniform elongation, the intragranular stress fields were in highly triaxial stress states, which cannot be determined from the grain-averaged stresses. The ability to determine intragranular stress tensor fields can facilitate the understanding and prediction of the deformation and failure of materials through multiscale modeling.