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Mutations in many synaptic genes are associated with autism spectrum disorders (ASD), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C mutation on human neurons has not been investigated. Here, we generated human knockin neurons with the NLGN3 R451C and NLGN3 null mutations. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses; meanwhile NLGN3 knockout neurons showed reduction in excitatory synaptic strengths. Moreover, overexpression of NLGN3 R451C recapitulated the synaptic enhancement in human neurons. Notably, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons corresponding to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASD., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Neuroligins are synaptic cell adhesion proteins with a role in synaptic function, implicated in neurodevelopmental disorders. The autism spectrum disorder-associated substitution Arg451Cys (R451C) in NLGN3 promotes a partial misfolding of the extracellular domain of the protein leading to retention in the endoplasmic reticulum (ER) and the induction of the unfolded protein response (UPR). The reduced trafficking of R451C NLGN3 to the cell surface leads to altered synaptic function and social behavior. A screening in HEK-293 cells overexpressing NLGN3 of 2662 compounds (FDA-approved small molecule drug library), led to the identification of several glucocorticoids such as alclometasone dipropionate, desonide, prednisolone sodium phosphate, and dexamethasone (DEX), with the ability to favor the exit of full-length R451C NLGN3 from the ER. DEX improved the stability of R451C NLGN3 and trafficking to the cell surface, reduced the activation of the UPR, and increased the formation of artificial synapses between HEK-293 and hippocampal primary neurons. The effect of DEX was validated on a novel model system represented by neural stem progenitor cells and differentiated neurons derived from the R451C NLGN3 knock-in mouse, expressing the endogenous protein. This work shows a potential rescue strategy for an autism-linked mutation affecting cell surface trafficking of a synaptic protein., (© 2024 The Authors. Traffic published by John Wiley & Sons Ltd.)

The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and selectively localized to the axon initial segment (AIS), the subcellular compartment triggering action potentials. Ectopic expression of LRRC37B in mouse CPNs in vivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. Molecularly, LRRC37B binds to the secreted ligand FGF13A and to the voltage-gated sodium channel (Nav) β-subunit SCN1B. LRRC37B concentrates inhibitory effects of FGF13A on Nav channel function, thereby reducing excitability, specifically at the AIS level. Electrophysiological recordings in adult human cortical slices reveal lower neuronal excitability in human CPNs expressing LRRC37B. LRRC37B thus acts as a species-specific modifier of human neuron excitability, linking human genome and cell evolution, with important implications for human brain function and diseases., Competing Interests: Declaration of interests B.L.-P., P.V., J.d.W., A.L., K.W., J.S., F.R., N.L, and K.K. are inventors on a PCT application related to this work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Protective immune responses against respiratory pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, are initiated by the mucosal immune system. However, most licensed vaccines are administered parenterally and are largely ineffective at inducing mucosal immunity. The development of safe and effective mucosal vaccines has been hampered by the lack of a suitable mucosal adjuvant. In this study we explore a class of adjuvant that harnesses mucosal-associated invariant T (MAIT) cells. We show evidence that intranasal immunization of MAIT cell agonists co-administered with protein, including the spike receptor binding domain from SARS-CoV-2 virus and hemagglutinin from influenza virus, induce protective humoral immunity and immunoglobulin A production. MAIT cell adjuvant activity is mediated by CD40L-dependent activation of dendritic cells and subsequent priming of T follicular helper cells. In summary, we show that MAIT cells are promising vaccine targets that can be utilized as cellular adjuvants in mucosal vaccines., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Emerging SARS-CoV-2 variants pose a threat to human health worldwide. SARS-CoV-2 receptor binding domain (RBD)-based vaccines are suitable candidates for booster vaccines, eliciting a focused antibody response enriched for virus neutralizing activity. Although RBD proteins are manufactured easily, and have excellent stability and safety properties, they are poorly immunogenic compared to the full-length spike protein. We have overcome this limitation by engineering a subunit vaccine composed of an RBD tandem dimer fused to the N-terminal domain (NTD) of the spike protein. We found that inclusion of the NTD (1) improved the magnitude and breadth of the T cell and anti-RBD response, and (2) enhanced T follicular helper cell and memory B cell generation, antibody potency, and cross-reactive neutralization activity against multiple SARS-CoV-2 variants, including B.1.1.529 (Omicron BA.1). In summary, our uniquely engineered RBD-NTD-subunit protein vaccine provides a promising booster vaccination strategy capable of protecting against known SARS-CoV-2 variants of concern., Competing Interests: The authors declare no conflict of interests, other than the filing of patent application number AU2021902667 entitled “Fusion Polypeptide”. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2023 The Authors.)

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuronal development and function are dependent in part on the several roles of the secreted glycoprotein Reelin. Endogenous proteases process this 400 kDa, modular protein, yielding N-terminal, central, and C-terminal fragments that each have distinct roles in Reelin's function and regulation. The C-terminal fragment comprises Reelin repeat (RR) domains seven and eight, as well as a basic stretch of 32 amino acid residues termed the C-terminal region (CTR), influences Reelin signaling intensity, and has been reported to bind to Neuropilin-1, which serves as a co-receptor in the canonical Reelin signaling pathway. Here, we present a crystal structure of RR8 at 3.0 Å resolution. Analytical ultracentrifugation and small-angle x-ray scattering confirmed that RR8 is monomeric and enabled us to identify the CTR as a flexible, yet compact subdomain. We conducted structurally informed protein engineering to design a chimeric RR8 construct guided by the structural similarities with RR6. Experimental results support a mode of Reelin-receptor interaction reliant on the multiple interfaces coordinating the binding event. Structurally, RR8 resembles other individual RRs, but its structure does show discrete differences that may account for Reelin receptor specificity toward RR6., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Although many neuronal membrane proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding (ES). Here, we show that the neuronal sheddome is detectable in human cerebrospinal fluid (hCSF) and is enriched in neurodevelopmental disorder (NDD) risk factors. Among shed synaptic proteins is the ectodomain of CNTNAP2 (CNTNAP2-ecto), a prominent NDD risk factor. CNTNAP2 undergoes activity-dependent ES via MMP9 (matrix metalloprotease 9), and CNTNAP2-ecto levels are reduced in the hCSF of individuals with autism spectrum disorder. Using mass spectrometry, we identified the plasma membrane Ca 2+ ATPase (PMCA) extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto enhances the activity of PMCA2 and regulates neuronal network dynamics in a PMCA2-dependent manner. Our data underscore the promise of sheddome analysis in discovering neurobiological mechanisms, provide insight into the biology of ES and its relationship with the CSF, and reveal a mechanism of regulation of Ca 2+ homeostasis and neuronal network synchrony by a shed ectodomain., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Reelin operates through canonical and non-canonical pathways that mediate several aspects of brain development and function. Reelin's dimeric central fragment (CF), generated through proteolytic cleavage, is required for the lipoprotein-receptor-dependent canonical pathway activation. Here, we analyze the signaling properties of a variety of Reelin fragments and measure the differential binding affinities of monomeric and dimeric CF fragments to lipoprotein receptors to investigate the mode of canonical signal activation. We also present the cryoelectron tomography-solved dimeric structure of Reelin CF and support it using several other biophysical techniques. Our findings suggest that Reelin CF forms a covalent parallel dimer with some degree of flexibility between the two protein chains. As a result of this conformation, Reelin binds to lipoprotein receptors in a manner inaccessible to its monomeric form and is capable of stimulating canonical pathway signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Dopamine (DA) transmission is critical to motivation, movement, and emotion. Unlike glutamatergic and GABAergic synapses, the development of DA synapses is less understood. We show that bassoon (BSN) clusters along DA axons in the core of nucleus accumbens (NAcc) were increased in neonatal stages and reduced afterward, suggesting DA synapse elimination. Remarkably, DA neuron-specific ablating neuregulin 3 (NRG3), a protein whose levels correlate with BSN clusters, increased the clusters and impaired DA release and behaviors related to DA transmission. An unbiased screen of transmembrane proteins with the extracellular domain (ECD) of NRG3 identified Caspr3 (contactin associate-like protein 3) as a binding partner. Caspr3 was enriched in striatal medium spiny neurons (MSNs). NRG3 and Caspr3 interact in trans, which was blocked by Caspr3-ECD. Caspr3 null mice displayed phenotypes similar to those in DAT-Nrg3 f/f mice in DA axonal BSN clusters and DA transmission. Finally, in vivo disruption of the NRG3-Caspr3 interaction increased BSN clusters. Together, these results demonstrate that DA synapse development is controlled by trans interaction between NRG3 in DA neurons and Caspr3 in MSNs, identifying a novel pair of cell adhesion molecules for brain circuit wiring., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cell adhesion generally involves formation of homophilic or heterophilic protein complexes between two cells to form transcellular junctions. Neural cell-adhesion members of the α/β-hydrolase fold superfamily of proteins use their extracellular or soluble cholinesterase-like domain to bind cognate partners across cell membranes, as illustrated by the neuroligins. These cell-adhesion molecules currently comprise the synaptic organizers neuroligins found in all animal phyla, along with three proteins found only in invertebrates: the guidance molecule neurotactin, the glia-specific gliotactin, and the basement membrane protein glutactin. Although these proteins share a cholinesterase-like fold, they lack one or more residues composing the catalytic triad responsible for the enzymatic activity of the cholinesterases. Conversely, they are found in various subcellular localisations and display specific disulfide bonding and N-glycosylation patterns, along with individual surface determinants possibly associated with recognition and binding of protein partners. Formation of non-covalent dimers typical of the cholinesterases is documented for mammalian neuroligins, yet whether invertebrate neuroligins and their neurotactin, gliotactin and glutactin relatives also form dimers in physiological conditions is unknown. Here we provide a brief overview of the localization, function, evolution, and conserved versus individual structural determinants of these cholinesterase-like cell-adhesion proteins. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Reelin is a secreted glycoprotein that is integral in neocortex development and synaptic function. Reelin exists as a homodimer with two chains linked by a disulfide bond at cysteine 2101, a feature that is vital to the protein's function. This is highlighted by the fact that only dimeric Reelin can elicit efficient, canonical signaling, even though a mutated (C2101A) monomeric construct of Reelin retains the capacity to bind to its receptors. Receptor clustering has been shown to be important in the signaling pathway, however direct evidence regarding the stoichiometry of Reelin-receptor binding interaction is lacking. Here we describe the construction and purification of a heterodimeric Reelin construct to investigate the stoichiometry of Reelin-receptor binding and how it affects Reelin pathway signaling. We have devised different strategies and have finalized a protocol to produce a heterodimer of Reelin's central fragment using differential tagging and tandem affinity chromatography, such that chain A is wild type in amino acid sequence whereas chain B includes a receptor binding site mutation (K2467A). We also validate that the heterodimer is capable of binding to the extracellular domain of one of Reelin's known receptors, calculating the K D of the interaction. This heterodimeric construct will enable us to understand in greater detail the mechanism by which Reelin interacts with its known receptors and initiates pathway signaling.

Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function.

The concept that exposure in utero to maternal anti-brain antibodies contributes to the development of autism spectrum disorders (ASD) has been entertained for over a decade. We determined that antibodies targeting Caspr2 are present at high frequency in mothers with brain-reactive serology and a child with ASD, and further demonstrated that exposure in utero to a monoclonal anti-Caspr2 antibody, derived from a mother of an ASD child, led to an-ASD like phenotype in male offspring. Now we propose a new model to study the effects of in utero exposure to anti-Caspr2 antibody. Dams immunized with the extracellular portion of Caspr2 express anti-Caspr2 antibodies throughout gestation to better mimic the human condition. Male but not female mice born to dams harboring polyclonal anti-Caspr2 antibodies showed abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as repetitive behaviors and impairments in novelty interest in the social preference test as adults. These data supporting the pathogenicity of anti-Caspr2 antibodies are consistent with the concept that anti-brain antibodies present in women during gestation can alter fetal brain development, and confirm that males are peculiarly susceptible.

COVID-19 is a new zoonotic disease caused by the SARS-CoV-2 virus. Since its emergence in Wuhan City, China, the virus has rapidly spread across the globe causing calamitous health, economic and societal consequences. It causes disproportionately severe disease in the elderly and those with co-morbidities, such as hypertension and diabetes. There is currently no proven treatment for COVID-19 and a safe and effective vaccine is at least a year away. The virus gains access to the respiratory epithelium through cell surface angiotensin converting enzyme 2 (ACE2). The receptor binding domain (RBD) of the virus is unlikely to mutate without loss of pathogenicity and thus represents an attractive target for antiviral treatment. Inhaled modified recombinant human ACE2, may bind SARS-CoV-2 and mitigate lung damage. This decoy strategy is unlikely to provoke an adverse immune response and may reduce morbidity and mortality in high-risk groups., Competing Interests: Dr Rolleston reports affiliation with South Pacific Sera Ltd outside the submitted work; and is the Chair of the Life Sciences Network. Dr Petousis-Harris reports grants from GSK outside the submitted work.

In the developing brain, cell-surface proteins play crucial roles, but their protein-protein interaction network remains largely unknown. A proteomic screen identified 200 interactions, 89 of which were not previously published. Among these interactions, we find that the IgLONs, a family of five cell-surface neuronal proteins implicated in various human disorders, interact as homo- and heterodimers. We reveal their interaction patterns and report the dimeric crystal structures of Neurotrimin (NTRI), IgLON5, and the neuronal growth regulator 1 (NEGR1)/IgLON5 complex. We show that IgLONs maintain an extended conformation and that their dimerization occurs through the first Ig domain of each monomer and is Ca 2+ independent. Cell aggregation shows that NTRI and NEGR1 homo- and heterodimerize in trans. Taken together, we report 89 unpublished cell-surface ligand-receptor pairs and describe structural models of trans interactions of IgLONs, showing that their structures are compatible with a model of interaction across the synaptic cleft., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Amyloid-β precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for secreted APP (sAPP). Here we show that the sAPP extension domain directly bound the sushi 1 domain specific to the γ-aminobutyric acid type B receptor subunit 1a (GABA B R1a). sAPP-GABA B R1a binding suppressed synaptic transmission and enhanced short-term facilitation in mouse hippocampal synapses via inhibition of synaptic vesicle release. A 17-amino acid peptide corresponding to the GABA B R1a binding region within APP suppressed in vivo spontaneous neuronal activity in the hippocampus of anesthetized Thy1-GCaMP6s mice. Our findings identify GABA B R1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABA B R1a function to modulate synaptic transmission., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand-receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein-protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand-receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand-receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning., (© 2019 Elsevier Inc. All rights reserved.)

The interaction between the T cell antigen receptor (TCR) expressed by natural killer T cells (NKT cells) and the antigen-presenting molecule CD1d is distinct from interactions between the TCR and major histocompatibility complex (MHC). Our molecular modeling suggested that a hydrophobic patch created after TCRα-TCRβ pairing has a role in maintaining the conformation of the NKT cell TCR. Disruption of this patch ablated recognition of CD1d by the NKT cell TCR but not interactions of the TCR with MHC. Partial disruption of the patch, while permissive to the recognition of CD1d, significantly altered NKT cell development, which resulted in the selective accumulation of adipose-tissue-resident NKT cells. These results indicate that a key component of the TCR is essential for the development of a distinct population of NKT cells.