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The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N -methyl-D-aspartate-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons. The analysis of AMPA-type glutamate receptor (AMPAR) trafficking is essential for understanding molecular mechanisms of learning and memory, but the analytical tools are currently limited. Here, the authors report a method that combines affinity-based receptor labeling and bioorthogonal click chemistry to quantify AMPAR distribution and trafficking under physiological conditions.

Changes in neural activity induced by learning and novel environments have been reported to lead to the formation of new synapses in the adult brain. However, the underlying molecular mechanism is not well understood. Here, we show that Purkinje cells (PCs), which have established adult-type monosynaptic innervation by climbing fibers (CFs) after elimination of weak CFs during development, can be reinnervated by multiple CFs by increased expression of the synaptic organizer C1ql1 in CFs or Bai3, a receptor for C1ql1, in PCs. In the adult cerebellum, CFs are known to have transverse branches that run in a mediolateral direction without forming synapses with PCs. Electrophysiological, Ca 2+ -imaging and immunohistochemical studies showed that overexpression of C1ql1 or Bai3 caused these CF transverse branches to elongate and synapse on the distal dendrites of mature PCs. Mature PCs were also reinnervated by multiple CFs when the glutamate receptor GluD2, which is essential for the maintenance of synapses between granule cells and PCs, was deleted. Interestingly, the effect of GluD2 knockout was not observed in Bai3 knockout PCs. In addition, C1ql1 levels were significantly upregulated in CFs of GluD2 knockout mice, suggesting that endogenous, not overexpressed, C1ql1-Bai3 signaling could regulate the reinnervation of mature PCs by CFs. Furthermore, the effects of C1ql1 and Bai3 overexpression required neuronal activity in the PC and CF, respectively. C1ql1 immunoreactivity at CF-PC synapses was reduced when the neuronal activity of CFs was suppressed. These results suggest that C1ql1-Bai3 signaling may mediate CF synaptogenesis in mature PCs, potentially in concert with neuronal activity.

A wide variety of systemic pathologies, including infectious and autoimmune diseases, are accompanied by joint pain or inflammation, often mediated by circulating immune complexes (ICs). How such stimuli access joints and trigger inflammation is unclear. Whole-mount synovial imaging revealed PV1 + fenestrated capillaries at the periphery of the synovium in the lining–sublining interface. Circulating ICs extravasated from these PV1 + capillaries, and nociceptor neurons and three distinct macrophage subsets formed a sentinel unit around them. Macrophages showed subset-specific responses to systemic IC challenge; LYVE1 + CX 3 CR1 + macrophages orchestrated neutrophil recruitment and activated calcitonin gene-related peptide + (CGRP + ) nociceptor neurons via interleukin-1β. In contrast, major histocompatibility complex class II + CD11c + (MHCII + CD11c + ) and MHCII + CD11c – interstitial macrophages formed tight clusters around PV1 + capillaries in response to systemic immune stimuli, a feature enhanced by nociceptor-derived CGRP. Altogether, we identify the anatomical location of synovial PV1 + capillaries and subset-specific macrophage–nociceptor cross-talk that forms a blood–joint barrier protecting the synovium from circulating immune challenges. Why joints are highly responsive to systemic inflammation is unknown. Hasegawa et al. sought to address this question, developing a whole-mount imaging system of the entire synovium to profile the vascular, neuronal and immune components.

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders. Visualizing neurotransmitter receptor traffic is fundamental to understanding brain functions, but the current imaging methods can compromise the process. Here, the authors synthesize small fluorescent probes to label endogenous glutamate receptors in cell cultures and brain tissues, without affecting their function.

Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)–interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This pathway is particularly enriched in nAChR α3 and β4 subunits, both of which are genetically linked to nicotine dependence. However, the cellular and subcellular expression of endogenous α3β4-containing nAChRs remains largely unknown because specific antibodies and appropriate detection methods were unavailable. Here, we successfully uncovered the expression of endogenous nAChRs containing α3 and β4 subunits in the MHb–IPN pathway using novel specific antibodies and a fixative glyoxal that enables simultaneous detection of synaptic and extrasynaptic molecules. Immunofluorescence and immunoelectron microscopy revealed that both subunits were predominantly localized to the extrasynaptic cell surface of somatodendritic and axonal compartments of MHb neurons but not at their synaptic junctions. Immunolabeling for α3 and β4 subunits disappeared in α5β4-knockout brains, which we used as negative controls. The enriched and diffuse extrasynaptic expression along the MHb–IPN pathway suggests that α3β4-containing nAChRs may enhance the excitability of MHb neurons and neurotransmitter release from their presynaptic terminals in the IPN. The revealed distribution pattern provides a molecular and anatomical basis for understanding the functional role of α3β4-containing nAChRs in the crucial pathway of nicotine dependence.

Ionotropic glutamate receptor (iGIuR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGIuR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGIuR δ2 (GIuD2) and presynaptic β-neurexin 1 (β-NRX1) via CbIn1, a C1q-like synaptic organizer. We show how CbIn1 hexamers \"anchor\" GIuD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for D-serine–dependent GIuD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber–Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGIuR function.

Although aberrations in the number and function of glutamate AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors are thought to underlie neuropsychiatric disorders, no methods are currently available for visualizing AMPA receptors in the living human brain. Here we developed a positron emission tomography (PET) tracer for AMPA receptors. A derivative of 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide radiolabeled with 11 C ([ 11 C]K-2) showed specific binding to AMPA receptors. Our clinical trial with healthy human participants confirmed reversible binding of [ 11 C]K-2 in the brain according to Logan graphical analysis ( UMIN000020975 ; study design: non-randomized, single arm; primary outcome: dynamics and distribution volumes of [ 11 C]K-2 in the brain; secondary outcome: adverse events of [ 11 C]K-2 during the 4–10 d following dosing; this trial met prespecified endpoints). In an exploratory clinical study including patients with epilepsy, we detected increased [ 11 C]K-2 uptake in the epileptogenic focus of patients with mesial temporal lobe epilepsy, which was closely correlated with the local AMPA receptor protein distribution in surgical specimens from the same individuals ( UMIN000025090 ; study design: non-randomized, single arm; primary outcome: correlation between [ 11 C]K-2 uptake measured with PET before surgery and AMPA receptor protein density examined by biochemical study after surgery; secondary outcome: adverse events during the 7 d following PET scan; this trial met prespecified endpoints). Thus, [ 11 C]K-2 is a potent PET tracer for AMPA receptors, potentially providing a tool to examine the involvement of AMPA receptors in neuropsychiatric disorders. A newly developed PET tracer allows visualization of AMPA receptors in the living human brain, providing a new tool to study their potential involvement in neurological or psychiatric disorders.

Various types of induced pluripotent stem (iPS) cells have been established by different methods, and each type exhibits different biological properties. Before iPS cell-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Here we show the directed neural differentiation of murine iPS cells and examine their therapeutic potential in a mouse spinal cord injury (SCI) model. \"Safe\" iPS-derived neurospheres, which had been pre-evaluated as nontumorigenic by their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse brain, produced electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro. Furthermore, when the safe iPS-derived neurospheres were transplanted into the spinal cord 9 d after contusive injury, they differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in remyelination and induced the axonal regrowth of host 5HT⁺ serotonergic fibers, promoting locomotor function recovery. However, the transplantation of iPS-derived neurospheres pre-evaluated as \"unsafe\" showed robust teratoma formation and sudden locomotor functional loss after functional recovery in the SCI model. These findings suggest that pre-evaluated safe iPS clone-derived neural stem/progenitor cells may be a promising cell source for transplantation therapy for SCI.

SignificanceLong-term depression (LTD) commonly affects learning and memory in various brain regions. Although LTD in the cerebellum absolutely requires δ2 glutamate receptors, its underlying mechanisms remain elusive. LTD is caused by endocytosis of AMPA receptors, which is triggered by activity-induced serine phosphorylation of the GluA2 subunit. Our work showed that this serine phosphorylation required prior dephosphorylation of the nearby tyrosine residue. By interaction with a tyrosine phosphatase, δ2 glutamate receptors regulated tyrosine dephosphorylation status of GluA2 to gate inducibility of LTD. These findings will provide better understanding of general mechanisms regulating AMPA receptor endocytosis during synaptic plasticity. Long-term depression (LTD) commonly affects learning and memory in various brain regions. Although cerebellar LTD absolutely requires the δ2 glutamate receptor (GluD2) that is expressed in Purkinje cells, LTD in other brain regions does not; why and how cerebellar LTD is regulated by GluD2 remains unelucidated. Here, we show that the activity-dependent phosphorylation of serine 880 (S880) in GluA2 AMPA receptor subunit, which is an essential step for AMPA receptor endocytosis during LTD induction, was impaired in GluD2-null cerebellum. In contrast, the basal phosphorylation levels of tyrosine 876 (Y876) in GluA2 were increased in GluD2-null cerebellum. An in vitro phosphorylation assay revealed that Y876 phosphorylation inhibited subsequent S880 phosphorylation. Conversely, Y876 dephosphorylation was sufficient to restore S880 phosphorylation and LTD induction in GluD2-null Purkinje cells. Furthermore, megakaryocyte protein tyrosine phosphatase (PTPMEG), which binds to the C terminus of GluD2, directly dephosphorylated Y876. These data indicate that GluD2 gates LTD by coordinating interactions between the two phosphorylation sites of the GluA2.

Myoblast fusion is tightly regulated during development and regeneration of muscle fibers. BAI3 is a receptor that orchestrates myoblast fusion via Elmo/Dock1 signaling, but the mechanisms regulating its activity remain elusive. Here we report that mice lacking BAI3 display small muscle fibers and inefficient muscle regeneration after cardiotoxin-induced injury. We describe two proteins that repress or activate BAI3 in muscle progenitors. We find that the secreted C1q-like1–4 proteins repress fusion by specifically interacting with BAI3. Using a proteomic approach, we identify Stabilin-2 as a protein that interacts with BAI3 and stimulates its fusion promoting activity. We demonstrate that Stabilin-2 activates the GPCR activity of BAI3. The resulting activated heterotrimeric G-proteins contribute to the initial recruitment of Elmo proteins to the membrane, which are then stabilized on BAI3 through a direct interaction. Collectively, our results demonstrate that the activity of BAI3 is spatiotemporally regulated by C1qL4 and Stabilin-2 during myoblast fusion. Myoblast fusion is an essential step in muscle growth and regeneration, and is regulated by the G-protein coupled receptor (GPCR) BAI3. Here Hamoud et al. show that the GPCR activity of BAI3 is spatiotemporally regulated during myoblast fusion, and identify C1qL4 and Stabilin-2 as, respectively, negative and positive regulators of its activity.