Published papers

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, Ca2+-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.

Neurons connect to each other via synapses to form neural circuits. Recent research has shown that neuropsychiatric disorders and neurological disorders, such as autism spectrum disorders and Alzheimer's disease, are synaptic diseases caused by abnormalities of synapses. Synaptic organizers are molecules responsible for synapse formation. Neuronal pentraxin 2 (NP2) is a synaptic organizer and a secreted protein that is expressed mainly in the hippocampus and cerebellum, and it contributes to synaptic plasticity. NP2 forms clusters with its family proteins, NP1 and neuronal pentraxin receptor, and binds to postsynaptic amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type receptors. In recent years, research has revealed the disease relevance of NP2. For example, it can be a biomarker of Alzheimer's disease, and its overexpression in the peripheral nervous system has been reported to cause chronic itch. However, the mechanism of NP2 function has not been well described at the molecular level. In this study, we developed a variable domain of a heavy-chain antibody (VHH) against NP2 to elucidate its molecular mechanism of action and to regulate its function of NP2. The obtained VHH N1 showed high specificity and affinity to NP2, and its binding mechanism was elucidated by X-ray crystallography. Furthermore, VHH N1 inhibited the binding of NP2 to amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type receptors, and this inhibitory activity was confirmed in cells. These results provide useful insights into the molecular mechanism of NP2 function and highlight the potential application of VHH N1 as a detection agent for NP2 or as a therapeutic agent for chronic itch.

Kainate (KA)-type glutamate receptors (KARs) are implicated in various neuropsychiatric and neurological disorders through their ionotropic and metabotropic actions. However, compared to AMPA- and NMDA-type receptor functions, many aspects of KAR biology remain incompletely understood. Our study demonstrates an important role of KARs in organizing climbing fiber (CF)-Purkinje cell (PC) synapses and synaptic plasticity in the cerebellum, independently of their ion channel or metabotropic functions. The amino-terminal domain (ATD) of the GluK4 KAR subunit binds to C1ql1, provided by CFs, and associates with Bai3, an adhesion-type G protein-coupled receptor expressed in PC dendrites. Mice lacking GluK4 exhibit no KAR-mediated responses, reduced C1ql1 and Bai3 levels, and fewer CF-PC synapses, along with impaired long-term depression and oculomotor learning. Remarkably, introduction of the ATD of GluK4 significantly improves all these phenotypes. These findings demonstrate that KARs act as synaptic scaffolds, orchestrating synapses by forming a KAR-C1ql1-Bai3 complex in the cerebellum.

Copyright © 2020 The Authors, Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory–inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer’s disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.

© 2020 Kim et al. Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that β-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive β-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents.

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Synapses are precisely established, maintained, and modified throughout life by molecules called synaptic organizers, which include neurexins and neuroligins (Nlgn). Despite the importance of synaptic organizers in defining functions of neuronal circuits, the cellular and subcellular localization of many synaptic organizers has remained largely elusive because of the paucity of specific antibodies for immunohistochemical studies. In the present study, rather than raising specific antibodies, we generated knock-in mice in which a hemagglutinin (HA) epitope was inserted in the Nlgn1 gene. We have achieved high-throughput and precise gene editing by delivering the CRISPR/Cas9 system into zygotes. Using HA-Nlgn1 mice, we found that HA-Nlgn1 was enriched at synapses between parallel fibers and molecular layer interneurons (MLIs) and the glomeruli, in which mossy fiber terminals synapse onto granule cell dendrites. HA immunoreactivity was colocalized with postsynaptic density 95 at these synapses, indicating that endogenous Nlgn1 is localized at excitatory postsynaptic sites. In contrast, HA-Nlgn1 signals were very weak in dendrites and somata of Purkinje cells. Interestingly, HA-immunoreactivities were also observed in the pinceau, a specialized structure formed by MLI axons and astrocytes. HA-immunoreactivities in the pinceau were significantly reduced by knockdown of Nlgn1 in MLIs, indicating that in addition to postsynaptic sites, Nlgn1 is also localized at MLI axons. Our results indicate that epitope-tagging by electroporation-based gene editing with CRISPR/Cas9 is a viable and powerful method for mapping endogenous synaptic organizers with subcellular resolution, without the need for specific antibodies for each protein.

Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as acrucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesionG-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.

D-Serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2. © 2011 Nature America, Inc. All rights reserved.

Cbln1 (a.k.a. precerebellin) is a unique bidirectional synaptic organizer that plays an essential role in the formation and maintenance of excitatory synapses between granule cells and Purkinje cells in the mouse cerebellum. Cbln1 secreted from cerebellar granule cells directly induces presynaptic differentiation and indirectly serves as a postsynaptic organizer by binding to its receptor, the δ2 glutamate receptor. However, it remains unclear how Cbln1 binds to the presynaptic sites and interacts with other synaptic organizers. Furthermore, although Cbln1 and its family members Cbln2 and Cbln4 are expressed in brain regions other than the cerebellum, it is unknown whether they regulate synapse formation in these brain regions. In this study, we showed that Cbln1 and Cbln2, but not Cbln4, specifically bound to its presynaptic receptor -α and β isoforms of neurexin carrying the splice site 4 insert [NRXs(S4+)] - and induced synaptogenesis in cerebellar, hippocampal and cortical neurons in vitro. Cbln1 competed with synaptogenesis mediated by neuroligin 1, which lacks the splice sites A and B, but not leucine-rich repeat transmembrane protein 2, possibly by sharing the presynaptic receptor NRXs(S4+). However, unlike neurexins/neuroligins or neurexins/leucine-rich repeat transmembrane proteins, the interaction between NRX1β(S4+) and Cbln1 was insensitive to extracellular Ca2+ concentrations. These findings revealed the unique and general roles of Cbln family proteins in mediating the formation and maintenance of synapses not only in the cerebellum but also in various other brain regions. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/ isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1Asn has a weaker affinity to glutamate than GRIA1Ser, both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1Asn have a slower luteinizing hormone (LH) surge than cows with GRIA1Ser. In addition, cows with GRIA1Asn possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1Ser. Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy. © 2010 Sugimoto et al.

Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor 52 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cblnl binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components.

The δ2 glutamate receptor (GluRδ2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)-Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2wt) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2wt, but not of a mutant GluD2 lacking the NTD (GluD2ΔNTD), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2 NTD-GluK2) did. Similarly, GluD2wt and GluD2 NTD-GluK2, but not GluD2ΔNTD, induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF-Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively. Copyright © 2009 Society for Neuroscience.

Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluRδ2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluRδ2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluRδ2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum. © The Authors (2009).

Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders. Copyright © 2008 Society for Neuroscience.

AP-4 is a member of the adaptor protein complexes, which control vesicular trafficking of membrane proteins. Although AP-4 has been suggested to contribute to basolateral sorting in epithelial cells, its function in neurons is unknown. Here, we show that disruption of the gene encoding the β subunit of AP-4 resulted in increased accumulation of axonal autophagosomes, which contained AMPA receptors and transmembrane AMPA receptor regulatory proteins (TARPs), in axons of hippocampal neurons and cerebellar Purkinje cells both in vitro and in vivo. AP-4 indirectly associated with the AMPA receptor via TARPs, and the specific disruption of the interaction between AP-4 and TARPs caused the mislocalization of endogenous AMPA receptors in axons of wild-type neurons. These results indicate that AP-4 may regulate proper somatodendritic-specific distribution of its cargo proteins, including AMPA receptor-TARP complexes and the autophagic pathway in neurons. © 2008 Elsevier Inc. All rights reserved.