Keiko Matsuda

Cbln1 Is a Ligand for an Orphan Glutamate Receptor δ2, a Bidirectional Synapse Organizer

Orphan No More The glutamate receptor δ2 (GluD2), another member of the ionotropic glutamate receptor family, has long been considered to be an orphan receptor because there are no known endogenous ligands. Nevertheless, GluD2 is essential for the normal development of cerebellar circuits. Using immunocytochemistry, binding assays, electrophysiology, and freeze-fracture electron microscopy, Matsuda et al. (p. 363) found that Cbln1, a soluble protein secreted from cerebellar granule cells, binds to the extracellular N terminus of GluD2 on Purkinje cells. Binding has two independent consequences: First, it leads to presynaptic differentiation and second, it causes postsynaptic clustering of several important synapse-specific molecules. Both events are needed for synapse formation between granule cells and Purkinje cells. A signaling complex serves as a synapse organizer that acts bidirectionally on both pre- and postsynaptic components. Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor δ2 (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 Cbln1 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.

Accumulation of AMPA Receptors in Autophagosomes in Neuronal Axons Lacking Adaptor Protein AP-4

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 beta 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.

Cbln family proteins promote synapse formation by regulating distinct neurexin signaling pathways in various brain regions

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.

D -Serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor

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 δ2 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.

Cbln1 Regulates Rapid Formation and Maintenance of Excitatory Synapses in Mature Cerebellar Purkinje Cells In Vitro and In Vivo

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.

Differential Regulation of Synaptic Plasticity and Cerebellar Motor Learning by the C-Terminal PDZ-Binding Motif of GluRδ2

The δ2 glutamate receptor (GluRδ2) is predominantly expressed in Purkinje cells and plays crucial roles in cerebellar functions: GluRδ2−/− mice display ataxia and impaired motor learning. In addition, long-term depression (LTD) at parallel fiber (PF)–Purkinje cell synapses is abrogated, and synapse formation with PFs and climbing fibers (CFs) is severely disturbed in GluRδ2−/− Purkinje cells. Recently, we demonstrated that abrogated LTD was restored in GluRδ2−/− Purkinje cells by the virus-mediated expression of the wild-type GluRδ2 transgene (Tgwt) but not by that of mutant GluRδ2 lacking the C-terminal seven residues to which several PDZ proteins bind (TgΔCT7). These results indicated that the C terminus of GluRδ2 conveys the signal(s) necessary for LTD. In contrast, other phenotypes of GluRδ2−/− cerebellum, especially morphological abnormalities at PF and CF synapses, could not be rescued by virus-mediated transient expression. Thus, whether these phenotypes are mediated by the same signaling pathway remains unclear. To address these issues and to further delineate the function of GluRδ2 in vivo, we generated transgenic mice that expressed TgΔCT7 on a GluRδ2−/− background. Interestingly, although TgΔCT7 restored abnormal PF and CF synapse formation almost completely, it could not rescue abrogated LTD in GluRδ2−/− Purkinje cells. Furthermore, although the gross motor discoordination of GluRδ2−/− mice was restored, the cerebellar motor learning underlying delayed eyeblink conditioning remained impaired. These results indicate that LTD induction and motor learning are regulated by signaling via the C-terminal end of GluRδ2, whereas other functions may be differentially regulated by other regions of GluRδ2.

Anterograde C1ql1 Signaling Is Required in Order to Determine and Maintain a Single-Winner Climbing Fiber in the Mouse Cerebellum

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 a crucial 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-adhesion G-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.

The N-Terminal Domain of GluD2 (GluRδ2) Recruits Presynaptic Terminals and Regulates Synaptogenesis in the Cerebellum In Vivo

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 (GluD2NTD–GluK2) did. Similarly, GluD2wt and GluD2NTD–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.

Characterization of a transneuronal cytokine family Cbln − regulation of secretion by heteromeric assembly

Cbln1, a member of the C1q and tumor necrosis factor superfamily, plays crucial roles as a cerebellar granule cell‐derived transneuronal regulator of synapse integrity and plasticity in Purkinje cells. Although other Cbln family members, Cbln2–Cbln4, have distinct spatial and temporal patterns of expression throughout the CNS, their biochemical and biological properties have remained largely uncharacterized. Here, we demonstrated that in mammalian heterologous cells, Cbln2 and Cbln4 were secreted as N‐linked glycoproteins, like Cbln1. In contrast, despite the presence of a functional signal sequence, Cbln3 was not secreted when expressed alone but was retained in the endoplasmic reticulum (ER) or cis‐Golgi because of its N‐terminal domain. All members of the Cbln family formed not only homomeric but also heteromeric complexes with each other in vitro. Accordingly, when Cbln1 and Cbln3 were co‐expressed in heterologous cells, a proportion of the Cbln1 proteins was retained in the ER or cis‐Golgi; conversely, some Cbln3 proteins were secreted together with Cbln1. Similarly, in wild‐type granule cells expressing Cbln1 and Cbln3, Cbln3 proteins were partially secreted and reached postsynaptic sites on Purkinje cell dendrites, while Cbln3 was almost completely degraded in cbln1‐null granule cells. These results indicate that like Cbln1, Cbln2 and Cbln4 may also serve as transneuronal regulators of synaptic functions in various brain regions. Furthermore, heteromer formation between Cbln1 and Cbln3 in cerebellar granule cells may modulate each others trafficking and signaling pathways; similarly, heteromerization of other Cbln family proteins may also have biological significance in other neurons.

Characterization of the δ2 Glutamate Receptor-binding Protein Delphilin SPLICING VARIANTS WITH DIFFERENTIAL PALMITOYLATION AND AN ADDITIONAL PDZ DOMAIN

The glutamate receptor δ2 (GluRδ2) is predominantly expressed at parallel fiber-Purkinje cell postsynapses and plays crucial roles in synaptogenesis and synaptic plasticity. Although the mechanism by which GluRδ2 functions remains unclear, its lack of channel activity and its role in controlling the endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors have suggested that GluRδ2 may convey signals by interacting with intracellular signaling molecules. Among several proteins that interact with GluRδ2, delphilin is unique in that it is selectively expressed at parallel fiber-Purkinje cell synapses and that, in addition to a single PDZ domain, it contains a formin homology domain that is thought to regulate actin dynamics. Here, we report a new isoform of delphilin, designated as L-delphilin, that has alternatively spliced N-terminal exons encoding an additional PDZ domain. Although original delphilin, designated S-delphilin, was palmitoylated at the N terminus, this region was spliced out in L-delphilin. As a result, S-delphilin was associated with plasma membranes in COS cells and dendritic spines in hippocampal neurons, whereas L-delphilin formed clusters in soma and dendritic shafts. In addition, S-delphilin, but not L-delphilin, facilitated the expression of GluRδ2 on the cell surface. These results indicate that, like PSD-95 and GRIP/ABP, delphilin isoforms with differential palmitoylation and clustering capabilities may provide two separate intracellular and surface GluRδ2 pools and may control GluRδ2 signaling in Purkinje cells.