Published papers

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 (Tg ΔCT7). 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. Copyright © 2008 Society for Neuroscience.

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 other's trafficking and signaling pathways; similarly, heteromerization of other Cbln family proteins may also have biological significance in other neurons. © The Authors (2007).

The glutamate receptor δ2 (GluR δ2) 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 δ-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 Ldelphilin. 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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

The number of each subclass of ionotropic glutamate receptors (iGluRs) at the spines is differentially regulated either constitutively or in a neuronal activity-dependent manner. The δ2 glutamate receptor (GluRδ2) is abundantly expressed at the spines of Purkinje cell dendrites and controls synaptic plasticity in the cerebellum. To obtain clues to the trafficking mechanism of the iGluRs, we expressed wild-type or mutant GluRδ2 in cultured hippocampal and Purkinje neurons and analyzed their intracellular localization using immunocytochemical techniques. Quantitative analysis revealed that deletion of the 20 amino acids at the center of the C terminus (region E) significantly reduced the amount of GluRδ2 protein at the spines in both types of neurons. This effect was partially antagonized by the inhibition of endocytosis by high dose sucrose treatment or coexpression of dominant negative dynamin. In addition, mutant GluRδ2 lacking the E region (GluRδ2ΔE), but not wild-type GluRδ2, was found to colocalize with the endosomal markers Rab4 and Rab7. Moreover, the antibody-feeding assay revealed that GluRδ2ΔE was internalized more rapidly than GluRδ2wt. These results indicate that the E region (more specifically, a 12-amino-acid-long segment of the E2 region) is necessary for rendering GluRδ2 resistant to endocytosis from the cell surface at the spines. Furthermore, insertion of the E2 region alone into the C terminus of the GluR1 subtype of iGluRs was sufficient to increase the amount of GluR1 proteins in the spines. Therefore, we propose that the E2 region of GluRδ2 is necessary, and also sufficient, to inhibit endocytosis of the receptor from postsynaptic membranes. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Of the two members of the δ subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knock-out mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knock-out mice, the density of PF synapses on interneuron somata was significantly reduced and the size and number of interneurons were significantly diminished. Therefore, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex; that is, GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function. © 2014 the authors.

Functions of ionotropic glutamate receptors (iGluRs) are tightly regulated by the intracellular trafficking of receptor proteins. Unlike other iGluRs that are considerably retained in the intracellular component, the δ2 glutamate receptor (GluRδ2) is efficiently expressed on the Purkinje cell surface. To understand the trafficking mechanism of iGluRs, we deleted various portions of the C-terminal intracellular domain of GluRδ2 and analysed the localization of the mutant proteins in heterologous cells and neurons. Biotinylation assays indicated that GluRδ2 lacking the C-terminal juxtamembrane region of 13 amino acids (region A) was not present on the cell surface. This mutant GluRδ2 was sensitive to endoglycosidase H, which digests unprocessed high-mannose oligosaccharides on proteins retained in the endoplasmic reticulum (ER) or cis-Golgi. Therefore, we concluded that region A is crucial for the transport of GluRδ2 beyond the trans-Golgi to the cell surface. Because the immunostaining pattern of GluRδ2 lacking region A in cultured hippocampal neurons completely overlapped the pattern of fluorescence emitted by ER-resident green fluorescent protein, region A is most likely necessary for GluRδ2's exit from the ER. Furthermore, this region is essential for the proper intracellular trafficking of GluRδ2 in Purkinje cells. Region A does not rely on a dihydrophobic motif or positively charged residues to participate in trafficking, but its function is dependent on the juxtamembrane position. Therefore, we propose that GluRδ2's efficient transport to the cell surface utilizes an unknown but general ER exit mechanism, which probably works in close relation to the membrane of heterologous cells and neurons.

The NMDA receptor 3B (NR3B) subunit is the most recently identified member of the NMDA receptor family. In heterologous cells, it has been shown to reduce the Ca2+ permeability of glutamatergic receptor complexes formed together with NR1 and NR2 subunits and to form the unique excitatory glycine receptor complex with the NR1 subunit. However, it is unclear whether NR3B protein is expressed in and exerts similar functions in neurons. In addition, it is not understood how NR3B interacts with NR1 and NR2 and how such an interaction may regulate the membrane trafficking of the NMDA receptor complex. Here we report that our analysis using an antibody specific for NR3B showed that the NR3B protein is selectively expressed in somatic motor neurons in the brainstem of adult mice. Coimmunoprecipitation and electrophysiological analyses demonstrated that NR3B, when exogenously introduced into hippocampal neurons, can coassemble with endogenous NR1 and NR2A and can reduce the Ca 2+ permeability of NMDA currents. In contrast, NR3B was not involved in the excitatory glycine response in neurons under our test conditions. Although NR1 or NR3B alone cannot be transported to the cell surface, coexpression of these subunits mutually supported transport of the NMDA receptor complex by interaction involving the specific regions of the C terminus of NR3B. These results indicate that NR3B may modulate the function of NMDA receptors in somatic motor neurons during adulthood by controlling membrane trafficking and by reducing Ca2+ permeability.