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Science 328 (5976): 363-368

Copyright © 2010 by the American Association for the Advancement of Science

Cbln1 Is a Ligand for an Orphan Glutamate Receptor {delta}2, a Bidirectional Synapse Organizer

Keiko Matsuda1, Eriko Miura1, Taisuke Miyazaki2, Wataru Kakegawa1, Kyoichi Emi1, Sakae Narumi1, Yugo Fukazawa3, Aya Ito-Ishida1,4, Tetsuro Kondo1,5, Ryuichi Shigemoto3, Masahiko Watanabe2, and Michisuke Yuzaki1,*

1 Department of Physiology, School of Medicine, Keio University, Tokyo 160-8582, Japan.
2 Department of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan.
3 Division of Cerebral Structures, National Institutes for Physiological Sciences, Okazaki 444-8787, Japan.
4 Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan.
5 Molecular Neurophysiology, Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan.


Figure 1
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Fig. 1. Cbln1 binds to the NTD of GluD2. (A and B) Cbln1 binding assay. Binding to HA-Cbln1 (red) on HEK293 cells (GFP, green) expressing GluD2, GluK2, or chimeric constructs was visualized by immunostaining (A) and quantified using immunoblot analysis (B). The mean amount of HA-Cbln1 bound to cells expressing GluD2 was defined as 100%. Three independent experiments. **P = 3.56 x 10–5. Scale bar, 50 µm. (C) Direct interaction between HA-Cbln1 and the NTD of GluD2. The NTD of iGluRs or the extracellular domain of CD4 was fused to Fc and coupled to protein G beads. Bound HA-Cbln1 was quantified using an immunoblot analysis. (D) Dose-dependent interaction between HA-Cbln1 and the NTD of GluD2 immobilized on beads. Each point represents the mean amount of bound HA-Cbln1 (filled circles) or HA-CS-Cbln1 (open circles) for three independent experiments, quantified using an immunoblot analysis. The line indicates a logistic equation. (E) Confocal images of Purkinje cells immunostained for calbindin (calb; green) and HA (red). Purkinje cell dendrites marked by the white boxes are enlarged below. Scale bars, 50 µm and 20 µm. (Right) HA-Cbln1 immunoreactivity along the dendrites is quantified. At least 27 cells for each group were analyzed in three independent experiments. **P = 2.37 x 10–16. (F) Wild-type (wt) and GluD2-null cerebella immunostained for Cbln1 (red) and calbindin (green). (Bottom) Enlarged images of the molecular layer. Arrows indicate Cbln1 immunoreactivity on the dendritic spines. Scale bars, 20 µm and 1 µm. (G) Postembedding immunogold EM images of endogenous Cbln1. Cbln1 and vesicular glutamate transporter 1 (a marker for parallel fiber) were immunolabeled with 10-nm (arrows) and 15-nm gold particles, respectively. Arrowheads indicate the edges of the PSD on Purkinje cell spines (sp). Scale bar, 100 nm.

 

Figure 2
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Fig. 2. Cbln1 and the NTD of GluD2 cooperatively induce synaptogenesis in vitro and in vivo. (A) HEK293 cells expressing GluD2, GluD2{triangleup}NTD, or GluK2 and GFP were cocultured with wild-type (wt) or cbln1-null cerebellar granule cells with or without HA-Cbln1 or HA-CS-Cbln1. HEK293 cells were immunostained for GFP (green) and synaptophysin (Syn; red) and the mean intensities of synaptophysin immunoreactivity in the GFP-positive area are quantified. Scale bar, 50 µm. At least 24 fields were analyzed in three independent experiments. **P = 9.22 x 10–22. (B) GluD2NTD and Cbln1 induced functional synapses of granule cells. GluD2NTD-Fc or HA-Fc was conjugated with protein G beads (green) and added to cbln1-null cerebellar granule cells with or without HA-Cbln1. Functional presynaptic terminals were labeled with FM4-64 (red), and the mean intensities in the bead areas are quantified. Arrows indicate FM4-64 fluorescence around the beads. Scale bar, 20 µm. At least 150 beads were analyzed for each group in two independent experiments. **P = 9.52 x 10–49. (C) EM analysis on the effect of HA-Cbln1. Two days after we injected HA-Cbln1, the percentage of normal synapses (asterisks) was counted in cbln1-null, GluD2-null, and cbln1/GluD2-null mice. Free spines and mismatched synapses are indicated by f and m, respectively. Scale bars, 500 nm. **P = 5.14 x 10–4. (D) Functional restoration of PF synapses. PF-evoked EPSC traces (middle traces) were recorded in cbln1/GluD2-null Purkinje cells transduced with Sin-GFP or Sin-GluD2-GFP after the subdural injection of HA-Cbln1 in acutely prepared cerebellar slices. GluD2 (red) is expressed in GFP (green)–positive Purkinje cell spines (arrows). Scale bars, 20 µm and 2 µm. The averaged input-output relationship of PF-EPSCs for each condition (n = 30 each) is summarized in the lower graph. **P = 0.0032 (+HA-Cbln1+GluD2 versus +cont.), **P = 0.001 (+HA-Cbln1+GluD2 versus +GluD2) at 200 µA.

 

Figure 3
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Fig. 3. Cbln1 is a direct presynaptic organizer. (A to C) HA-Cbln1 or HA-CS-Cbln1 was conjugated with beads and added to cbln1-null granule cells at 8 DIV as illustrated in the schematic diagram. (A) HA-Cbln1 immobilized on beads was sufficient for the accumulation of presynaptic terminals. (Left) Confocal images of granule cells (13 DIV) immunostained for synapsin I (red) and beads (green). Regions marked by the white boxes are enlarged below. Scale bars, 50 µm and 20 µm. The averaged intensity of synapsin I in the bead area is quantified on the right. At least 30 fields were analyzed in three independent experiments. **P = 1.05 x 10–11. (B) HA-Cbln1 immobilized on beads was sufficient for the accumulation of functional presynaptic terminals. At 13 DIV, functional presynaptic terminals were labeled with FM4-64 (red). Scale bar, 20 µm. (C) Synaptic terminals were directly induced by HA-Cbln1–coated beads. Synapsin I–immunopositive terminals (red) were induced around HA-Cbln1–coated beads (arrowheads), which were located at extrasynaptic sites lacking endogenous AMPA receptors (pan GluA, green). Scale bar, 20 µm.

 

Figure 4
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Fig. 4. Cbln1 directly promotes the clustering of GluD2 and other postsynaptic molecules. (A to C) Control or HA-Cbln1–conjugated beads were added to cbln1-null Purkinje cells at 7 DIV, as illustrated in the schematic diagram. (A) HA-Cbln1 immobilized on beads caused the clustering of endogenous GluD2 (red) in Purkinje cells (calbindin; green) at 10 DIV. Dendrites marked by the white boxes in the top panels are enlarged below. Scale bars, 50 µm and 20 µm. (B) HA-Cbln1–coated beads (blue) caused the clustering of GluD2 (green) and indicated postsynaptic proteins (red) in cbln1-null Purkinje cells. Arrowheads indicate accumulated GluD2 around the beads. Scale bar, 20 µm. (C) Accumulation of postsynaptic molecules induced by HA-Cbln1–coated beads requires GluD2. HA-Cbln1–conjugated beads (blue) were added to cbln1-null or cbln1/GluD2-null Purkinje cells and immunostained for calbindin (green) and each postsynaptic marker (red). Scale bar, 20 µm. (D) The density of endogenous GluD2 was reduced in cbln1-null synapses in vivo. GluD2 immunopositive particles (blue) were visualized using the SDS-FRL method in the molecular layer of the wild-type or cbln1-null cerebellum. The density of GluD2 in intact synapses accompanied by the presynaptic protoplasmic face is plotted as an occurrence histogram. At least three replicates were analyzed for two mice. **P = 2.55 x 10–12. (E) Proposed mechanism for Cbln1-GluD2 signaling as a bidirectional synaptic organizer.

 


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