Elucidation of molecular mechanisms that regulate synapse formation is prerequisite for the understanding of neural wiring, higher brain functions and mental disorders. Despite the wealth of information, the fundamental questions about how glutamatergic synapses are formed in the mammalian brain remain unanswered. In cerebellum, there is clear in vivo evidence that GluRδ2, a member of the δ-type glutamate receptor (GluR), plays an essential role in cerebellar Purkinje cell (PC) synapse formation (Kashiwabuchi et al., 1995; Kurihara et al., 1997; Takeuchi et al., 2005). The cerebellum receives two excitatory afferents, the climbing fiber (CF) and the mossy fiber-parallel fiber (PF) pathway, both converging onto PCs that are the sole neurons sending outputs from the cerebellar cortex. GluRδ2 is selectively expressed in cerebellar PCs (Araki et al., 1993; Lomeli et al., 1993) and is exclusively localized at PF-PC synapses (Takayama et al., 1996; Landsend et al., 1997). A significant number of PC spines lack synaptic contacts with PF terminals and some of residual PF-PC synapses show mismatching between pre- and postsynaptic specializations in conventional and conditional GluRδ2 knockout mice (Kashiwabuchi et al., 1995; Kurihara et al., 1997; Takeuchi et al., 2005). These studies indicate that the formation and maintenance of PF-PC synapses are critically dependent on GluRδ2 in vivo. The synaptogenic activity of GluRδ2 is reproduced in vitro using primary cultures of cerebellar granule cells (GCs). The extracellular N-terminal domain (NTD) of GluRδ2 is essential and sufficient to induce synapse formation in vitro (Uemura and Mishina, 2008). Thus, it is likely that GluRδ2 regulates synapse formation by direct interaction between its NTD and presynaptic protein(s). Here, I show that the NTD of GluRδ2 interacts with presynaptic neurexins (NRXNs) through Cbln1. According to the immunocytochemistry, β-NRXNs interacted, in the presence of Cbln1, with GluRδ2 expressed on the surface of HEK 293T cells. And in a protein pull-down assay, Cbln1 was coprecipitated with NTD of GluRδ2 but not with that of GluRα1 or GluRα2 and it was also coprecipitated with extracellular domain (ECD) of NRXN1β. The kinetic analysis by Surface Plasmon Resonance (SPR) binding assay showed both dissociation constant (KD) values, 16.5nM for Cbln1-GluRδ2 and 0.17nM for Cbln1-NRXN1β, indicating that they interact with high affinities. These suggest that Cbln1 directly binds to the N-terminal domain of GluRδ2 and also to the extracelllar domain of NRXN1β with high affinities, acting as a divalent ligand to link postsynaptic GluRδ2 and presynaptic NRXNs in the cerebellar synapse (Figure 1).

Next, I examined the stoichiometry for the assembly of GluRδ2-Cbln1-NRXN1β triad. NTD of GluRδ2 (GluRδ2-NTD) was firstly examined for its own stoichiometry. Native-PAGE and Gel-filtration assay showed that the GluRδ2-NTD exists as a homodimer with non-disulfide interaction. Cbln1 well known molecule as a hexamer interacted with the GluRδ2-NTD. One molecule of the Cbln1 participated in the interaction with one dimeric GluRδ2-NTD. On the other hand, when the hexameric Cbln1 interacted with NRXN1β which is known as a monomer, it bound to two NRXN1βs. With the mutation assay, I examined which regions of Cbln1 are important for the interaction with each GluRδ2 or NRXN1β. The results indicated that N-terminus of Cbln1 is relatively important to interact with NRXN1β-ECD but its C-terminus is for GluRδ2-NTD, although the interaction properties of Cbln1 to NRXN1β or GluRδ2 seemed to be regulated by its overall structure. GluRδ2 exists as a tetramer form in the plasmamembrane because the GluRδ subfamily belongs to the ionotropic GluR family and positions between the classical AMPA/kainate and NMDA subtypes from the amino acid sequence identity, (Yamazaki et al., 1992; Araki et al., 1993; Lomeli et al., 1993). Thus, the tetrameric GluRδ2, according to the present results, interacts with two Cbln1s and four NRXNs (Figure 2).

Altogether, the present results suggest that GluRδ2 interacts with NRXNs through Cbln1 for synapse formation between Purkinje cell and that parallel fiber in cerebellum and the interaction stoichiometry of NRXN-Cbln1-GluRδ2 is 4:2:1.

本研究は小脳のシナプス形成過程において重要な役割を演じていると考えられるδ2-type glutamate receptor(GluRδ2)のシナプス形成における役割を明らかにするため、GluRδ2の結合相手を調べ、その複合体の化学的性質の解明を試みたものであり、下記の結果を得ている。

1. ポストシナプスに存在するGluRδ2のN-terminal domain(NTD)に結合する蛋白質をクロスリンカーを用いて架橋した。取れた蛋白質を質量分析にかけ、蛋白質の同定をした結果、分泌蛋白であるCbln1とプレシナプスの膜蛋白質が得られた。得られた蛋白質を用いてCell-surface binding assayおよびPull-down assayを行い、GluRδ2はCbln1を介してプレシナプスのNeurexinと結合する事が明らかとなった。

2. シナプス結合分子、Neurexin, Cbln1, GluRδ2の複合体の化学的結合形式をゲルろ過とCalorimetryを利用して調べた。その結果、一分子のGluRδ2は二分子のCbln1と結合してさらに四分子のNeurexinと結合することが示された。

以上、本論文ではシナプス形成におけるシナプス接着分子の新しい結合様式の発見した。その中で、Cbln1とGluRδ2のノクアウトマウスのPhenotypeを説明する分子メカニズムを本研究で明らかにした。さらに、本研究でNeurexin-Cbln-GluRδ2複合体の化学的結合形式が解明され、三者複合体のシナプス形成における役割も提案できた。本研究はこれまで未知に等しかった、小脳でのシナプス形成と新しい形式のシナプス結合様式を明らかにするものであり、脳の神経ネットワークの形成機構の解明に重要な貢献をすると考えられ、学位の授与に値するものと考えられる。